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PMC11699263 | Section title: Introduction Educational score: 4.08517599105835 Domain: biomedical Document type: Study Language: en The anterior region of the maxilla undergoes many surgical interventions. Dental implant placement, surgeries of supernumerary impacted teeth and cysts, and orthognathic surgeries are some of these interventions in the anterior maxilla. 1 However, the most important of all these is the increasing demand for dental implants. Thus, a more precise anatomical investigation of this segment is essential due to the presence of canalis sinuosus (CS) in the anterior maxilla. 2 CS is a neurovascular canal about 2 mm in diameter, which carries a branch of nerves of the infraorbital canal, the anterior superior alveolar nerve (ASA), and related vessels. 3 , 4 The infraorbital nerve is a branch of the maxillary nerve, which is the second branch of the fifth cranial nerve, i.e., the trigeminal nerve. The skin distribution of the infraorbital nerve extends to the upper lip, cheeks, lower eyelids, outer nose, and nasal cavity. 5 Section title: Introduction Educational score: 4.033580303192139 Domain: biomedical Document type: Other Language: en On cone-beam computed tomography (CBCT) images, CS is a curved bone canal originating laterally from the infraorbital canal. It passes through an internal and anterior course to reach the maxillary anterior region, passes through the lateral wall of the nose, and is placed in the marginal part of the nasal cavity floor; then, lateral canals branch off, eventually opening next to the incisor canal in the palate. 6 CS detection is important because damage to such structures might cause sensory disorders. In addition, such structures may be mistaken for other anatomical structures or lesions, leading to unnecessary or incorrect procedures. 7 Therefore, accurately identifying the anatomy of the face, mouth, and jaws and using radiographic images, specifically CBCT in surgical procedures, are necessary to avoid the destruction of blood vessels and nerves in this area. 8 Section title: Introduction Educational score: 3.9246859550476074 Domain: biomedical Document type: Study Language: en The anatomy of the CS 9 - 13 and its accessory canals 14 - 19 has been evaluated in numerous studies. A systematic review study conducted in 2023 considered CS and its accessory canals as anatomic structures due to their high prevalence. 20 However, a few studies have quantitatively evaluated the canal’s relationship with adjacent structures. 4 Therefore, this study investigated the exact position of CS relative to adjacent teeth and its distance from adjacent anatomical structures in CBCT images of an Iranian population. Section title: Methods Educational score: 2.560286521911621 Domain: biomedical Document type: Study Language: en The present retrospective study was conducted on 400 CBCT images of the anterior maxilla of Iranian patients referred to a private dental and maxillofacial radiology center in Urmia, Iran. Section title: Methods Educational score: 2.529785633087158 Domain: biomedical Document type: Study Language: en The inclusion criteria: (1) a chronological age of 12−90, (2) no history of systemic disease due to osteoporosis, (3) and good diagnostic quality of images. Section title: Methods Educational score: 2.4778454303741455 Domain: biomedical Document type: Study Language: en The exclusion criteria: the images of edentulous patients and patients with dentoalveolar fractures, pathologic conditions, dental implants, or bone grafting in the anterior maxilla. Section title: Methods Educational score: 3.323244333267212 Domain: biomedical Document type: Study Language: en The CBCT images were captured using an 8-cm field of view of Planmeca Promax 3D mid (Helsinki, Finland) with the following conditions: voxel size: 200 μm; time: 12 s; mA: 10; kVp: 90. Section title: Methods Educational score: 4.143873691558838 Domain: biomedical Document type: Study Language: en Two observers investigated all the images twice with a time interval of one month. The first observer was an experienced periodontist, and the second observer was an experienced oral and maxillofacial radiologist. Observations and measurements were carried out by Planmeca Romexis software version 3.8.1. The presence of CS was defined in axial and sagittal planes, and its clear extension was confirmed in the coronal sections. CS diameter was measured on the axial plane. Canals with a diameter of > 1 mm and a definite extension to the infraorbital canal were considered . Slice thickness and interval were 0.5 in all the sections. The axial plane in which the CS had the largest diameter was chosen to measure the distance of the CS to the adjacent teeth and the nasopalatine canal. The tooth with the closest distance to the CS was considered the main tooth; then, the mesial, mid, or distal position of the main tooth related to the CS was determined. The distances of CS from the nasal floor, ridge crest, buccal cortical plate, and the distance of canal extension from the main tooth apex were measured on the cross-sections perpendicular to the axial plane at the CS site . Section title: Methods Educational score: 3.862691640853882 Domain: biomedical Document type: Study Language: en Intra- and inter-observer reliability was evaluated in 10% of the CBCT images after two weeks using the intraclass correlation coefficient (ICC). Section title: Statistical analysis Educational score: 3.9116480350494385 Domain: biomedical Document type: Study Language: en The data were analyzed with SPSS 22 using descriptive statistics, i.e., maximum, minimum, mean, and standard deviation. The ICC value was ˃0.80 for both intra- and inter-observer reliabilities. Pearson’s correlation coefficient was used to determine the relation between two quantitative variables. Fisher’s exact test was used to compare CS distribution in terms of sex and location. T-test was used to compare distance measurements between males and females or between the right and left sides. P ≥ 0.05 was considered statistically insignificant. Section title: Results Educational score: 4.008119583129883 Domain: biomedical Document type: Study Language: en CBCT images of 400 patients were investigated, with 185 males (46.3%) and 215 (53.7%) females. The mean age of the patients was 43.06 ± 13.60, with a range of 20–86 years. Forty-two patients (10.5%) had at least one CS with a clear extension towards the infraorbital canal, with a diameter of > 1 mm; 20 (47.6%) were female, and 22 (53.4%) were male. In 13 patients (30.95%), the canal was bilateral, and in 29 (69.05%), the canal was unilateral. In 25 (86.2%) patients, the canal was on the right side, and in 4 (13.80%), the canal was on the left side; generally, 55 canals were observed in images. Section title: Results Educational score: 4.024277687072754 Domain: biomedical Document type: Study Language: en Table 1 shows the mean diameters of CS in both sexes. There was no significant difference in the frequency of cases between the two genders ( P > 0.05). No significant correlation was observed between CS diameter and age ( P = 0.101) or sex ( P = 0.284). CS was more frequent on the right side than on the left side, but this difference was not significant ( P > 0.05) ( Table 2 ). The CS had the closest distance to the lateral incisor in most cases. The mid-position of the CS compared to the main tooth was significantly more common than other positions ( P = 0.04). Section title: Results Educational score: 4.029425621032715 Domain: biomedical Document type: Study Language: en The average distances of CS from the nasal floor, ridge crest, buccal cortical plate, main tooth apex, and nasopalatine canal were higher in males than in females; this difference was not statistically significant except for the buccal cortical plate distance and perpendicular distance to the nasopalatine canal ( Table 3 ). The distance of CS from neighboring structures on the right and left sides was not statistically significant ( P > 0.05). Section title: Discussion Educational score: 2.428687572479248 Domain: biomedical Document type: Other Language: en CS is a branch of the infraorbital nerve, which is the maxillary division of the trigeminal nerve. Damage to this canal during surgery can lead to complications such as bleeding and paresthesia. CS can be detected in special imaging modalities such as thin sections of CBCT. 4 Section title: Discussion Educational score: 2.6807286739349365 Domain: biomedical Document type: Study Language: en This study evaluated the location and distance of CS relative to adjacent structures in the Iranian population because of anatomical variations between different populations. Section title: Discussion Educational score: 4.029975891113281 Domain: biomedical Document type: Study Language: en The prevalence of CS in this study was 10%, which was close to a study by de Oliveira-Santos et al. (15.7%) 21 ; this prevalence was about 35% in a study by Manhães Júnior et al, 4 66.5% in a study by Aoki et al, 13 88% in a study by Wanzeler et al, 5 and 98.5% in a study by Yeap et al. 22 However, the prevalence of CS in a study by Gurler et al 23 was 100%. The possible reasons for this discrepancy include differences in the slice thickness of the CBCT images, 23 the software used, sample size, and the content of studies. 11 In addition, in this study, cases traceable to the infraorbital canal with a diameter of > 1 mm were selected, which could be another reason for the difference. Section title: Discussion Educational score: 3.7062504291534424 Domain: biomedical Document type: Study Language: en The canal was bilateral in 13 patients (30.95%) of the 42 patients with CS. The frequency percentage of bilateral cases of CS was various in other literature as follows: de Oliveira-Santos et al. 21 reported 21.4%, Manhães et al 4 reported 24.3%, Aoki et al 13 reported 54.1%, and Gurler et al 23 reported 100%. Section title: Discussion Educational score: 2.656949043273926 Domain: biomedical Document type: Study Language: en There was no significant difference in the overall frequency of CS on both sides ( P = 0.41), consistent with the Manhães et al. study. Section title: Discussion Educational score: 3.530846357345581 Domain: biomedical Document type: Study Language: en Gender distribution of CS was not statistically significant, consistent with studies by Gurler et al, 23 Von Arks et al, 6 Machado et al, 19 and Wanzeler et al. 5 In a study by Anatoly et al, 24 the prevalence of CS was significantly higher in females, while this prevalence was significantly higher in males in Aoki et al. study. 13 Section title: Discussion Educational score: 4.01983642578125 Domain: biomedical Document type: Study Language: en The mean (SD) diameter of CS was 1.06 (0.26) mm, which was significantly ( P = 0.284) higher in males [mean (SD) diameter in males = 1.10 (0.31) vs. females = 1.02 (0.28)]. In the study by Aoki et al, 13 this difference was not significant, either. Gurler et al. 23 reported a significantly higher mean diameter of CS in males ( P = 0.001). Section title: Discussion Educational score: 3.786699056625366 Domain: biomedical Document type: Study Language: en Because of the difficulty of some cases in precisely attributing the CS to the specific tooth, 16 Beyzade et al 25 modified the classification used by Oliveira-Santos et al. 21 For the same reason, in this study, CS’s distance to the neighboring teeth was determined in axial plane in which the CS had the most diameter, at first. The position of the CS with regard to the closest tooth was determined at the second. CS was most related to the mid-position of lateral incisors. CS was more commonly associated with this tooth in other populations. 4 , 11 , 22 , 24 Section title: Discussion Educational score: 4.034453392028809 Domain: biomedical Document type: Study Language: en In this study, CS-to-the-buccal cortical plate and CS-perpendicular-to-nasopalatine canal distances were significantly higher in males, so the CS location was more palato-lateral. In the Manhães Júnior et al 4 study, the nasal cavity distance was higher than in our study, and the ridge crest and the buccal cortical plate distances were lower than in our study, so the CS was located in a more buccal position and closer to the crest in Manhães Júnior and colleagues’ study. Section title: Conclusion Educational score: 2.330476999282837 Domain: biomedical Document type: Study Language: en The prevalence, diameter, and most of the linear distances of the CS to the adjacent structures were not significantly different between the two genders. However, CS had a significantly more palato-lateral position in males. There were variations in the prevalence, location, and linear distances between different populations. Section title: Acknowledgments Educational score: 0.850701630115509 Domain: other Document type: Other Language: en The authors would like to thank Dr. Majid Abdolrahimi for his cooperation in writing the manuscript. Section title: Competing Interests Educational score: 0.9564493298530579 Domain: other Document type: Other Language: en The authors declare that they have no competing interests. Section title: Consent for Publication Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. Section title: Data Availability Statement Educational score: 0.9841994643211365 Domain: other Document type: Other Language: en The data that support our results are available upon reasonable request from the corresponding author, Maryam Mostafavi. Section title: Ethical Approval Educational score: 0.9524725079536438 Domain: other Document type: Other Language: en This study was approved by the Ethics Committee of Urmia University of Medical Sciences with the ethical number of IR.UMSU.REC.1397.064. There was no conflict with ethical considerations. | Study | biomedical | en | 0.999996 |
PMC11699265 | Section title: Introduction Educational score: 2.377279043197632 Domain: biomedical Document type: Other Language: en Using dental implants to address esthetic zones brought a new challenge for clinicians. The effective osseointegration of the implant, harmony between the final restoration and the neighboring teeth, and the health of the surrounding soft and hard tissues are the aims of such restoration. 1 Section title: Introduction Educational score: 4.118165969848633 Domain: biomedical Document type: Study Language: en Four factors should be addressed when evaluating an implant site in an esthetic zone: smile line, soft-tissue morphology, tooth morphology, and osseous architecture. 2 The placement of an implant in an appropriate connection to the intended restoration is critical for the best esthetic and functional outcomes, which would require sufficient alveolar bone volume and position. 3 - 5 Various surgical methods, such as distraction osteogenesis, 6 guided tissue regeneration, 7 and graft procedures, have been suggested to maintain or repair the alveolar ridge. 8 These techniques can be used to treat the ridge defect at the time of extraction or later. Orthodontic forced eruption (OFE) is another method of soft and hard tissue augmentation. According to Heithersay 9 and Ingber, 10 orthodontic treatment causes the periodontal ligament to be pulled during eruption, which increases bone volume and causes osteoblastic activity to occur where the periodontal attachment is located. 11 The gingiva and bone linked by the periodontal ligaments migrate in the same direction of the tooth’s coronal movement. 12 Additionally, this method may be used to move the root, creating room and anchoring for an implant. In 1993, Salama and Salama 13 suggested modifying the forced eruption method. By forced orthodontic extrusion of “hopeless” teeth and their periodontal structures, this novel technique, known as “orthodontic extrusive remodeling,” was employed to improve the soft and hard tissue profiles of possible implant sites. 13 Hence, the present study aimed to assess the effect of site preparation of dental implants using OFE in hopeless teeth. Section title: Methods Educational score: 3.899223566055298 Domain: biomedical Document type: Review Language: en This systematic review was conducted according to the PRISMA (Preferred Reporting Items for Systemic Reviews and Meta-Analyses) guidelines statement. Section title: Focused question Educational score: 3.1928226947784424 Domain: biomedical Document type: Study Language: en The following focused question was formulated to outline the search strategy: “What is the effect of orthodontic extrusion on site development for implant placement in the esthetic zone?” with “site development” referring to the optimization of the implant spatial positioning and minimizing the need for adjunctive regenerative treatments. Section title: Search strategy Educational score: 3.486844778060913 Domain: biomedical Document type: Study Language: en A search of electronic databases, including Scopus, PubMed/MEDLINE, and Google Scholar, was performed from January 2000 to November 2023. The search was limited to English-language studies with available full texts. Furthermore, a hand search was conducted to assess the studies that were not electronically available. The search was performed based on the following keywords: [orthodontic AND extrusion] AND [dental AND implant], [orthodontic AND extrusion] AND [site AND development], [orthodontic AND extrusion] AND [dental AND implant] AND/OR [[soft OR hard] AND tissue AND [regeneration OR augmentation]]. Section title: Selection criteria Educational score: 3.2416486740112305 Domain: biomedical Document type: Study Language: en All the clinical studies that used orthodontic extrusion for further implant placement in the esthetic zone were included. Abstracts, letters, and reviews were excluded. Studies were excluded if orthodontic extrusion was performed solely or adjacent to a dental implant. De-duplicating was done manually, and finally, eligible studies were included, and their full texts were obtained. Section title: Selection criteria Educational score: 3.7534923553466797 Domain: biomedical Document type: Study Language: en Two independent reviewers assessed the full texts, and the following data were extracted and further classified in a table: type of study, number of patients, gender, age, number of tooth/teeth, type of jaw (maxilla/ mandible), orthodontic movement procedure, implant characteristics, follow-up, and outcome. Any disagreement between reviewers was resolved following discussion. Section title: Results Educational score: 4.0214762687683105 Domain: biomedical Document type: Review Language: en Initially, 57 articles were identified via a search through the above-mentioned databases. After removing duplicate investigations, 37 studies were further considered. After evaluating titles, 7 studies, and after assessing the titles with their corresponding abstracts, 7 more studies were excluded. The full texts of the 23 remaining studies were evaluated based on the predetermined inclusion/exclusion criteria; consequently, 15 studies were included in the current systematic review. Figure 1 shows the strategy flowchart of the present investigation. Section title: Study characteristics Educational score: 3.4494388103485107 Domain: biomedical Document type: Study Language: en All the included studies were case reports. Sixteen individuals have been documented in the literature, where orthodontic extrusion was used to establish implant sites in the esthetic zone. Patients’ age varied from 22 to 57 years and consisted of 11 women and 5 men. A total of 21 teeth underwent extrusion and further extraction and implant placement. All these teeth were maxillary anteriors, and central incisors were the most common. Section title: Study characteristics Educational score: 4.005043029785156 Domain: biomedical Document type: Study Language: en The orthodontic extrusion period varied noticeably between studies, from the shortest period of 3 months 14 to the longest one of 12 months. 15 In two studies, implant insertion was performed 6 15 and 7 16 months after tooth extraction, and in 8 studies, an additional bone grafting procedure was performed before or simultaneous with implant placement. 14 , 16 - 22 The most chosen implant diameter was 3.75 × 11 mm in two studies. 23 , 24 Section title: Study characteristics Educational score: 2.2185781002044678 Domain: biomedical Document type: Study Language: en Among 8 studies mentioning follow-up periods, the shortest time was 11.5 months, 19 and the longest period was 10 years. 16 Table 1 shows more details about the included studies. Section title: Discussion Educational score: 3.905914306640625 Domain: biomedical Document type: Review Language: en Dental implant placement has become the gold standard treatment for replacing missing teeth for a long time. 29 , 30 Bone and tissue loss following inflammatory disease in periodontium could lead to departure from normal alveolar morphology and further difficulties for implant placement where hopeless teeth exist. 24 , 31 OFE has been recommended as the only non-surgical adjunctive way to enhance hard and soft tissue conditions for implant placement. 31 , 32 Hence, the present review evaluated the effect of OFE for implant placement in the esthetic zone. Section title: Discussion Educational score: 3.8560597896575928 Domain: biomedical Document type: Review Language: en Almost all the 15 studies included in our review reported positive outcomes of OFE from clinical, radiographic, and esthetic aspects. Continuous light force was the preferred approach in most of the studies. According to Kim and colleagues’ study, this light force could induce the stretch of gingival and periodontal fibers and further formation of new bone and gingiva in the coronal part. 24 Section title: Discussion Educational score: 3.8883159160614014 Domain: biomedical Document type: Study Language: en In multiple studies, noticeable advantages of OFE for hopeless teeth before implant placement were mentioned: first, the hopeless teeth could aid in oral rehabilitation procedures. Second, the patient’s discomfort would decrease since the hopeless teeth solve esthetic issues, and third, by following this treatment, periodontal ligaments’ capacity as a distraction osteogenesis means would be used by stimulation of biological potential of periodontal ligament and modifying the morphology of an intrabony defect to a desirable one. 11 , 28 , 33 , 34 Section title: Discussion Educational score: 4.056697845458984 Domain: biomedical Document type: Study Language: en However, this treatment modality is not without complications; esthetic problems due to the presence of wires and brackets, phonetic discomfort, difficulty in orthodontic force control, undesirable external root resorption of adjacent teeth, gingival recession, and buccal bone dehiscence could be considered the drawbacks of the above-mentioned technique. 24 , 28 As was observed in Kim and colleagues’ study, the radiographic evaluation revealed apical root resorption in the lateral incisor adjacent to the targeted tooth. This phenomenon mainly contributed to a greater magnitude of force than intended. 24 Section title: Discussion Educational score: 2.543225049972534 Domain: biomedical Document type: Study Language: en On the other hand, OFE has not always provided enough bone, so further surgical regenerative procedures were needed in multiple studies 14 , 16 , 17 , 19 - 22 , 28 to provide acceptable hard tissue morphology. Section title: Discussion Educational score: 3.400836706161499 Domain: biomedical Document type: Other Language: en To precisely perform case selection, good plaque control, resolution of periodontitis, presence of at least 1/3 to 1/4 of apical attachment, and feasibility of adequate stabilization period must be considered. 35 Section title: Discussion Educational score: 3.950728416442871 Domain: biomedical Document type: Study Language: en Magnitude, duration, and retention period varied in the studies. In Joo’s study, 1 mm/month of movement had similar results to 1 mm/week of coronal displacement. 27 However, Isola’s study emphasized using low and controlled ( < 100 g) forces to obtain < 1 mm/month movement. 33 The retention period was reported to be 6‒12 weeks, 27 while in Alsahhaf and Att’s study, a minimum of 3‒6 months was suggested. 30 This variation in OFE procedures necessitates careful examination and individualized treatment planning. Section title: Discussion Educational score: 3.7158093452453613 Domain: biomedical Document type: Review Language: en The current review has its limitations. First, the number of available studies in the literature is limited, which does not allow for a comprehensive conclusion to be drawn. The lack of a long-term follow-up negatively affects the reliability of the outcomes. Moreover, all the studies included in this review are case reports or case series that will negatively affect the quality of the generated evidence, and the interpretation of the results should be carried out with caution. Section title: Conclusion Educational score: 3.51171875 Domain: biomedical Document type: Other Language: en In conclusion, OFE of hopeless teeth seems an acceptable alternative to enhance soft and hard tissue conditions for future implant placement. Nevertheless, additional interventions such as submerged healing and guided bone regeneration should be considered in special cases to achieve the best outcome in the esthetic zone. Section title: Competing Interests Educational score: 0.939154863357544 Domain: other Document type: Other Language: en The authors of the current manuscript declare no conflicts of interest regarding the publication of the presented paper. Section title: Data Availability Statement Educational score: 0.8937035202980042 Domain: biomedical Document type: Other Language: en The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Section title: Ethical Approval Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. | Other | biomedical | en | 0.999998 |
PMC11699266 | Section title: Introduction Educational score: 3.9235098361968994 Domain: biomedical Document type: Review Language: en Despite the evidence showcasing the high success rate of dental implants in oral rehabilitation, healthcare providers must be aware of possible complications after implant placement. 1 , 2 Peri-implant mucositis affects the soft tissue surrounding implants and can progress into peri-implantitis if left untreated. Peri-implantitis is characterized by bleeding on probing (BoP) and/or suppuration, increased probing depth and/or marginal recession alongside progressive radiographic bone loss compared to previous visits . 3 The average prevalence rate of peri-implantitis is 22%, with a range of 1%‒47%. 4 Section title: Introduction Educational score: 4.058259963989258 Domain: biomedical Document type: Study Language: en The incidence of peri-implantitis is associated with the accumulation of bacterial plaque primarily consisting of the microorganisms involved in periodontitis. 3 , 5 However, Porphyromonas gingivalis , Tannerella forsythia , and Treponema denticola are found at higher concentrations in samples obtained from peri-implantitis patients. 6 Factors such as smoking, untreated periodontitis, irregular maintenance, and diabetes mellitus have been identified as risk factors for peri-implantitis. 7 - 10 Local factors such as excess cement, incorrect prosthesis seating, implant malpositioning, implant micro- and macro-design, abutment connection type, and excessive mechanical loads can all contribute to disease progression. 5 , 11 Section title: Introduction Educational score: 4.028098106384277 Domain: biomedical Document type: Review Language: en Peri-implantitis treatments primarily aim to eradicate tissue inflammation, stop disease progression and bone loss, regenerate lost supportive tissues, and restore osseointegration. 12 These treatments encompass diverse surgical and non-surgical approaches, such as mechanical debridement, application of antiseptics, antibiotic therapy, surgical flaps, and resective or regenerative surgeries. 13 - 19 Resective surgery is ideal for shallow defects, while deeper intrabony defects are better suited to regenerative strategies. 20 , 21 A regenerative approach would be preferred if the bony defect has a minimum depth of 3 mm, is enclosed by three or four walls, and sufficient keratinized mucosa is present. 22 In cases where a failing implant is predicted to have a poor prognosis or the aforementioned treatment strategies do not lead to success, explantation is the inevitable choice. 23 Section title: Introduction Educational score: 3.9847140312194824 Domain: biomedical Document type: Review Language: en The regenerative approach involves surface decontamination and the use of bone grafts with or without a barrier membrane. 24 When selecting a treatment strategy, decisions must be made concerning the decontamination method, graft material, barrier membrane, and surgical technique. Decontamination can be achieved through mechanical or chemical methods, such as using acids, antiseptics, abrasives, and lasers. 25 - 28 Moreover, different graft materials can be used, including allografts, autografts, xenografts, etc. 28 , 29 If necessary, a range of resorbable or non-resorbable membranes can be used to cover the graft. 30 Given the multiple biomaterials and techniques reported in the literature, numerous protocols can be used to manage peri-implantitis. 24 , 31 However, the consensus is that no specific biomaterial or treatment protocol for peri-implantitis has proven superior to others. 32 Section title: Introduction Educational score: 3.9401938915252686 Domain: biomedical Document type: Review Language: en Hence, this systematic review aimed to compare various surgical regenerative interventions for peri-implantitis management based on their clinical and radiographic enhancements. Section title: Methodology and protocol registration Educational score: 2.1345646381378174 Domain: biomedical Document type: Study Language: en The study adhered to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines, 33 and the protocol was registered in PROSPERO (International Prospective Register of Systematic Reviews) under the ID CRD42021288572. Section title: Focused question Educational score: 2.9946486949920654 Domain: biomedical Document type: Other Language: en When treating peri-implantitis patients, which surgical regenerative protocols lead to more significant improvements in clinical and radiographic parameters during a minimum follow-up duration of 12 months? Section title: Eligibility criteria Educational score: 2.3387746810913086 Domain: biomedical Document type: Study Language: en A PICO-style search strategy was designed as follows: Section title: Eligibility criteria Educational score: 2.686943292617798 Domain: biomedical Document type: Study Language: en Only randomized and non-randomized clinical trials meeting the following criteria were included: Section title: Eligibility criteria Educational score: 1.9660454988479614 Domain: biomedical Document type: Study Language: en The exclusion criteria were as follows: Section title: Search strategy Educational score: 3.070861339569092 Domain: biomedical Document type: Study Language: en In January 2022, the initial search was performed in electronic databases, including PubMed, Embase, Web of Science, Scopus, Google Scholar, Cochrane CENTRAL, and ProQuest (for grey literature). An updated search was also conducted in May 2022. The search terms used in these electronic databases included the following: ((peri-implantitis) OR (peri-implant disease) OR (peri-implant disease)) AND ((regenerative medicine) OR (biomaterial) OR (regenerative surgery) OR (surgical regeneration) OR (bone graft) OR (bone substitute) OR (membrane) OR (growth factor)) AND ((treatment) OR (management) OR (therapy)). Section title: Search strategy Educational score: 2.2550270557403564 Domain: biomedical Document type: Study Language: en Notably, the search query was tailored to the search guidelines of each database. The reference lists of the included studies were also reviewed to uncover any pertinent studies that might have been overlooked. Additionally, a manual search was conducted in journals related to dental implants and peri-implant diseases to identify any articles that might have been missed in the electronic search. Section title: Screening and data extraction Educational score: 3.6730189323425293 Domain: biomedical Document type: Study Language: en The results were imported into EndNote X20 software (Clarivate Company, Philadelphia, USA), and the duplicates were removed. The titles and abstracts of the remaining articles were independently screened by two reviewers who were unaware of each other’s decisions. After omitting irrelevant results, the full texts of the remaining articles were meticulously read and compared against the inclusion and exclusion criteria. In case of disagreements between the two reviewers, a third reviewer was consulted to reach an agreement. Section title: Screening and data extraction Educational score: 1.5843929052352905 Domain: biomedical Document type: Study Language: en The data extraction was limited to the following items: Section title: Risk of bias assessment Educational score: 4.10297966003418 Domain: biomedical Document type: Study Language: en Two authors independently performed the risk of bias assessment concurrent with data extraction. The Cochrane risk of bias tool for randomized clinical trials was used. 34 The tool evaluates bias across five distinct domains: randomization process, deviations from intended interventions, missing outcome data, outcome measurement, and selection of reported results. A study was deemed to have a “low risk of bias” if all domains displayed a low risk. Conversely, the presence of high risk in even one domain classified the study as having a “high risk of bias.” If a study presented some concerns in at least one domain but did not manifest a high risk in any domain, it was categorized as having “some concerns.” Section title: Data analysis Educational score: 4.070626258850098 Domain: biomedical Document type: Study Language: en The standardized mean difference (SMD) was computed for each outcome measure (BoP, PD, PI, BL, and BG) using Cohen’s d or Hedges’ g. Meta-analysis was performed using the random-effects restricted maximum likelihood (REML) method in Stata version 17 (Stata Corp., College Station, Texas, USA). Potential sources were examined through meta-regression analysis to assess the presence of heterogeneity. Section title: Study selection Educational score: 4.026196002960205 Domain: biomedical Document type: Study Language: en The initial search in electronic databases, hand-search, and update search yielded 5457 results, which were reduced to 4737 after deduplication. Further screening of titles and abstracts led to the exclusion of 4638 studies, leaving 99 potentially relevant studies. After a thorough examination of the full texts, 15 studies were chosen for data extraction . The remaining 84 studies were excluded for various reasons, such as not providing a disease definition or providing an unclear definition, a follow-up duration < 12 months, an undesired study design, not employing a regenerative strategy, and exhibiting a high risk of bias. Section title: Study characteristics Educational score: 4.063554763793945 Domain: biomedical Document type: Study Language: en The 15 remaining studies included 12 original randomized clinical trials and three long-term follow-up studies from these original trials. The total number of patients who received treatments for peri-implantitis was 455 (507 implants), with a mean age of 63.13 ± 10.72 years (ranging from 54.4 to 73.5 years). The examined implants were in function for 7.06 ± 3.08 years on average (ranging from 4.82 to 14 years). Ten studies included smoking patients, 29 , 35 - 43 one excluded smokers, 44 and one did not report smoking status. 45 Smokers comprised 31.85% of the participants in studies reporting smoking status (ranging from 15% to 69.6%). Section title: Outcome measures Educational score: 4.089651107788086 Domain: biomedical Document type: Study Language: en BoP was reported in 11 studies, 29 , 35 - 43 , 45 and the initial measurements showed a minimum of 15.4% and a maximum of 100%. The pre- and postoperative PDs were measured in 12 studies. 29 , 35 - 45 The baseline PD ranged from 4.9 mm to 7.6 mm. PI was reported in 10 studies, but two different indices were used. Seven studies used the O’Leary index, 29 , 35 , 38 - 40 , 42 , 43 and three used Silness & Löe. 36 , 37 , 45 The baseline measurements showed a minimum of 13% and a maximum of 45% through the former index and a minimum of 0.5 and a maximum of 1.21 through the latter. In three out of seven studies reporting BL, the implant shoulder was considered the coronal reference point, 38 , 40 , 44 and four studies did not clarify their reference points. 35 , 39 , 42 , 43 The baseline BL ranged from 3.91 mm to 5.3 mm in the first group and 3.6 mm to 5.6 mm in the second group. The reports of 11 studies evaluating BG showed a minimum value of 0.2 mm 29 , 42 and a maximum of 3.58 mm. 35 However, some interventions led to bone loss, with a maximum loss of 1.9 mm in a study by Andersen et al ( Tables 1 and 2 ). 41 Six studies included clinical and radiographic measurements for follow-up periods exceeding one year ( Table 3 ). Section title: Components of treatment Educational score: 3.9380266666412354 Domain: biomedical Document type: Study Language: en Peri-implant bone defects were filled with various materials, including xenografts, autografts, alloplasts, growth factors, etc. ( Table 2 ). 29 , 35 - 46 Aghazadeh et al 29 reported a greater bone fill through xenograft insertion compared to autograft. In another study, Polymeri et al 38 found no significant difference between the two types of xenografts, namely EndoBon and Bio-Oss. Section title: Components of treatment Educational score: 3.9297802448272705 Domain: biomedical Document type: Study Language: en In six studies, collagen or concentrated growth factor (CGF) membranes were used to cover the grafting materials. 29 , 36 , 37 , 40 , 43 , 45 Isler et al 37 compared collagen and CGF membranes for covering similar bone substitutes. The results demonstrated significant improvements with both modalities, but using collagen membranes resulted in superior outcomes. Among the seven studies mentioning the merging status following the treatment, five selected non-submerged healing, 29 , 35 , 38 , 40 , 45 and two opted for submerged healing. 37 , 41 Section title: Components of treatment Educational score: 4.024986743927002 Domain: biomedical Document type: Study Language: en The decontamination phase of the treatments involved a combination of mechanical and chemical techniques. Mechanical methods included plastic curettes, Ti curettes/brushes, ultrasonic devices, sonic devices, and implantoplasty. 35 - 37 , 39 - 41 , 44 , 45 Chemical agents such as saline, H 2 O 2 , NaCl, ozone, and EDTA were employed during chemical debridement. 29 , 35 - 45 A comparison of decontamination methods was conducted in the study by De Tapia et al, 40 which revealed that the additional use of a Ti brush resulted in a significant PD reduction. One study used an Er:YAG laser to decontaminate the peri-implant site. 45 However, laser application failed to obtain significantly superior outcomes compared to conventional decontamination via plastic curettes. In terms of postoperative care, the most frequently prescribed medications included ibuprofen, amoxicillin, azithromycin, metronidazole, and CHX. 29 , 35 - 45 Section title: Components of treatment Educational score: 3.899935007095337 Domain: biomedical Document type: Study Language: en Of the six studies reporting bone gain or loss, 35 , 39 , 41 - 44 one showed a deterioration of 1.9 mm, 41 while another showed a maximum BG of 3.63 mm during the first year. 35 The former study used porous Ti granules (PTG) as the bone substitute without membrane coverage, while the latter observed BG solely through curettage without graft or membrane materials. Section title: Meta-analysis results Educational score: 1.966585636138916 Domain: biomedical Document type: Study Language: en After categorizing the comparisons made within the included studies, they were classified into: Section title: Meta-analysis results Educational score: 4.049182891845703 Domain: biomedical Document type: Study Language: en Due to the limited number of included studies and the diversity in the interventions they examined, only the six articles in the first category were sufficient to conduct a meta-analysis. Consequently, additional comparisons between different bone substitutes, membranes, healing status, and decontamination methods could not be established. The results of the meta-analyses for six parameters at baseline and one-year follow-up can be found in Figures 3 and 4 . Notably, the study by Jepsen et al 35 reported each parameter separately for the mesial and distal aspects. Thus, a separate meta-analysis was conducted for this study to obtain a single value for each parameter and avoid biased weighting compared to the other studies. Section title: Meta-analysis results Educational score: 4.086602210998535 Domain: biomedical Document type: Study Language: en At baseline, no statistically significant difference was observed between the studies regarding BoP, PD, PI, or BL . Regarding BoP at the one-year follow-up, the overall effect size for implementing bone grafts was 0.04 (95% CI: -0.26‒0.35) . However, this intervention did not result in a significantly lower BoP compared to not using bone grafts. The PD analysis at the one-year follow-up indicated that using bone substitutes in regenerative treatments did not show a significant advantage over approaches without these materials . In this regard, the overall effect size was -0.08 (95% CI: -0.42‒0.27). An overall effect size of 0.37 (95% CI: 0.08‒0.65) was obtained for bone substitutes regarding PI at one year . It was concluded that using bone grafts, regardless of their type, significantly boosted the decrease in PI values ( P = 0.01). When it came to BL comparison between the studies at one year , it was observed that using a bone graft during surgical regeneration had a significant positive impact on BL improvements, with an overall effect size of -0.44 (95% CI: -0.84 to -0.03). Similar to BoP and PD, bone grafts did not significantly influence the amount of BG following a one-year interval . The overall effect size equaled 0.16 (95% CI: -0.68‒1.01) for BG. Section title: Meta-analysis results Educational score: 4.02194881439209 Domain: biomedical Document type: Study Language: en The P values for the test of θ i = θ j were > 0.05 for all parameters at baseline, indicating that the studies were homogeneous at baseline. At the one-year follow-up, the BoP, PD, and PI analyses showed homogeneity, while the P values for BL and BG were ≤ 0.05 ( P = 0.05 and P = 0.00, respectively), indicating that the studies were heterogeneous in terms of these two parameters. Section title: Risk of bias assessment Educational score: 3.4564590454101562 Domain: biomedical Document type: Study Language: en The risk of bias assessment revealed that five studies had a low risk of bias, and seven raised some concerns ( Table 4 ). In addition, one study exhibited a high risk of bias and, as a consequence, was omitted due to exclusion criteria. Section title: Main findings Educational score: 3.9333817958831787 Domain: biomedical Document type: Review Language: en This systematic review aimed to compare the clinical and radiographic outcomes of different regenerative protocols for peri-implantitis treatment. The findings revealed significant improvements in PI and BL one year after using bone grafts. However, using bone substitutes did not significantly affect the BoP, PD, and BG values. Various factors, including the decontamination method, postoperative care, and graft type, can also impact treatment outcomes alongside the surgical approach. Section title: Bleeding on Probing Educational score: 4.029755592346191 Domain: biomedical Document type: Study Language: en Renvert et al. 43 recorded the lowest BoP (8.3%) one year after peri-implantitis treatment. This favorable outcome was achieved through decontamination with 3% H 2 O 2 and saline. Similarly, a significant decrease in BoP was recorded in the study by Leonhardt et al. 49 after applying H 2 O 2 for decontamination. In contrast, the highest BoP (83%) was observed in the study by Andersen et al. 41 after using a Ti curette accompanied by 24% EDTA gel. EDTA does not possess antimicrobial properties per se, and the additional usage of other chemicals, such as CHX, has been suggested for improved decontamination. 50 Ramanauskaite et al. 15 reported that regenerative interventions alongside conventional peri-implantitis treatment did not significantly enhance BoP changes. Supporting this finding, Daugela et al. 31 showed that a regenerative strategy could not improve BoP reduction significantly, whether a barrier membrane was used or not. Section title: Probing Depth Educational score: 4.077219486236572 Domain: biomedical Document type: Study Language: en One year after peri-implantitis treatment, decontamination with 3% H 2 O 2 and saline in conjunction with a xenograft as the bone substitute resulted in the most favorable PD (2.6 mm). 42 In line with this finding, Roccuzzo et al. 51 achieved significantly reduced PDs by using xenograft for bone substitution. However, their decontamination phase involved 24% EDTA and 1% CHX gels. On the contrary, Emanuel et al. 44 reported the least favorable PD (5.43 mm) following chemical decontamination with ultrasonic and saline. Luengo et al. 52 reported that ultrasonic decontamination yielded less favorable results than air-polishing or Ti brushes, particularly when cleaning the implant threads within the apical third. Based on our findings, the use of bone grafts did not significantly differ from treatment approaches without bone substitution in terms of PD changes. Consistently, Li et al. 53 concluded that the additional use of bone grafts did not significantly alter the changes in PD. When comparing regenerative, resective, and access flap procedures, it was found that PD reductions were relatively similar. 18 On the contrary, Ramanauskaite et al. 15 observed a greater PD reduction in studies using regenerative techniques along with conventional peri-implantitis treatment. Section title: Plaque Index Educational score: 4.009331703186035 Domain: biomedical Document type: Study Language: en At the one-year follow-up, the lowest PIs were observed in the studies by Isehed et al. 43 (0%) and Isler et al. 36 (0.22 via the Silness-Löe index). The former performed open flap debridement (OFD) using an ultrasonic device, Ti instruments, and saline irrigation. In the latter, decontamination was executed using Ti curette, saline, and ozone DTA, while the bone defect was filled with xenograft and CGF. In several studies, ozone therapy has been shown to improve PI and PD. 54 - 56 McKenna et al. 57 reported that ozone application significantly decreased PI in patients with peri-implant mucositis, which aligns with the results of Isler et al. 36 Section title: Plaque Index Educational score: 4.023899078369141 Domain: biomedical Document type: Study Language: en On the other hand, PI was the highest in the studies by Renvert et al. 42 (25%) and Schwarz et al. 45 (1.1 via the Silness-Löe index). The former executed chemomechanical decontamination using 3% H 2 O 2 , saline, and Ti curettes, while the latter used implantoplasty, saline, and plastic curettes for decontamination, as well as xenograft covered with collagen membrane for bone substitution. According to Monje et al., 58 incorporating implantoplasty into resective or reconstructive strategies of peri-implantitis treatment did not significantly improve clinical parameters, including PI. Furthermore, incorporating implantoplasty into regenerative treatment did not necessarily result in a marked amelioration in clinical measurements. 59 Also, regenerative treatments may not necessarily be superior to non-regenerative methods in PI reduction. However, it should be remembered that employing a barrier membrane in regenerative protocols would give rise to significant PI enhancement. 18 Section title: Bone Level Educational score: 4.041348934173584 Domain: biomedical Document type: Study Language: en The highest one-year BL change (3.57 mm) was obtained in a study by Jepsen et al. 35 This change resulted from decontamination with Ti curette, 3% H 2 O 2 , and saline, as well as bone substitution with PTG. In another study, using PTGresulted in greater improvements in periodontal indices compared to a xenograft. 60 The most undesirable BL change (-0.33 mm) occurred in a study by Emanuel et al., 44 in which ultrasonic and saline were used for decontamination, and D-PLEX 500 was used to fill the bony defect. D-PLEX 500 is a biodegradable, prolonged-release antibiotic-formulated bone graft that contains β-tricalcium phosphate granules coated with doxycycline hyclate. In contrast to the mentioned finding, De Tapia et al. 40 concluded that implementing β-tricalcium phosphate as the bone substituting material would significantly enhance BL. Sanz-Martín et al. 18 compared regenerative, resective, and access flap treatment methods and concluded that a regenerative approach could lead to more significant BL gains. Section title: Bone Gain Educational score: 3.979492425918579 Domain: biomedical Document type: Study Language: en Jepsen et al. 35 reported the highest BG at one year, with 3.58 mm in the control group. This study utilized OFD using a Ti curette, a Ti brush, 3% H 2 O 2 , and saline. On the contrary, the least favorable outcome (1.9 mm bone loss) was found in a study by Andersen et al., 41 following OFD with a Ti curette and EDTA gel and bone substitution using PTG. Conversely, Jepsen et al. 35 concluded that employing PTG outperformed OFD regarding defect fill. Moreover, Guler et al. 60 reported significant superiority for PTG over xenograft placement. It can be assumed that the undesirable outcomes in the study of Andersen et al. 41 might be attributed to factors other than bone graft material. Section title: Bone Gain Educational score: 3.903712511062622 Domain: biomedical Document type: Review Language: en Based on our findings, bone grafts did not significantly affect the amount of BG. Another systematic review exploring various surgical regenerative treatments reported the greatest increase in marginal BL in three studies using enamel matrix derivative (EMD), platelet-derived growth factor (PDGF), bovine-derived xenograft, and PTG. Notably, xenografts and PTGs can appear radiopaque, making it difficult to distinguish them from regenerated bone. 31 Overall, combining regenerative measures with conventional surgical peri-implantitis treatments would achieve greater defect fill. 15 However, the complete resolution of a bony defect following guided bone regeneration (GBR) cannot be predicted with certainty. 61 Section title: Bone Gain Educational score: 4.057458400726318 Domain: biomedical Document type: Review Language: en Diverse materials, such as xenografts, autografts, etc., were used in the reviewed studies to fill bony defects. Some research outside this review combined bovine hydroxyapatite with nanocrystalline calcium sulfate, resulting in enhanced and stable outcomes. 62 Mandelaris and DeGroot 63 used a bone graft made of mineralized freeze-dried bone allograft (FDBA) and xenograft, paired with recombinant human platelet-derived growth factor (rhPDGF). Wen et al. 64 combined FDBA and mineralized bovine and autogenous bone, proving their efficacy in peri-implantitis reconstructive procedures. The application of platelet-rich fibrin has also demonstrated successful resolution of bony defects. 65 , 66 Kadkhodazadeh et al. 67 successfully managed extensive peri-implant defects by employing a Ti mesh, autogenous bone, FDBA, and acellular dermal matrix. Augmentation in bone height and attachment level can be achieved by impregnating bone grafts with tetracycline, vancomycin, or tobramycin during the GBR of peri-implantitis-affected sites. Local application of antibiotics would be advantageous concerning the absence of side effects associated with systemic administration. 68 , 69 Section title: Barrier Membranes Educational score: 4.074251174926758 Domain: biomedical Document type: Review Language: en After intrabony defect debridement, various types of cells can proliferate within the defect, including epithelial cells, connective tissue cells, bone cells, and periodontal ligament (PDL) cells. Barrier membranes can be employed to selectively allow bone cells to occupy the defect and provide physical stability for the bone substitute. 70 Among the six studies using barrier membranes, collagen membranes and CGF were the two options. 29 , 36 , 37 , 40 , 43 , 45 However, there is ongoing debate regarding the benefits of covering bone grafts with membranes. Isler et al. 37 compared two different barrier membranes, CGF and collagen, along with the same bone substitute. Collagen membranes were reported to yield more satisfactory results at the one-year follow-up. Monje et al. 71 depicted that adding a resorbable cross-linked barrier to allograft did not impact the results of defect filling. Chan et al. 72 suggested that applying barrier membranes in conjunction with graft materials may enhance outcomes compared to grafts alone. In contrast, Daugela et al. 31 showed that the additional use of barrier membranes did not significantly improve clinical outcomes. In essence, current knowledge does not necessarily support the superiority of using barrier membranes over not using them. Section title: Barrier Membranes Educational score: 3.933255910873413 Domain: biomedical Document type: Study Language: en Despite the previously mentioned materials for bone graft coverage, Dong et al. 73 reported encouraging outcomes after applying a nanofiber barrier membrane made up of magnesium oxide as the antibacterial agent alongside parathyroid hormone as the pro-osteogenic drug. In a case report, a non-resorbable Ti-reinforced polytetrafluoroethylene membrane was coupled with an absorbable collagen membrane to achieve immobility in the reconstructed region and enhanced wound healing. 63 Human amnion-chorion membranes tested for GBR showed promising results after peri-implantitis treatment. 74 Section title: Barrier Membranes Educational score: 3.920515775680542 Domain: biomedical Document type: Study Language: en Membrane exposure is a potential complication after GBR, reducing the success rate extensively. 75 Garcia et al. 76 noted that barrier exposure during the treatment of peri-implant defects would decrease healing chances by 27%. Alarmingly, human studies report exposure rates up to 87.6%. 72 Despite their potential benefits, barrier membranes can be costly, time-consuming, and technically sensitive, which might not justify their use in specific configurations such as three-wall defects. 72 Section title: Decontamination Educational score: 3.8994877338409424 Domain: biomedical Document type: Study Language: en In addition to conventional decontamination techniques, the Er:YAG laser was applied in one study. 45 At the 2- and 7-year follow-ups, laser-treated subjects did not exhibit significant differences in BoP or CAL reduction. 45 , 47 However, plastic curette debridement demonstrated a significantly greater reduction in both BoP and CAL after four years. 48 There is limited research regarding the advantages of using lasers to treat peri-implantitis. Chala et al. 77 found that the benefits of applying lasers are confined to a short-term follow-up of three months. Even the short-term clinical benefits of the Er:YAG laser for surface decontamination were refuted in another study. 78 Section title: Decontamination Educational score: 3.883800745010376 Domain: biomedical Document type: Study Language: en Additional use of ozone alongside saline has yielded improved clinical and radiographic outcomes. 36 Ozone has also diminished bacterial adhesion to Ti and zirconia surfaces in vitro without inhibiting osteoblast proliferation. 79 In brief, the techniques employed for implant decontamination did not significantly impact the results following surgical regenerative procedures, and none exhibited superiority over others. 78 , 80 Additionally, the decontamination technique must be tailored to implant surface characteristics for optimal biofilm removal. 81 Section title: Postoperative Care Educational score: 3.9352762699127197 Domain: biomedical Document type: Review Language: en Antibiotics, namely amoxicillin and metronidazole, were the most common options. Although the prescription of these two antibiotics has been shown to be beneficial for peri-implantitis treatment, 78 the efficacy of local or systemic administration of metronidazole remains unclear. 82 CHX, an antiseptic, was widely used in almost all studies. The combination of azithromycin, ibuprofen, and CHX resulted in satisfactory periodontal improvements, 42 , 43 while amoxicillin in conjunction with metronidazole was another proper choice for postoperative care. 35 , 38 Overall, a personalized evaluation must be performed before the prescription of systemic antibiotics due to insufficient evidence supporting the integration of this drug delivery route into the standard treatment protocol. 83 Section title: Postoperative Care Educational score: 3.9457569122314453 Domain: biomedical Document type: Review Language: en Although some research favors non-submerged healing, a consensus report advocates for submerged healing, as it stimulates protected physiological wound closure. 32 Keeping the suprastructure in place during surgical treatment may negatively affect the efficacy of postoperative oral hygiene maintenance, intraoperative decontamination, flap design, and numerical measurements. 32 The non-submerged approach has also satisfied clinicians regarding clinical and radiographic improvements following peri-implantitis therapy. 84 The debate continues since any relationship between the success of peri-implantitis treatment and the postoperative merging status has been refuted. 85 Section title: Other Factors Educational score: 4.077006816864014 Domain: biomedical Document type: Review Language: en Regardless of the materials and techniques implemented throughout peri-implantitis treatment, other factors such as implant location, defect morphology, and implant surface characteristics can be differential. 21 , 36 , 86 - 88 In addition to the higher prevalence of peri-implantitis within the upper jaw, 89 maxillary implants are more responsive to regenerative treatments. 36 Although Roccuzzo et al. 90 reported no significant association between defect configuration and defect resolution, Aghazadeh et al. 86 observed enhanced defect fill in four-wall and deeper defects. Also, Schwarz et al. 21 found a higher likelihood of resolution for circumferential defects than for dehiscence-type defects. A review of animal studies highlighted the crucial role of surface characteristics in peri-implantitis progression and treatment outcomes as opposed to the onset of the disease. In detail, treated surfaces represented the minimum BL and most desirable outcomes. 87 Furthermore, improvements were more pronounced around sandblasted and acid-etched implants than Ti plasma-sprayed implants after regenerative treatment. 88 Re-osseointegration has also been reported to occur more frequently around smooth-surface implants than around moderately rough implants. 91 Section title: Other Factors Educational score: 4.024306774139404 Domain: biomedical Document type: Study Language: en Peri-implantitis risk factors can be divided into five categories, including factors associated with the patient, implant design, implant site, prosthesis, and clinician. 92 Achieving satisfactory long-term outcomes becomes possible when the primary cause is accurately identified and addressed. A 3% recurrence rate for peri-implantitis has been reported following surgical intervention, potentially resulting in a 36% implant loss in the long term. 93 Factors such as deep residual PD, recessed marginal BL, and implant surface modification during surgical peri-implantitis treatment were identified as contributors to disease recurrence at a surgically treated site. 94 Despite the limited number of histological examinations on re-osseointegration following regenerative treatment on previously contaminated implant surfaces, 95 re-osseointegration seems feasible given that an effective decontamination method and suitable regenerative strategy are employed. 91 , 96 Section title: Other Factors Educational score: 4.066736221313477 Domain: biomedical Document type: Review Language: en Some argue that peri-implantitis is more of a foreign-body reaction than a bacterial-triggered disease such as periodontitis, suggesting that peri-implant bone loss can be traced to an osteolytic immune reaction. As in most cases, a physiologic balance is often established between osteoblast and osteoclast activity, making the long-term survival of implants feasible. However, when other factors, such as genetic variations, smoking, excessive cement, bacterial contamination, and technical issues, are added to the foreign-body reaction, the equilibrium gets disrupted, leading to bone loss. 97 , 98 Skeptics of this theory argue that there is insufficient evidence to solidify the pivotal role of foreign-body reactions in the pathogenesis of peri-implantitis. They assert that dental plaque biofilm is the principal causative agent of peri-implantitis, which should be the focus of both preventative and therapeutic measures. 99 Section title: Limitations Educational score: 3.1987507343292236 Domain: biomedical Document type: Study Language: en The vast variability in peri-implantitis treatment components affected the reliability of inter-study comparisons and prevented the establishment of a standardized protocol. Differing disease definitions and outcomes may have also added heterogeneity. Soft tissue parameters and factors such as smoking or genetics were not addressed. Lastly, the full text of one study was unavailable, so data from its 7-year follow-up was used instead. 41 Section title: Conclusion Educational score: 3.931004047393799 Domain: biomedical Document type: Review Language: en Following the comparison between various surgical regenerative protocols in peri-implantitis treatment, it was concluded that employing bone grafts did not significantly improve the parameters of BoP, PD, and BG, yet PI and BL showed significant enhancements. Decontamination predominantly relied on Ti instruments and chemicals such as H 2 O 2 . A variety of bone substitutes, including xenografts and CGF, were employed. Approximately half of the studies utilized collagen or CGF membranes, while others opted for none. Postoperative care involved a mix of antibiotics, CHX, and analgesics. Given the diverse materials and peri-implantitis definitions, more standardized trials are needed to establish a standardized protocol. Section title: Consent for Publication Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. Section title: Competing Interests Educational score: 0.9564492702484131 Domain: other Document type: Other Language: en The authors declare that they have no competing interests. Section title: Data Availability Statement Educational score: 0.8140149116516113 Domain: biomedical Document type: Other Language: en The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Section title: Ethical Approval Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. | Review | biomedical | en | 0.999997 |
PMC11699267 | Section title: Introduction Educational score: 3.789306640625 Domain: biomedical Document type: Review Language: en Periodontal regenerative treatment aims to provide suitable conditions for periodontal regeneration. Gingival recession can compromise esthetics and lead to root surface caries and tooth hypersensitivity. Several techniques have been suggested for root coverage, including pedicle flap, free gingival graft, guided tissue regeneration, and allografts. Autogenous grafts are procured from the palate or alveolar ridge and have limitations such as donor site morbidity and limited availability. Section title: Introduction Educational score: 3.9369847774505615 Domain: biomedical Document type: Other Language: en Acellular dermal matrix (ADM) is an alternative to autogenous grafts. ADM has applications for root coverage, augmentation of keratinized tissue around teeth and implants, and treatment of gingival recession. 1 It eliminates the need for autogenous grafts and subsequent pain and discomfort. However, the absence of vasculature and cells in ADM slows down the unity and blending of the graft with the host tissue compared to autogenous grafts. Also, allograft requires cell attachment and anastomosis of the vasculature for maturity and reorganization. 2 Section title: Introduction Educational score: 3.8363165855407715 Domain: biomedical Document type: Other Language: en Tissue engineering enables the fabrication of structures with the desired shape using biomaterials and progenitor cells and also allows cell proliferation and differentiation on suitable scaffolds. 3 ADM also serves as a temporary matrix for tissue regeneration, enhances the adhesion and proliferation of cells, and plays a key role in the transfer of MSCs to the defect site. 4 Section title: Introduction Educational score: 3.7264740467071533 Domain: biomedical Document type: Other Language: en An ideal scaffold must be biocompatible, easy to use, and easily fixed at the site. Also, it should have interconnected porosities to allow the growth and proliferation of mesenchymal stem cells (MSCs) and angiogenesis. Moreover, it should have osteoconductive and osteoinductive properties. 5 Section title: Introduction Educational score: 3.904129981994629 Domain: biomedical Document type: Study Language: en The strength and stability of the scaffold also play an important role in the proliferation and differentiation of MSCs. 6 The size of porosities in the scaffold also affects the attachment, proliferation, and differentiation of MSCs. 7 Large pores provide less surface for the attachment of cells, and numerous pores increase the number of attachments. 8 Section title: Introduction Educational score: 3.097522258758545 Domain: biomedical Document type: Other Language: en MSCs are commonly used for cell therapy and tissue engineering due to their self-renewal property and differentiation ability. 9 These cells can be isolated from different human tissues. 10 Section title: Introduction Educational score: 2.9479401111602783 Domain: biomedical Document type: Study Language: en Many studies are available on MSCs’ attachment, proliferation, and morphology on different commercially available scaffolds; however, studies on the MSCs’ behavior on ADMs produced in Iran are scarce. This study compared the biological behavior of MSCs on two types of commercial ADM scaffolds commonly used for root coverage. Section title: Methods Educational score: 4.013183116912842 Domain: biomedical Document type: Study Language: en The present in vitro study was conducted on two types of ADM scaffolds, namely scaffold type I (CenoDerm®, Tissue Regeneration Corporation, Tehran, Iran) and scaffold type II (Acellular Dermis, Iranian Tissue Product Co., Tehran, Iran). Of each scaffold, 26 samples were evaluated in this study. Also, 26 empty wells served as controls 11 (78 samples). One of the samples in each group was used to evaluate the morphologic characteristics of the cells. The scaffolds were coded to blind the operator to the group allocation of scaffolds. Section title: Cell isolation and culture Educational score: 4.156728744506836 Domain: biomedical Document type: Study Language: en MSCs isolated from a sample of the buccal fat pad were seeded and cultured. The tissue specimens were immersed in sterile phosphate-buffered saline (PBS) (Sigma, USA) supplemented with 100-U/mL penicillin (Sigma, USA), 100-μg/mL streptomycin (Sigma, USA), and 2-mg/mL collagenase type IV (Sigma, USA) and incubated at 37°C for 90 minutes. After filtering the cell suspension using a 70-μm filter (SPL, Korea), they were cultured in a 75-cm 2 cell culture flask (SPL, Korea) containing alpha modification of Eagle’s medium (SPL, Korea) supplemented with 100-μg/mL streptomycin, 15% fetal bovine serum (SPL, Korea), 100-U/mL penicillin, 200-mM L-glutamine and 100-mM ascorbic acid 2-phosphate (Sigma, USA). The cells were incubated with 5% CO 2 and 95% air at 37 °C for 24 hours. After this period, unattached cells were rinsed off with PBS. The medium was refreshed every three days. Section title: Preparation of scaffold and cell seeding Educational score: 4.142394065856934 Domain: biomedical Document type: Study Language: en Twenty-six rectangular pieces from each scaffold group, measuring 1.5 × 1 cm, with 0.2‒0.6 mm thickness, were rinsed with sterile saline solution (SPL, Korea) in 500-mL flasks for 10 minutes according to the manufacturer’s instructions. The samples were adapted to the bottom of 52 wells in six plates (SPL, Korea). Scaffolds I and II were placed in five wells on each of the five plates. Five empty wells were also considered as the control group in each plate. The sixth plate containing one sample of each scaffold and one empty well as control was used to assess cell morphology. The cell suspension with a density of 16,000 cells/mL was added to the scaffolds and control wells and incubated at 37 °C and 5% CO 2 for 12, 24, and 84 hours and 7 days. In total, two plates were used for cell attachment assessment using 6,4-diamidino-2-phenylindole (DAPI) staining and methyl thiazole tetrazolium (MTT) assay at 12 hours, and 3 plates were used to assess cell proliferation using the MTT assay at 24 and 84 hours and 7 days. 12 Five replicates were performed in every assessment at each time interval. One plate was used for cell morphology assessment at 24 hours. Section title: Assessment of cell attachment Educational score: 4.041369438171387 Domain: biomedical Document type: Study Language: en DAPI staining: The cell fixation was performed by 12 hours of incubation with 2.5% glutaraldehyde (Sigma, USA) and stained with 50 μg/mL of DAPI stain (Sigma, USA) for 30 minutes. The samples were washed with PBS to eliminate unattached cells. Then, the cells were observed under a fluorescence microscope at a 290 nm wavelength and counted in five points (four points at the corners and one at the center). 12 This was repeated for five samples in all the three groups. Section title: Assessment of cell attachment Educational score: 3.568964958190918 Domain: biomedical Document type: Study Language: en MTT assay: Optical density (OD) was measured 12 hours after culture to determine the primary attached cells in all the groups with five repetitions. 12 Section title: Assessment of cell viability and proliferation Educational score: 4.129219055175781 Domain: biomedical Document type: Study Language: en The cell viability and proliferation on the scaffolds and the control group were assessed 24 and 84 hours and 7 days after culture using the MTT assay. In this way, 200 μL of RPM1640 and 20 μL of fresh MTT solution (5 mg/mL) (Sigma, USA) were added to the cell culture wells, followed by incubation at 37 °C under 5% CO 2 for 4 hours. 12 Tetrazolium salt present in MTT was absorbed by biologically active cells, resulting in formation of purple formazan crystals, which were dissolved by adding isopropanol (Sigma, USA), including 0.1-N HCL (150 mL/well). The OD of the solution was read by a microplate spectrophotometer (SPL, Korea) by decreasing the wavelength from OD690 to OD570. 13 For assessment of cell proliferation and attachment, cell viabilityat each time interval was performed separately for five samples of each group, and determined based on a linear diagram representing the correlation between OD and cell number. Section title: Assessment of cell morphology Educational score: 4.114706993103027 Domain: biomedical Document type: Study Language: en To assess cell morphology, the cells were cultured on scaffolds and a control group and incubated for 24 hours. Then, they were washed twice with PBS, fixed with 2.5% glutaraldehyde for one hour at room temperature, and dehydrated with six graded concentrations of ethanol (from 50% to 100%), and hexamethyldisilazane (Sigma, USA). The samples were then gold-coated and evaluated under a scanning electron microscope (SEM; Nikon, Japan) at × 1000 magnification. One sample of each group was scanned under the SEM. Two parameters were assessed, including scaffold surface area covered with cells (in square micrometers) and roundness of the cells (smaller-to-larger diameter ratio of the cells). 14 Cell morphology assessment was performed on one sample of each group. Section title: Statistical analysis Educational score: 3.9456593990325928 Domain: biomedical Document type: Study Language: en Cell proliferation and attachment experiments were performed in five replicates. All the results were statistically analyzed using SPSS 25 (SPSS Inc., USA). Means ± standard deviations were used for adhesion and proliferation data analysis. The Mann-Whitney test was used to statistically analyze cell attachment, while ANOVA was applied to assess the proliferation of cells. Post hoc Tukey tests were applied for pairwise comparisons in cases of significant differences. A P value of < 0.05 with a 95% confidence interval was considered statistically significant. Section title: Results Educational score: 3.6440279483795166 Domain: biomedical Document type: Study Language: en Cell attachment was assessed in 30 samples using the MTT assay and DAPI staining (five samples from each group for each test) at 12 hours. Section title: Results Educational score: 3.8411190509796143 Domain: biomedical Document type: Study Language: en In the MTT assay, the scaffold II group had the highest cell attachment, followed by the control group. ANOVA showed no significant difference in cell attachment between the three groups ( P = 0.4). Section title: Results Educational score: 3.754638433456421 Domain: biomedical Document type: Study Language: en In DAPI staining , the highest attachment was noted in scaffold I, followed by the control group. ANOVA showed that the difference between the three groups was not significant ( P = 0.4) ( Table 1 ). Section title: Results Educational score: 3.904759168624878 Domain: biomedical Document type: Study Language: en In the assessment of cell proliferation, 45 samples were evaluated at 24 and 84 hours and 7 days in all the groups (5 samples in each group) with MTT assay ( Table 2 ). Section title: Results Educational score: 4.099632740020752 Domain: biomedical Document type: Study Language: en Over time, cell proliferation increased in all the three groups. At 24 hours, the highest proliferation rate was noted in the scaffold II group, followed by scaffold I. ANOVA showed that the proliferation rate was significantly higher in scaffolds I and II groups compared to the control group ( P < 0.001). Pairwise comparisons by Tukey’s test showed that the difference between the two scaffolds was not significant ( P = 0.8). Section title: Results Educational score: 3.9460606575012207 Domain: biomedical Document type: Study Language: en At 84 hours, the highest proliferation rate was noted in the control group, followed by the scaffold I group. ANOVA showed no significant difference in this regard between the control and scaffold groups ( P = 0.2) or between the two scaffold groups ( P = 0.9). Section title: Results Educational score: 3.799180269241333 Domain: biomedical Document type: Study Language: en At seven days, the highest proliferation rate was noted in the control group, followed by the scaffold I group. The difference in this regard between the three groups was statistically significant ( P = 0.01). Section title: Results Educational score: 3.8971548080444336 Domain: biomedical Document type: Study Language: en In all the three groups, the proliferation rate increased over time (shown by the MTT assay) such that in the control group, multiple comparisons revealed significant differences in the proliferation rate over time ( P < 0.001). Section title: Results Educational score: 3.2609119415283203 Domain: biomedical Document type: Study Language: en In scaffold I, ANOVA showed that the difference in the proliferation rate was statistically significant over time ( P = 0.01). In scaffold II, ANOVA showed that the proliferation rate difference was not significant over time ( P = 0.2). Section title: Results Educational score: 3.572887659072876 Domain: biomedical Document type: Study Language: en In the three groups, the highest proliferation rate was noted at 84 hours and 7 days (the highest cell count was noted in the control group, with the lowest in the scaffold II group). Section title: Results Educational score: 3.9180028438568115 Domain: biomedical Document type: Study Language: en Figure 2 shows the OD of MSCs of all the groups at all time intervals. Assessment of cell morphology under SEM at 24 hours revealed greater cell expansion with more appendages in the control group, followed by the scaffold I group compared to the scaffold II group . Section title: Discussion Educational score: 4.082511901855469 Domain: biomedical Document type: Study Language: en In this study, two commonly used scaffolds, CenoDerm and Acellular Dermis, were used. Attachment (at 12 hours) and proliferation (at 24 and 84 hours and 7 days) of MSCs cultured on these scaffolds were assessed using the MTT assay plus DAPI staining and MTT assay, respectively. Morphological properties of cells were also evaluated under SEM at 24 hours. The results showed no significant difference between these scaffolds concerning cell attachment at 12 hours. However, better results were achieved with CenoDerm at 24 hours and 7 days concerning cell morphological properties and cell proliferation, respectively. Section title: Discussion Educational score: 3.348478078842163 Domain: biomedical Document type: Other Language: en Cell attachment is the first response of the cell to scaffold. 15 Primary cell attachment to scaffold depends on the size and amount of porosities, water, and protein absorption 16 and plays an essential role in the proliferation of cells. Section title: Discussion Educational score: 3.8372747898101807 Domain: biomedical Document type: Study Language: en According to Pabst et al, 17 an autologous scaffold enhancing proliferation of human gingival fibroblasts, endothelial cells, osteoblasts, and oral keratinocytes in vitro can also show higher angiogenic properties in vivo. Thus, scaffolds might show more favorable behaviors in vitro and have higher applicability in vivo. Section title: Discussion Educational score: 4.022439002990723 Domain: biomedical Document type: Study Language: en In the present study, the MTT assay showed no significant difference at 12 hours in cell attachment between the three groups. Similarly, Ma et al 18 used an MTT assay and showed proper attachment of fibroblasts to bilayer dermal equivalent. They indicated that using bilayer dermal equivalent also resulted in optimal regeneration in vivo. Thus, the results of MTT can be generalized to the clinical setting. Section title: Discussion Educational score: 4.05483865737915 Domain: biomedical Document type: Study Language: en Hussein et al 19 also assessed the attachment of fibroblasts to scaffolds with different sterilization methods using the MTT assay and DAPI staining, with both tests showing similar results. Thus, DAPI staining can confirm the results of the MTT assay in vitro. Also, the results of DAPI staining in vitro can be generalized to the clinical setting. In the present study, to confirm the results of the MTT assay, DAPI staining was also performed after 12 hours, which showed the same results, and both showed no significant difference between the three groups in cell attachment. Regarding the current study analysis, it might be concluded that the attachment of cells was the same in two types of scaffolds. Thus, they probably have the same efficacy for use in the clinical setting regarding attachment of MSCs. Section title: Discussion Educational score: 3.342289686203003 Domain: biomedical Document type: Study Language: en SEM showed morphological differences, demonstrating the superiority of scaffold I to scaffold II, which indicates the more biologically active cells. 20 Greater expansion of cells in the control group might be due to the smoother surface of wells in the control group compared to the porous surface of scaffolds. 21 Section title: Discussion Educational score: 4.091872215270996 Domain: biomedical Document type: Study Language: en The current study assessed the proliferation of MSCs in the three groups after 24 and 84 hours and 7 days using the MTT assay. It showed that the proliferation rate significantly increased over time, which was not significant in scaffold II between time intervals. At 24 or 84 hours, there were no significant differences between the scaffold groups in this respect. At 7 days, the significantly lowest cell population rate was noted in the scaffold II group. Overall, cell proliferation in the scaffold I group was higher than in the scaffold II group. The higher proliferation rate in the control group at each time interval might be attributed to the smooth surface of the plate compared to the porous surface of scaffolds. As previously confirmed, surface topography affects the attachment and differentiation of cells. 22 Osteoblasts have a greater attachment to rougher surfaces, while fibroblasts and MSCs better adhere to smoother surfaces. 23 - 25 Section title: Discussion Educational score: 2.9419796466827393 Domain: biomedical Document type: Study Language: en In assessing the proliferation rate and morphological properties, scaffold I showed better results than scaffold II. Thus, it might also be superior for clinical use because evidence shows that the results of the MTT assay can be generalized to the clinical setting. Section title: Discussion Educational score: 3.486013650894165 Domain: biomedical Document type: Study Language: en We assessed MSC morphology, attachment, and proliferation, which are essential parameters in wound healing and repair. Using both DAPI and MTT assays simultaneously was a strength of our study. Also, no previous study has compared these two scaffolds concerning MSC behavior. The study was performed blindly, and each measurement was repeated five times. Section title: Discussion Educational score: 3.9896256923675537 Domain: biomedical Document type: Study Language: en Hydrophilicity, pore size, biocompatibility, mechanical properties, composition, and solvent or toxic compounds in the scaffold all affect cell seeding. 13 The structure of biomaterials in the cellular matrix is also important and affects cell behaviors such as attachment, proliferation, and differentiation. 26 Attachment and proliferation of cells on scaffolds depend on the availability of nutrients, porosity, and interconnection between pores. 27 The Strength and density of the scaffold also affect cell morphology. 28 According to the above, further studies are recommended to compare these scaffold structural properties and the efficacy of these scaffolds in MSCs’ behavior in clinical situations. Section title: Conclusion Educational score: 4.099188804626465 Domain: biomedical Document type: Study Language: en Both scaffolds showed similar efficacy in attachment of MSCs in vitro, but the proliferation of MSCs after 7 days was higher on scaffold I compared to scaffold II. Also, MSCs on scaffold I were more active, expanded more, and had more cellular appendages. Scaffold I was superior to scaffold II in terms of proliferation and morphology of MSCs in vitro. Section title: Acknowledgments Educational score: 0.9430283904075623 Domain: other Document type: Other Language: en As the authors of this paper, we thank the Nanotechnology Department of Tehran University of Medical Sciences for allowing us to conduct our experiments in their laboratory. We also thank Amirkabir University for SEM analyses. Section title: Competing Interests Educational score: 0.8786384463310242 Domain: other Document type: Other Language: en The authors declare no competing interests. Section title: Consent for Publication Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. Section title: Data Availability Statement Educational score: 0.8846843838691711 Domain: biomedical Document type: Other Language: en All data from the current study are available upon request from the corresponding author. Section title: Ethical Approval Educational score: 1.3150831460952759 Domain: biomedical Document type: Other Language: en Not applicable. The study was conducted in vitro without human participation. Therefore, the study did not have any ethical registrations. | Review | biomedical | en | 0.999995 |
PMC11699300 | Section title: Introduction Educational score: 3.953913927078247 Domain: biomedical Document type: Review Language: en Schizophrenia is a complex psychiatric disorder characterized by positive (e.g., delusions, hallucinations), negative (e.g., social withdrawal, lack of motivation), and cognitive (e.g., impairments in processing speed, working memory, and executive function) symptoms . Cognitive impairment associated with schizophrenia (CIAS) predicts poor functional outcomes , is likely a barrier to medication adherence and is associated with substantial burden on patients' daily lives . To relieve the burden on patients and reduce the financial and social burden of illness, CIAS must be adequately treated. However, there are currently no approved pharmacotherapies specifically targeting cognitive symptoms in schizophrenia . Section title: Introduction Educational score: 3.9893596172332764 Domain: biomedical Document type: Review Language: en Positive, negative, and cognitive symptoms of schizophrenia have been linked with N -methyl- d -aspartate (NMDA) receptor hypofunction in the glutamatergic pathway, which is involved in synaptic plasticity and cognition . While studies of multiple compounds, including glutamatergic agents (i.e., D-cycloserine, cycloserine, glycine, and glycine transporter inhibitors) have suggested small beneficial effects on cognition, their clinical relevance is debatable . Section title: Introduction Educational score: 3.926693916320801 Domain: biomedical Document type: Study Language: en Computerized Cognitive Training (CCT) refers to software designed to improve cognition by promoting activity-dependent neuroplasticity practice by engaging cognitive functions . CCT may be combined with psychosocial programs in cognitive remediation therapy ; . Although only a few studies have demonstrated some beneficial effects for pharmacological interventions , evidence suggests CCT interventions may have beneficial effects on the glutamatergic system in patients with schizophrenia . It is possible that, alongside CCT, pharmacological interventions may act synergistically to improve cognition. There has been limited testing of glutamatergic agents administered in combination with CCT (referred to as Pharmacologically Augmented Cognitive Training [PACT]) as a potential facilitating strategy . Section title: Introduction Educational score: 4.116117000579834 Domain: biomedical Document type: Study Language: en Iclepertin is a novel, potent, and selective glycine transporter-type-1 (GlyT1) inhibitor in development for the treatment of CIAS that increases glycine levels in the synaptic cleft . Glycine acts as a co-agonist for NMDA receptors in excitatory glutamatergic neurotransmission . Increased synaptic glycine concentration by GlyT1 inhibition may normalize NMDA receptor hypofunction in patients with schizophrenia, strengthening glutamatergic signaling and synaptic plasticity, resulting in cognitive improvement . Phase I studies with iclepertin have demonstrated its safety and tolerability in healthy volunteers in single and multiple doses with a favorable pharmacokinetic profile . A Phase II proof-of-concept trial demonstrated improved cognition in adults with schizophrenia after 2—25 mg iclepertin treatment versus placebo at Week 12 . Section title: Introduction Educational score: 3.826638698577881 Domain: biomedical Document type: Other Language: en The current Phase II trial assessed the efficacy and safety of PACT using 10 mg iclepertin combined with self-administered at-home CCT. We hypothesized that the low-level cognitive stimulation provided by the at-home CCT would augment the effects of iclepertin in treating the symptoms of CIAS in patients with schizophrenia on stable antipsychotic treatment. Section title: Study design, randomization, and blinding Educational score: 4.069466590881348 Domain: biomedical Document type: Other Language: en A detailed description of the study design, including full eligibility and exclusion criteria, methodology, and a complete list of endpoints has been published previously . Briefly, in this Phase II, 12-week, multicenter, multinational, randomized, double-blind, placebo-controlled, parallel-group exploratory trial , patients with schizophrenia across approximately 40 centers in 6 countries (USA, UK, Canada, Australia, France, New Zealand) were randomized 1:1 to receive iclepertin 10 mg or placebo for 12 weeks . 10 mg was chosen as the dose level to be investigated in the present study based on the results from a proof of mechanism trial where central target engagement of iclepertin was demonstrated by a dose-dependent increase of glycine in rat and human cerebrospinal fluid (CSF). This study demonstrated a mean increase of 50 % in CSF glycine after multiple doses of 10 mg iclepertin in healthy volunteers . Fig. 1 Study design. aDuring screening, patients received CCT (HappyNeuron Pro) for a 2-week run-in period, and those with sufficient adherence to CCT (completion of at least 2 h per week for 2 weeks during the run-in period) were eligible for randomization. bApproximately 30 h over 12 weeks; target of approximately 2.5 h/week. CCT, computerized cognitive training; QD, once daily; R, randomization; S, screening. Fig. 1 Section title: Study design, randomization, and blinding Educational score: 4.040556907653809 Domain: clinical Document type: Other Language: en During screening, patients received CCT (HappyNeuron Pro) for a 2-week run-in, and those with sufficient adherence to CCT (completion of at least 2 h per week for 2 weeks during the run-in period) were randomized in a blinded fashion via Interactive Response Technology at Visit 2. All randomized patients received access to an adjunctive self-administered at-home CCT for the full duration of the 12-week treatment period. Previous studies have demonstrated similar efficacy for cognitive performance with home-based self-administered training compared with office-based interventions ; at-home training was chosen to evaluate its feasibility in a patient population who may have difficulty accessing office-based programs . Target CCT compliance was 2–3 h per week, based on the results of a large meta-analysis . The minimum compliance threshold was at least 1 h per week and any less than this in a given week was recorded as a ‘CCT low compliance’ protocol deviation and triggered a follow-up from the CCT coach. Patients who completed no CCT training for three consecutive weeks or more despite follow-up from the CCT coach were classed as ‘CCT non-compliance’ protocol deviations. The trial was conducted in accordance with the Declaration of Helsinki, the International Council for Harmonization of Good Clinical Practice guidelines, applicable regulatory requirements, and BI standard operating procedures. The trial protocol and informed consent form were reviewed by the Independent Ethics Committees and/or Institutional Review Boards of the participating centers. Section title: Patients Educational score: 4.04974365234375 Domain: clinical Document type: Other Language: en The trial included patients with schizophrenia (18–50 years of age) who were clinically stable outpatients, with no hospitalizations for worsening of schizophrenia or symptom exacerbation within 3 months before randomization. Patients could have no more than moderate positive symptoms (per the Positive and Negative Syndrome Scale [PANSS]), had to be on stable antipsychotic treatment of 1–2 antipsychotics (except for clozapine) for ≥3 months and be on their current dose for ≥30 days before randomization. All patients had to demonstrate compliance with CCT during the run-in period and have a consistent study partner to provide reliable input into the Schizophrenia Cognition Rating Scale (SCoRS) and PANSS evaluation. Section title: Computerized cognitive training Educational score: 4.136253356933594 Domain: biomedical Document type: Study Language: en The CCT program available through HappyNeuron Pro was adapted for this trial to include 22 different training tasks to train major cognitive functions often impaired in patients with schizophrenia, including processing speed, selective attention, working memory, visual memory, verbal memory, and executive functions. Task difficulty was rated from 1 to 10 for each exercise and was titrated so that progression to increasingly difficult tasks followed successful completion of previous exercises. The first time a patient completed an exercise, they began at level 1. Patients were required to successfully (≥80 % accuracy) complete this exercise twice to move up a difficulty level at their next attempt, while two consecutive failures (<60 % accuracy) would result in the patient moving down a level for their next attempt. Progression scores were generated by the HappyNeuron Pro algorithm: individual progression scores for each exercise were calculated as an average from the last two attempts of that exercise, with attempted level of difficulty adjusted for accuracy (missing attempts were scored zero). Overall progression scores were calculated as an average of all 22 individual exercise progression scores. Participants were allowed to train on their preferred tasks and training engagement was measured across the training protocol. Section title: Primary endpoint Educational score: 4.143522262573242 Domain: biomedical Document type: Study Language: en The primary efficacy endpoint was the change from baseline in the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Consensus Cognitive Battery (MCCB) neurocognitive composite T-score after 12 weeks. The MCCB comprises 10 tests assessing 7 cognitive domains, including speed of processing, attention/vigilance, working memory, verbal learning, visual learning, reasoning and problem solving, and social cognition . Social cognition is distinct from non-social cognition (i.e., neurocognition) , thus the MCCB neurocognitive composite T-score was calculated without the social cognition domain. Several studies have shown that the MCCB is sensitive to CCT effects in participants with schizophrenia . Section title: Primary endpoint Educational score: 2.978645086288452 Domain: biomedical Document type: Study Language: en A pre-specified subgroup analysis of the primary endpoint by cumulative CCT compliance in terms of average hours per week during treatment period (<median vs ≥ median) is also reported. Section title: Secondary endpoints Educational score: 3.9885685443878174 Domain: biomedical Document type: Study Language: en The key secondary endpoints were the change from baseline after 12 weeks in the SCoRS interviewer total score and the PANSS total score. The SCoRS is a 20-item interview-based assessment of cognitive deficits and the degree to which they affect day-to-day functioning that has shown preliminary evidence of sensitivity to treatment. The PANSS is a 30-item rating scale to assess the presence and severity of psychopathology . Section title: Selected exploratory endpoints Educational score: 3.4406187534332275 Domain: biomedical Document type: Study Language: en The change from baseline in the Virtual Reality Functional Capacity Assessment Tool (VRFCAT) adjusted total time T-score was an exploratory endpoint. The VRFCAT is a functional capacity measure in which participants are assessed for their ability to perform routine activities of daily living. The VRFCAT has been shown to improve with a social-cognition-focused CCT intervention in a study focused on social cognition . Section title: Correlation between CCT engagement and cognitive improvement Educational score: 4.090941429138184 Domain: biomedical Document type: Study Language: en In a post hoc analysis, linear regressions of change from baseline at Week 12 in MCCB neurocognitive T-score, SCoRS interviewer total score, and VRFCAT adjusted total time T-score with CCT training time were conducted. Additionally, linear regressions of the change from baseline in these cognitive measures at Week 12 with the CCT progression score by quartiles of CCT overall training time (≤11.65, >11.65 ≤ 19.38, >19.38 ≤ 29.83, >29.83) were performed. Section title: Safety Educational score: 3.7525839805603027 Domain: biomedical Document type: Other Language: en Safety was assessed as the percentage of patients with adverse events (AEs), serious AEs (SAEs), clinically relevant abnormalities of physical examination, vital signs, electrocardiogram, and laboratory tests. Additional safety assessments were protocol-specified AEs of special interest (AESIs), worsening of disease state (assessed by PANSS) and assessment of suicidality through electronic Columbia Suicide Severity Rating Scale (eC-SSRS). Section title: Statistical analysis and sample size Educational score: 4.095998764038086 Domain: biomedical Document type: Study Language: en A detailed description of the planned statistical analysis has been previously reported . In summary, a sample size of 200 patients was considered sufficient to achieve the aims of this exploratory trial, which was not powered for any formal hypothesis testing. Demographics, baseline characteristics, and safety analyses were performed on the treated set . For the primary, secondary, and VRFCAT (exploratory) endpoints, analysis was carried out on the full analysis set. The changes from baseline after 6 and 12 weeks in MCCB neurocognitive composite T-score were analyzed by a restricted maximum likelihood (REML) estimation of a mixed model for repeated measures (MMRM) adjusting for treatment by visit, baseline by visit, change from screening to baseline, and age strata. Within-patient correlation was modelled with an unstructured variance-covariance matrix. The same MMRM analysis was performed for each pre-defined subgroup with ≥25 patients. For SCoRS and VRFCAT endpoints, an analysis of covariance was performed, whereas REML estimation of a MMRM was performed for PANSS; these analyses were adjusted for age strata and the corresponding baseline value . Post hoc exploratory regression analysis of associations between the cognitive measures, CCT training time, and CCT progression score were only performed in patients receiving placebo. Section title: Patient disposition and demographics Educational score: 3.9777443408966064 Domain: biomedical Document type: Other Language: en Of 406 screened patients, 200 were randomized (iclepertin group: iclepertin 10 mg + CCT [ n = 99]; placebo group: placebo + CCT [ n = 101]) and 154 (77.0 %) completed the trial medication. A total of 46 patients (23.0 %) prematurely discontinued treatment; the discontinuation rate was similar across groups . Most patients were randomized in the USA (148/200 [74.0 %]), 136 (68.0 %) were male, and 98 (49.0 %) were white. The VRFCAT scores were relatively high at baseline for both treatment groups ( Table 1 ). There was no substantial difference in alcohol and nicotine use between treatment groups. The mean exposure to trial medication and CCT was comparable for both treatments ( Supplementary Table 1 ). Section title: Primary endpoint Educational score: 4.187772274017334 Domain: biomedical Document type: Study Language: en The adjusted mean (standard error [SE]) change from baseline in MCCB neurocognitive composite T-score after 12 weeks was 1.6 (0.6) points for iclepertin and 2.5 (0.6) points for placebo. The MMRM analysis did not show a treatment difference between iclepertin 10 mg + CCT and placebo + CCT groups . However, the adjusted mean [SE] change from baseline in the MCCB neurocognitive composite T-score at Week 12 indicated a slight improvement for the iclepertin 10 mg + CCT group, albeit not reaching statistical significance ( Table 2 ). Fig. 2 Patient disposition. a Of the 176 patients who did not meet the inclusion/exclusion criteria, 47 patients were unable to properly use the CCT device or were non-compliant with the requirements of the at-home CCT during the 2-week run-in period. AEs, adverse events; CCT, computerized cognitive training; COVID-19, coronavirus disease 2019; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. Fig. 2 Table 1 Patient demographics and baseline assessments. Table 1 Baseline Characteristic Placebo + CCT ( n = 101) Iclepertin 10 mg + CCT ( n = 99) Total ( N = 200) Demographics Male, n (%) 71 (70.3) 65 (65.7) 136 (68.0) Female, n (%) 30 (29.7) 34 (34.3) 64 (32.0) Race, n (%) White 51 (50.5) 47 (47.5) 98 (49.0) Black or African American 40 (39.6) 44 (44.4) 84 (42.0) Asian 5 (5.0) 4 (4.0) 9 (4.5) Other ⁎ 5 (5.0) 2 (2.0) 7 (3.5) Age (years), mean (SD) 38.5 (7.7) 37.9 (8.1) 38.2 (7.9) Time since diagnosis of schizophrenia (years), mean (SD) 13.9 (8.4) 13.1 (8.4) 13.5 (8.4) Baseline assessments MCCB neurocognitive composite T-score, mean (SD) 34.0 (11.3) 33.5 (12.0) 33.7 (11.6) SCoRS interviewer total score, mean (SD) † 34.8 (8.9) 36.3 (8.6) 35.5 (8.7) PANSS total score, mean (SD) 63.3 (14.5) 65.8 (14.5) 64.5 (14.5) VRFCAT summary score, mean (SD) 44.9 (15.2) 45.1 (13.9) 45.0 (14.6) Antipsychotic concomitant therapies Patients with 1 antipsychotic, n (%) 79 (78.2) 78 (78.8) 157 (78.5) Patients with 2 antipsychotics, n (%) 20 (19.8) 20 (20.2) 40 (20.0) Patients with oral antipsychotics, n (%) 78 (77.2) 75 (75.8) 153 (76.5) CCT, computerized cognitive training; MCCB, MATRICS Consensus Cognitive Battery; PANSS, Positive and Negative Syndrome Scale; SCoRS, Schizophrenia Cognition Rating Scale; SD, standard deviation; VRFCAT, Virtual Reality Functional Capacity Assessment Tool. ⁎ Includes American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, or those in multiple racial categories. † Baseline was available for 94 and 91 patients in the placebo + CCT arm and iclepertin + CCT arm, respectively. Table 2 Change from baseline in MCCB neurocognitive composite T-score by treatment groups at Weeks 6 and 12 (FAS). Table 2 Week 6 Week 12 (EoT) Placebo + CCT ( n = 86) Iclepertin 10 mg + CCT ( n = 88) Placebo + CCT ( n = 86) Iclepertin 10 mg + CCT ( n = 88) Change from baseline Adjusted mean (SE) 1.8 (0.5) 1.8 (0.5) 2.5 (0.6) 1.6 (0.6) 95 % CI (0.8, 2.7) (0.9, 2.8) (1.2, 3.7) (0.4, 2.8) Iclepertin vs placebo Adjusted mean difference (SE) 0.1 (0.7) −0.9 (0.9) 95 % CI (−1.3, 1.4) (−2.6, 0.8) p -value 0.9099 0.3101 Data was analyzed by a MMRM model using an unstructured within-subject variance-covariance matrix and included the following fixed effects: categorical factors of planned treatment, visit, planned treatment by visit interaction, and age group as well as continuous covariates of baseline value, baseline by visit interaction, and change from screening to baseline value. Increases in MCCB scores indicate improvements. CCT, computerized cognitive training; CI, confidence interval; EoT, end of treatment; FAS, full analysis set; MCCB, MATRICS Consensus Cognitive Battery; MMRM, mixed models for repeated measures; SE, standard error. Section title: Primary endpoint Educational score: 4.111724376678467 Domain: biomedical Document type: Study Language: en While the subgroup analysis by CCT training adherence was generally consistent with the overall analysis, a slight trend towards a higher improvement in the placebo arm was observed for the subgroup of patients with cumulative CCT compliance ≥ median . Fig. 3 Treatment effects versus placebo at Week 12 for MCCB neurocognitive composite T-score overall, and by cumulative CCT compliance (FAS). The adjusted mean difference between iclepertin and placebo groups was obtained by a REML estimation of a MMRM model with unstructured variance-covariance matrix fitted to each subgroup level. CCT, computerized cognitive training; CI, confidence interval; FAS, full analysis set; MCCB, MATRICS Consensus Cognitive Battery; MMRM, mixed model for repeated measures; REML, restricted maximum likelihood. Fig. 3 Section title: Secondary endpoints and selected exploratory endpoints Educational score: 4.204132556915283 Domain: biomedical Document type: Study Language: en There was no treatment difference between iclepertin and placebo groups in the SCoRS interviewer total score or PANSS total score at Week 12 ( Table 3 A and 3B ). Similar to the primary endpoint, the adjusted mean [SE] change from baseline in the SCoRS interviewer total score at Week 12 indicated a slight improvement (not statistically significant) for the iclepertin 10 mg + CCT group ( Table 3 A ). Table 3 Change from baseline in (A) SCoRS interviewer total score at Week 12 (FAS), (B) PANSS total score at Week 6 and Week 12 (FAS), and (C) VRFCAT total time T-score at Week 12 (FAS). Table 3 A. SCoRS Placebo + CCT ( n = 75) Iclepertin 10 mg + CCT ( n = 82) Change from baseline at Week 12 (EoT) Adjusted mean (SE) −3.1 (0.7) −2.5 (0.6) 95 % CI (−4.4, −1.8) (−3.7, −1.3) Iclepertin vs placebo Adjusted mean difference (SE) 0.6 (0.9) 95 % CI (−1.2, 2.4) p-value 0.5237 B. PANSS Week 6 Week 12 (EoT) Placebo + CCT (n = 85) Iclepertin 10 mg + CCT (n = 88) Placebo + CCT ( n = 85) Iclepertin 10 mg + CCT (n = 88) Change from baseline Adjusted mean (SE) −2.0 (1.0) −0.6 (0.9) −2.1 (1.0) −2.7 (1.0) 95 % CI (−3.9, −0.1) (−2.5, 1.2) (−4.1, −0.1) (−4.7, −0.8) Iclepertin vs placebo Adjusted mean difference (SE) 1.3 (1.3) −0.7 (1.4) 95 % CI (−1.3, 4.0) (−3.5, 2.2) p-value 0.3194 0.6420 C. VRFCAT Placebo + CCT ( n = 65) Iclepertin 10 mg + CCT ( n = 62) Change from baseline at Week 12 (EoT) Adjusted mean −2.6 −3.4 95 % CI (−5.0, −0.2) (−5.8, −1.0) Iclepertin vs placebo Adjusted mean difference (SE) −0.8 (1.7) 95 % CI (−4.2, 2.6) p-value 0.6542 Data was analyzed using the ANCOVA model which included the following fixed effects: categorical factors of planned treatment and age group as well as a continuous covariate of baseline value. Decreases in SCoRS rating indicates improvements. Data was analyzed by a MMRM model using an unstructured within-subject variance-covariance matrix and included the following fixed effects: categorical factors of planned treatment, visit, planned treatment by visit interaction, and age group as well as a continuous covariates of baseline value and baseline by visit interaction. Decreases in PANSS scores indicate improvements. Data was analyzed by an ANCOVA model which included the following fixed effects: categorical factors of planned treatment and age group as well as a continuous covariate of baseline value. Decreases in VRFCAT summary scores indicate worsening performance. ANCOVA, analysis of covariance; CCT, computerized cognitive training; CI, confidence interval; EoT, end of treatment; FAS, full analysis set; MMRM, mixed models for repeated measures; PANSS, Positive and Negative Syndrome Scale; SCoRS, Schizophrenia Cognition Rating Scale; SD, standard deviation; SE, standard error; VRFCAT, Virtual Reality Functional Capacity Assessment Tool. Section title: Secondary endpoints and selected exploratory endpoints Educational score: 3.823279857635498 Domain: biomedical Document type: Study Language: en There was also no treatment difference between iclepertin and placebo groups in the VRFCAT adjusted total time T-score . However, the adjusted mean [SE] change from baseline in the VRFCAT adjusted total time T-score at Week 12 indicated a slight deterioration for the iclepertin 10 mg + CCT group ( Table 3 C ). Section title: Correlation between CCT training time and cognitive improvement Educational score: 4.139424800872803 Domain: biomedical Document type: Study Language: en In placebo patients, there was no correlation between CCT overall training time and the change from baseline at Week 12 in MCCB neurocognitive composite T-score (estimated slope, SE: 0.08 [0.05] per hour) or VRFCAT adjusted total time T-score (−0.17 [0.12] per hour), while the data suggest a negative correlation between CCT overall training time and changes from baseline at Week 12 in SCoRS interviewer total score . Fig. 4 CCT Training time versus change from baseline at Week 12 in (A) MCCB neurocognitive composite T-score, (B) SCoRS interviewer total score, and (C) VRFCAT adjusted total time T-score. CCT, computerized cognitive training; MCCB, MATRICS Consensus Cognitive Battery; PANSS, Positive and Negative Syndrome Scale; SCoRS, Schizophrenia Cognition Rating Scale; VRFCAT, Virtual Reality Functional Capacity Assessment Tool. The CCT progression score of an attempt is based on the level attempted adjusting for accuracy: level×10 for 80 % or higher accuracy, (level – 0.5) × 10 for 60 % to < 80 % accuracy, (level – 1.5) × 10 for < 60 % accuracy, and 0 for no attempts, for exercise levels that progress from 1 to 10. Fig. 4 Section title: Correlation between CCT training time and cognitive improvement Educational score: 4.092662811279297 Domain: biomedical Document type: Study Language: en The linear regression of change from baseline in cognitive measures at Week 12 on CCT progression score was also analyzed by subgroups of CCT overall training time quartiles. Generally, no pattern of correlation is suggested by the data, except a positive correlation between the CCT progression score and the MCCB neurocognitive composite T-score at Week 12 in patients with >19.38 to 29.83 h of overall CCT training, and a negative correlation between the CCT progression score and the SCoRS interviewer total score at Week 12 in patients with ≤11.65 h of overall CCT training. Section title: Safety Educational score: 4.025550365447998 Domain: biomedical Document type: Other Language: en Overall, 39/99 (39 %) patients in the iclepertin group and 57/101 (56 %) patients in the placebo group reported ≥1 AE, and most AEs were mild to moderate ( Table 4 ). Between treatment groups, similar proportions of AEs led to discontinuation of trial medication. There were very few severe AEs and SAEs, and no AESIs or deaths. Table 4 Overall summary of adverse events (treated set). Table 4 Category of AE, N (%) Iclepertin 10 mg + CCT (n = 99) Placebo + CCT (n = 101) Any AE 39 (39.4) 57 (56.4) Severe AEs 1 (1.0) 3 (3.0) Investigator-defined drug-related AEs 17 (17.2) 21 (20.8) AEs leading to discontinuation of trial medication 3 (3.0) 4 (4.0) AESIs 0 0 SAEs a 1 (1.0) 2 (2.0) Fatal 0 0 Life-threatening 0 0 Persisting or significant disability/incapacity 0 0 Requiring or prolonging hospitalization 1 (1.0) 1 (1.0) Congenital anomaly or birth defect 0 0 Other 0 1 (1.0) An adverse event was defined as any untoward medical occurrence. An SAE was any AE which fulfilled one of the following criteria: resulted in death, was life-threatening, required inpatient hospitalization, required prolonging of existing hospitalization, resulted in a persistent/significant disability/incapacity, or deemed serious based on appropriate medical judgment. AE, adverse events; AESI, adverse events of special interest; CCT, computerized cognitive training; SAE, serious adverse events. a A patient could be counted in >1 seriousness criterion. Section title: Safety Educational score: 3.899264097213745 Domain: clinical Document type: Other Language: en Overall, no clinically relevant changes in laboratory parameters and vital signs were observed. A similar number of patients in both groups (iclepertin [ n = 8]; placebo [ n = 7]) had disease worsening indicated by the PANSS positive score ≥ 5 and 4 patients in each treatment group with PANSS total score ≥ 15. In the iclepertin and placebo groups, there were 7 and 8 patients, respectively, with ≥1 event of suicidal ideation during treatment. No events of C-SSRS type 4 or 5, suicidal behavior, or self-injury were reported. Section title: Discussion Educational score: 4.060291290283203 Domain: biomedical Document type: Study Language: en Results from this Phase II study to assess the efficacy and safety of iclepertin versus placebo for the treatment of CIAS demonstrated that iclepertin 10 mg had a favorable safety profile and was well tolerated. Consistent with previous Phase I and II trials, the iclepertin group had lower incidence of AEs versus placebo (39 % versus 56 %), and headache was the most common AE . However, the primary and secondary efficacy endpoints were not met in this trial. After 12 weeks of treatment, no difference was observed between the iclepertin and placebo groups in the MCCB neurocognitive composite T-score, SCoRS interviewer total score, or PANSS total score. Further, results from the exploratory VRFCAT endpoint also indicated no improvements in functional capacity. Section title: Discussion Educational score: 3.682767629623413 Domain: biomedical Document type: Study Language: en Although the present study failed to show positive efficacy results, 3 Phase III trials are ongoing to examine the efficacy, safety, and tolerability of iclepertin in improving cognition and functioning in a larger group of patients, over a longer treatment period (26 weeks); however, these trials do not include an adjunctive CCT: CONNEX-1, CONNEX-2, and CONNEX-3 . Section title: Discussion Educational score: 3.988070487976074 Domain: biomedical Document type: Study Language: en It is unclear whether the failure to meet efficacy endpoints in the current study truly indicates that iclepertin does not affect CIAS in its own right, or if there are methodological considerations which may have contributed to the lack of positive results. In the previous Phase II proof-of-concept trial that demonstrated improved cognition in adults with schizophrenia after 2—25 mg iclepertin the effect size seen was significant, but numerically small. It is possible that in the current study the sample size was too small and baseline VRFCAT scores too high to detect iclepertin monotherapy effects. Results from the larger, ongoing Phase III study are expected soon and should help answer this question. Section title: Discussion Educational score: 4.130871772766113 Domain: biomedical Document type: Study Language: en Similarly, it is difficult to say whether the results of the current study indicate iclepertin has no augmenting effect on CCT. Use of CCT to improve cognition is a relatively recent development, and there are minimal comparative data regarding efficacy of specific CCT programs. Although similar studies have demonstrated comparable efficacy of home-based and office-based interventions , it is possible that providing an office-based training environment and observation by healthcare professionals could have a slightly higher impact on training engagement and subsequent improvement in outcomes. Most previous studies using the HappyNeuron Pro CCT program deployed the training system as part of a comprehensive rehabilitation program, which includes training in areas such as strategic monitoring, bridging, and functional adaptation skills in order to achieve large and durable effects on functional competence and real-world functioning . In contrast, the intention of using CCT in the present study was not to effect cognitive change per se, but rather to provide a low level of cognitive stimulation to augment the effects of pharmacological treatment, in the most standardized possible way. Further to this, providing CCT in the home setting allowed us to evaluate the feasibility of at-home training in this patient population who may have had difficulty accessing traditional training programs . These differences mean a direct efficacy and mechanism of action comparison to the use in the current study is not possible . In two recent meta-analyses of cognitive training for the treatment of psychosis, at-home HappyNeuron Pro was not used in any of the cited articles . The lack of available efficacy data for HappyNeuron Pro alone makes the present results difficult to interpret, and it is difficult to speculate as to whether larger effects may have been seen from using a different CCT program. In the subgroup analysis of change from baseline in the MCCB neurocognitive composite score, a slight tendency towards greater improvement in the placebo + CCT group was observed for the subgroup of patients with total CCT compliance ≥ median 2.17 h/week. However, there was no correlation between CCT training time and cognitive outcomes. These mixed findings suggest no strong association between CCT and efficacy outcomes and add to the difficulty of differentiating treatment effects from CCT effects. Section title: Discussion Educational score: 4.099555969238281 Domain: biomedical Document type: Study Language: en Another factor that complicated the interpretation of the trial results was the absence of a non-CCT placebo arm. A recent publication reported results from a similar trial to the current study, wherein the impact of a GlyT1 inhibitor was investigated in combination with computer-based cognitive training (HappyNeuron Pro) to treat CIAS in a cross-over trial with no non-CCT placebo arm, and found no augmentation of the effects of cognitive training . One possible explanation offered by the authors is that it may be difficult to interpret the augmenting effects of two interventions in the context of one another, and an additional non-CCT placebo arm may help to produce clearer results , a consideration which may also be relevant in the present study. In contrast, a three-arm study comparing computerized cognitive training alone, in-person, group delivered functional adaptation skills training alone, or a combination of both (which met the criteria for being designated as “cognitive remediation”) and had no inactive placebo control arm, found statistically significant cognitive gains in both CCT and combined treatment arms, as well as gains in everyday functioning that were significant in the combined treatment arm . Section title: Discussion Educational score: 4.178988933563232 Domain: biomedical Document type: Study Language: en A potential limitation of this study is the high scores on VRFCAT assessment at baseline. The VRFCAT total time T-scores of ∼45 were observed in both treatment groups in this study; while average VRFCAT total time T-scores of 49.7 and 32.5 have previously been obtained from healthy volunteers without cognitive impairment, and patients with schizophrenia, respectively . While these may appear to be in contrast with the MCCB baseline scores of ∼34.0 (both treatment groups), which were in the expected, impaired range, it is important to remember that the MCCB is a paper and pencil test, while the VRFCAT is completed digitally. It is plausible that participants with advanced technological skills may have been more inclined to volunteer for a computer training study, meaning the technological performance of the population may have outstripped their general cognitive ability. This could partly explain the lack of efficacy observed in the VRFCAT endpoint and suggests a potential selection bias of patients with a high level of functional capacity. Another limitation of this study is that the trial site selection was restricted, given that the CCT program was only available in English and French languages. Finally, the study was exploratory in nature, which may affect its interpretation, and not powered to detect differences between the treatment groups beyond the primary measure. Section title: Discussion Educational score: 4.104948997497559 Domain: biomedical Document type: Study Language: en Despite the lack of positive efficacy results, the present study had several strengths. Foremost, this was the largest trial of its kind to combine CCT and pharmacotherapy for schizophrenia in 200 patients with CIAS. The recent publication by Surti TS et al. examined the effects of a GlyT1 inhibitor in combination with CCT in a smaller sample (71 patients randomized) . The successful implementation of the at-home CCT component represents an additional strength of the current study; on average, patients completed approximately 21.3 h of CCT in the iclepertin group and 19.4 h in the placebo group over a duration of 12 weeks. The adherence results presented here compare favorably to previous home-based training interventions . Section title: Conclusions Educational score: 3.9713757038116455 Domain: biomedical Document type: Study Language: en To our knowledge, this trial is the largest of its kind combining daily pharmacotherapy for CIAS with at-home CCT. Even though efficacy could not be demonstrated, the safety results of iclepertin 10 mg were consistent with previous clinical studies and no new risks were identified. Three multinational Phase III trials (CONNEX-1, −2, and − 3) are ongoing to further investigate the efficacy and safety of iclepertin in improving cognition and daily functioning (without utilizing an adjunctive CCT) in patients with schizophrenia. Section title: Conclusions Educational score: 1.817330241203308 Domain: biomedical Document type: Other Language: en The following is the supplementary data related to this article. Supplementary Table 1 Exposure to trial medication and CCT–TS. Supplementary Table 1 Section title: CRediT authorship contribution statement Educational score: 0.9586703777313232 Domain: other Document type: Other Language: en Philip D. Harvey: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Conceptualization. Sean McDonald: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. Eric Fu: Writing – review & editing, Writing – original draft, Validation, Methodology, Formal analysis, Data curation. Corey Reuteman-Fowler: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Conceptualization. Section title: Author contributions Educational score: 0.924649178981781 Domain: other Document type: Other Language: en SMD and PH were involved in the conception and design of the study. SMD and EF collected the data. CRF and PH were involved in data analysis, interpretation, and dissemination. All authors were involved in the preparation and review of the manuscript and approved the final version submitted for publication. Section title: Authors' statement Educational score: 0.9919755458831787 Domain: other Document type: Other Language: en This paper received editorial assistance funded by Boehringer-Ingelheim. Section title: Authors' statement Educational score: 1.0417566299438477 Domain: other Document type: Other Language: en The authors of the paper acknowledge that they are solely responsible for the article content. Section title: Funding Educational score: 0.7623989582061768 Domain: other Document type: Other Language: en This study was funded by Boehringer Ingelheim International GmbH . Section title: Declaration of competing interest Educational score: 1.0467950105667114 Domain: other Document type: Other Language: en CRF, SM, and EF are employees of Boehringer Ingelheim. PDH has received consulting fees or travel reimbursements from Alkermes, Boehringer Ingelheim, Karuna Therapeutics, Minerva Neuroscience, and Sunovion/DSP Pharma during the past year. He is Chief Science Officer of i-Function, Inc. (whose products were not used in this trial) and receives royalties from WCG Endpoint Solutions (owner of the VRFCAT) for the Brief assessment of Cognition. PDH does not receive royalties for the sale or use of VRFCAT. | Review | biomedical | en | 0.999995 |
PMC11699310 | Section title: Introduction Educational score: 1.4310652017593384 Domain: other Document type: Other Language: en Since the ancient time, domestic horses Equus caballus L. have been an essential part of economics of Kazakhstan as both a source of food and saddle/working animals. The area of Kazakhstan is arguably one of the place where horse domestication could take place historically, as the most ancient traces of horse have been discovered in this region [ , , , ]. The environment of the Central Asian steppes and the traditional ways of horse husbandry of local nomadic peoples have formed a specific type of domestic horse, namely Kazakh horse. The traditional Kazakh horses include three main types Zhabe, Adai, and Naiman, which originated from different regions of the country. Based on them, more derivative breeds were developed; the most known of them include Kostanay, Kushum, Mugalzhar and other breeds [ , , ]. Section title: Introduction Educational score: 1.1841888427734375 Domain: other Document type: Other Language: en The most known horses of the Kazakh breed are Zhabe horses. Zhabe horses have originated from southern parts of Aktobe region and distributed all over Kazakhstan. Their origin remains disputable; main hypotheses include the origination from wild Asian horses or Mongolian horse breed, or that they gradually evolved under combination of multiple factors of the natural and artificial selection . Supposedly, Kazakh horses have been influenced by Mongolian, Karabair, Arabian, and Akhal-Teke horse breeds; the modern influence (20th century) include also the Thoroughbred, Orlov Trotter and Don horses . Zhabe horses are considered to be a typical Kazakh horse landrase as they are the most widespread and not specialized for some specific conditions and/ar application (comparing to horses of Naiman type better adopted to mountain regions , or Adai horses which have been specialized as saddle horse ). Section title: Introduction Educational score: 1.5253254175186157 Domain: other Document type: Other Language: en Zhabe are characterized by rugged head, thick neck, wide body with straight back, deep chest, and muscled croup; skin is dense; hair colors varying from light gray to dark bay or red. Zhabe horses have relatively high body weight (400–500 kg) for their size (height at withers 142–144 cm, chest circumference 178–180 cm, cannon bone circumference 18.8–19 cm . Young Zhabe horses intensively accumulate living weight during pasturing and produce high quality meat regardless of their population and regional lineage . Their body properties and good milk productivity make Zhabe horses an important resource for meat and milk production. Zhabe horses are well adapted to the traditional nomadic husbandry practices based on seasonal transhumance and pasturing ; such way of horse breeding have been developing since Saka-Skythian historic period (mid-first millennium BC) . Section title: Introduction Educational score: 4.05194091796875 Domain: biomedical Document type: Study Language: en The traditional methods of horse breeding have been based on simple selection and crossing of animals with desirable traits. Although such methods are still in use in horse selection programs, their sole usage limits the breeding progress. Modern practices imply the use of the data on genetic mechanisms underlying economically import traits such as productivity. Genomic and marker associated selection (MAS) became an important field of animal breeding as the increasing data on genetic polymorphism stimulate studies not only elucidating genetic properties of breeds but also endorsing them for use in selection practice. The study of breeds, using molecular techniques is very important and useful for their characterizing [ , , ]. Conservation of genetic diversity in animal species requires the proper performance of conservation superiorities and sustainable handling plans that should be based on universal information on population structures, including genetic diversity resources among and between breeds . Genetic diversity is an essential element for genetic improvement, preserving populations, evolution and adapting to variable environmental situations . On the other hands, determination of gene polymorphism is important in farm animals breeding in order to define genotypes of animals and their associations with productive, reproductive and economic traits [ , , ]. Section title: Introduction Educational score: 4.103096008300781 Domain: biomedical Document type: Study Language: en Considering meat productivity, the living mass and size of an animal are the primary traits of interest. Body mass is a multifactorial quantitative trait affected by both genetic and ambiance factors. For example, heritability of weight and body measures have been shown previously [ , , ]. Particularly, heritability of Zhabe body mass and measurements have been estimated . The more detailed studied on genetic factors affecting horse body parameters involve the advances of horse genomics. The availability of continuously updated reference assembly of horse genome (current version EquCab3.0 ) assists studies on genetic bases of various traits of interest involving thousands and millions of SNPs . Genome wide association studies (GWAS) have identified genes LCORL/NCAPG, HMGA2, ZFAT, and LASP1 as the major genetic factors associated with body size in horses [ , , , ]. Section title: Introduction Educational score: 2.1932125091552734 Domain: biomedical Document type: Study Language: en To date, despite their prominent economical role in the country, only limited number of studies on the molecular genetic background of Kazakh horses including Zhabe have been conducted. The most used molecular tool is microsatellite markers, or short tandem repeats (STR). For example, STR-markers were used to investigate genetic structure of Adai horses and Mugalzhar horse breed . Section title: Introduction Educational score: 4.101596832275391 Domain: biomedical Document type: Study Language: en Our previous study was the biggest to date and involved total 2020 horses of six traditional types and breeds of Kazakh horses analyzed using 80,000 SNP microarray . This study have revealed the surprising absence of genetic differentiation between studied breeds. The observed genetic structure was an evidence for existence of broadly defined landrace corresponding to Kazakh horses without notable genetic demarcation between rational types and breeds. It has been also hypothesized that the traditional nomadic ways of horse breeding on the territory of contemporary Kazakhstan could have lead to formation of Kazakh horse landrace without strong pressure of artificial selection . The present article is focused on horses of Zhabe type and uses previously published data combined with newly genotyped sample of Zhabe horses to provide more details on the genetic structure is type of Kazakh horses including GWAS for body weight and size, the traits associated with meat productivity. A special attention was paid to population genetic parameters potentially reflecting the way of horse selection. Section title: Sample collection and DNA isolation Educational score: 4.007037162780762 Domain: biomedical Document type: Study Language: en Genetic material (hairs from tails and/or manes) of Zhabe horses was collected at private farms “Alakol Asyl” (Zhetysu region) and “Akzhar” (Pavlodar region); additionally, samples of white horses “Zhetysu Asyly” were collected at A. Sophin's private farm (Zhetysu region) ( Table 1 ). All collected materials were stored at 4 °C until further use. DNA was isolated from hair follicles using the kit ‘‘DNK-Extran2” (Syntol, Russian Federation) following the manufacturer's protocol and quantified using Qubit 4 Fluoremeter with the Qubit dsDNA Broad Range reagent (Thermo Fisher Scientific, USA) for downstream analysis. Table 1 Summary of samples and data used in the study. Table 1 Zhabe horses, Kazakhstan Population code Husbandry Region Number of individuals with available data Final number of individuals used for analysis Data source Genotype Phenotype (weight, size) 37 Alakol Asyl Zhetysu a 421 427 384 Present study 39 b A. Sophin Zhetysu a 42 88 42 25 Akzhar Pavlodar 81 c 173 64 , present study 33 Agro-Damu Pavlodar 153 186 152 3 Kalka Zhambyl 143 163 141 28 Zhaksylyk Almaty 86 122 86 34 Anar Pavlodar 76 77 76 32 Sayakhat Almaty 48 0 47 29 Kyzylsok Almaty 46 46 46 Total 1096 1282 1038 Foreign breeds Breed code Breed name Breed origin Number of genotypes Sample origin Data source AKTK Akhat-Teke Turkmenistan 19 USA, Russia ARR Arabian Middle East 24 USA CSP Caspian Persia 19 USA MON Mongolian Mongolia 19 Mongolia TB_UK Thoroughbred UK 19 UK TB_US 17 USA a Former part of Almaty region. b Horses of the derivative “Zhetysu Asyly” breed. c Including 17 newly analyzed samples. Section title: Sample collection and DNA isolation Educational score: 2.0978403091430664 Domain: biomedical Document type: Study Language: en Phenotypic data including body weight, height at the withers (HW), oblique body length (OBL), chest circumference (CC), and cannon bone circumference (CBC) were taken and provided by the horses owners prior to sample collection. Section title: SNP genotyping Educational score: 4.129613876342773 Domain: biomedical Document type: Study Language: en SNP genotyping of collected samples was performed using Equine80k SNP array on the iScan system (Illumina, USA) according to the manufacturer's protocol. Genotype calling and primary data filtering were performed using GenomeStudio software (Illumina, USA). The primary filtering criteria included call rate ≥0.9, median GC score ≥0.8 for samples; call frequency ≥0.95 and GT score ≥0.7 for SNP; indel markers included into array were excluded. PLINK1.9 software was used for further data filtering to exclude SNPs with minor allele frequencies <0.05 and those deviating from Hardy-Weinberg equilibrium with a P-value threshold of 1 × 10 −5 . Additional sample filtering was performed based on data availability for particular individuals when needed. Section title: Data analysis Educational score: 4.086246013641357 Domain: biomedical Document type: Study Language: en Newly obtained genotyping data were combined with previously reported data on Zhabe horses . Previously obtained unfiltered data were merged with current results and filtered (SNP call rate ≥0.9, minor allele frequency ≥0.05, Hardy-Weinberg equilibrium under p- value threshold 10 −5 ). The obtained dataset was used for genetic structure analysis using ADMIXTURE software and GWAS using PLINK1.9. ADMIXTURE run was set for K numbers of clusters from 2 to 20 with ten-fold cross-validation error estimate; results were vizualized using CLUMPAK web server . Section title: Data analysis Educational score: 4.052318572998047 Domain: biomedical Document type: Study Language: en For comparative analyzis the dataset on Zhabe horses was further merged with data on foreign horse breeds and filtered anew with the same criteria. The following breeds were chosen: Akhal-Teke, Caspian, Arabian, Mongolian, and Thorougbred (two populations)( Table 1 ). The analysis included principal component analysis and evaluation of linkage disequilibrium (LD), expected and observed heterozygosity, and pairwise between population Wright's F ST using PLINK1.9 and R . Neighbor net for pairwise F ST between Zhabe populations and foreign breeds was calculated and plotted using R package phangorn . Additionally, ADMIXTURE analysis was performed for the combined dataset with the same running conditions. Section title: Data analysis Educational score: 4.182552337646484 Domain: biomedical Document type: Study Language: en Genome wide association study was conducted using general linear model implemented in PLINK1.9. Sex and age of animals in years were used as covariates of the model. Animals without available phenotypic data (e.g. population 32 “Sayakhat”) or identified as outlying genotypes by population structure analysis were excluded from the analysis. The four size measurements (HW, OBL, CC, and CBC) were normalized by subtracting the mean and dividing by standard deviation and then used to perform principal component analysis using built-in R functions. The first principal component was further used in GWAS as a synthetic variable describing animal body size. GWAS was performed separately for animal body weight and size using --linear command of PLINK with age and sex as covariates (model Y = b 0 + b 1 X ADD + b 2 X AGE + b 3 X SEX + e, where Y – phenotype , X ADD – genotypes encoded as additive model (0, 2 for homozygote, 1 for heterozygote), X AGE – covariate of age, X SEX – covariate for sex, b n – regression coefficients, e – residual error term). P- values were estimated using 1000 Monte-Carlo permutation test with adaptive number of permutations (PLINK's--perm command) and used to construct Manhattan plot using ‘ggplot2’ R package. Variant Effect Predictor and DAVID tools were used to annotate significant SNPs. Results for SNPs associated with body weight and size were combined and filtered to include only variants with available annotations and exclude variants with duplicate annotations. Section title: Results Educational score: 3.61747670173645 Domain: biomedical Document type: Study Language: en As a final result of genotyping and data filtering, genotypes of 443 horses have been obtained including 401 samples of Zhabe horses and 42 samples of white horses ‘Zhetysu Asyly’, in addition to previously obtained data ( Table 1 ). The final combined dataset included 1038 individuals with 43,422 SNPs. The combined dataset including five foreign breeds consisted of 24,764 SNP after additional filtering. Section title: Results Educational score: 4.253612041473389 Domain: biomedical Document type: Study Language: en Calculated values of expected/observed heterozygosity and pairwise between population F ST are shown in Table 2 . Among Kazakh horses, the lowest hetezygosity level was in population 39 containing Zhetysu Asyly horses; expected value per chromosome 0.28 (SD 0.010), observed value 0.308 (SD 0.12). Other populations corresponding attributed Zhabe horses displayed expected values from 0.314 in population 34 to 0.334 in population 3, and observed values from 0.317 in populations 33 and 34 to 0.342 in population 32; the former population had the biggest deviation between expected and observed heterozygosity whereas other populations had only slightly differing values. All Kazakh horse populations had low F ST values not exceeding 0.010 between each other except for population 39 with F ST from 0.027 to 0.034. The foreign breeds displayed higher coefficients when compared to Zhabe horses: from 0.006 to 0.030 for Mongolian horses to 0.095–0.106 for the Thoroughbred horses. Thus, the Mongolian horses displayed more similarity to Zhabe horses, and the Thoroughbred horses were the most dissimilar. As it can be seen in the neighbor net plot of F ST , populations of Zhabe horses form close group except for popultaion 3 which demonstrated shift towards Thoroughbred horses. Population 39 (Zhetysu Asyly) formed independent branch distant from Zhabe populations. Table 2 Population genetic parameters calculated for combined dataset (24,764 SNP). Table 2 Population N H Echr H Ochr F ST 25 28 29 32 33 34 37 39 3 AKTK ARR CSP MON TB_UK TB_US 25 64 0.319 ± 0.009 0.325 ± 0.009 – 0.001 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.030 ± 0.007 0.008 ± 0.002 0.044 ± 0.006 0.057 ± 0.006 0.029 ± 0.004 0.008 ± 0.003 0.099 ± 0.017 0.096 ± 0.017 28 86 0.318 ± 0.008 0.318 ± 0.010 0.001 ± 0.001 – 0.004 ± 0.001 0.005 ± 0.001 0.002 ± 0.001 0.002 ± 0.001 0.003 ± 0.001 0.030 ± 0.007 0.008 ± 0.002 0.045 ± 0.007 0.058 ± 0.007 0.029 ± 0.004 0.007 ± 0.002 0.101 ± 0.018 0.098 ± 0.017 29 46 0.318 ± 0.009 0.326 ± 0.009 0.003 ± 0.001 0.004 ± 0.001 – 0.004 ± 0.001 0.003 ± 0.001 0.006 ± 0.001 0.004 ± 0.001 0.030 ± 0.007 0.007 ± 0.001 0.044 ± 0.006 0.057 ± 0.007 0.029 ± 0.005 0.008 ± 0.003 0.095 ± 0.015 0.092 ± 0.016 32 47 0.320 ± 0.009 0.342 ± 0.011 0.003 ± 0.001 0.005 ± 0.001 0.004 ± 0.001 – 0.006 ± 0.001 0.004 ± 0.001 0.006 ± 0.001 0.034 ± 0.007 0.011 ± 0.001 0.047 ± 0.006 0.059 ± 0.006 0.032 ± 0.004 0.012 ± 0.002 0.098 ± 0.017 0.095 ± 0.018 33 152 0.321 ± 0.009 0.317 ± 0.010 0.003 ± 0.001 0.002 ± 0.001 0.003 ± 0.001 0.006 ± 0.001 – 0.002 ± 0.001 0.002 ± 0.000 0.028 ± 0.007 0.006 ± 0.001 0.042 ± 0.006 0.055 ± 0.006 0.026 ± 0.004 0.006 ± 0.002 0.097 ± 0.016 0.094 ± 0.016 34 76 0.314 ± 0.009 0.317 ± 0.009 0.003 ± 0.001 0.002 ± 0.001 0.006 ± 0.001 0.004 ± 0.001 0.002 ± 0.001 – 0.004 ± 0.001 0.030 ± 0.006 0.010 ± 0.002 0.048 ± 0.006 0.061 ± 0.007 0.031 ± 0.005 0.007 ± 0.002 0.106 ± 0.018 0.104 ± 0.017 37 384 0.321 ± 0.008 0.319 ± 0.009 0.003 ± 0.001 0.003 ± 0.001 0.004 ± 0.001 0.006 ± 0.001 0.002 ± 0.000 0.004 ± 0.001 – 0.027 ± 0.007 0.008 ± 0.002 0.043 ± 0.006 0.057 ± 0.007 0.027 ± 0.004 0.005 ± 0.002 0.101 ± 0.017 0.099 ± 0.017 39 42 0.298 ± 0.010 0.308 ± 0.012 0.030 ± 0.007 0.030 ± 0.007 0.030 ± 0.007 0.034 ± 0.007 0.028 ± 0.007 0.030 ± 0.006 0.027 ± 0.007 – 0.032 ± 0.006 0.069 ± 0.009 0.079 ± 0.011 0.054 ± 0.007 0.034 ± 0.007 0.122 ± 0.017 0.120 ± 0.018 3 141 0.334 ± 0.008 0.324 ± 0.008 0.008 ± 0.002 0.008 ± 0.002 0.007 ± 0.001 0.011 ± 0.001 0.006 ± 0.001 0.010 ± 0.002 0.008 ± 0.002 0.032 ± 0.006 – 0.037 ± 0.006 0.050 ± 0.006 0.028 ± 0.004 0.015 ± 0.003 0.064 ± 0.012 0.060 ± 0.011 AKTK 19 0.300 ± 0.009 0.299 ± 0.012 0.044 ± 0.006 0.045 ± 0.007 0.044 ± 0.006 0.047 ± 0.006 0.042 ± 0.006 0.048 ± 0.006 0.043 ± 0.006 0.069 ± 0.009 0.037 ± 0.006 – 0.067 ± 0.009 0.058 ± 0.008 0.056 ± 0.009 0.097 ± 0.016 0.094 ± 0.017 ARR 24 0.299 ± 0.011 0.294 ± 0.016 0.057 ± 0.006 0.058 ± 0.007 0.057 ± 0.007 0.059 ± 0.006 0.055 ± 0.006 0.061 ± 0.007 0.057 ± 0.007 0.079 ± 0.011 0.050 ± 0.006 0.067 ± 0.009 – 0.059 ± 0.008 0.074 ± 0.01 0.109 ± 0.017 0.107 ± 0.018 CSP 18 0.297 ± 0.009 0.309 ± 0.013 0.029 ± 0.004 0.029 ± 0.004 0.029 ± 0.005 0.032 ± 0.004 0.026 ± 0.004 0.031 ± 0.005 0.027 ± 0.004 0.054 ± 0.007 0.028 ± 0.004 0.058 ± 0.008 0.059 ± 0.008 – 0.036 ± 0.004 0.110 ± 0.015 0.107 ± 0.015 MON 19 0.298 ± 0.009 0.306 ± 0.011 0.008 ± 0.003 0.007 ± 0.002 0.008 ± 0.003 0.012 ± 0.002 0.006 ± 0.002 0.007 ± 0.002 0.005 ± 0.002 0.034 ± 0.007 0.015 ± 0.003 0.056 ± 0.009 0.074 ± 0.01 0.036 ± 0.004 – 0.116 ± 0.020 0.113 ± 0.020 TB_UK 19 0.306 ± 0.010 0.321 ± 0.014 0.099 ± 0.017 0.101 ± 0.018 0.095 ± 0.015 0.098 ± 0.017 0.097 ± 0.016 0.106 ± 0.018 0.101 ± 0.017 0.122 ± 0.017 0.064 ± 0.012 0.097 ± 0.016 0.109 ± 0.017 0.110 ± 0.015 0.116 ± 0.02 – 0.003 ± 0.008 TB_US 17 0.310 ± 0.012 0.323 ± 0.016 0.096 ± 0.017 0.098 ± 0.017 0.092 ± 0.016 0.095 ± 0.018 0.094 ± 0.016 0.104 ± 0.017 0.099 ± 0.017 0.12 ± 0.018 0.060 ± 0.011 0.094 ± 0.017 0.107 ± 0.018 0.107 ± 0.015 0.113 ± 0.02 0.003 ± 0.008 – N – number of individual samples. H E chr – expected heterozygosity per chromosome, mean ± standard deviation. H Ochr – observed heterozygosity per chromosome, mean ± standard deviation. F ST – Wright's fixation index between populations. Fig. 1 Plot of linkage disequilibrium and pairwise F st of Zhabe horses; A) Linkage disequilibrium decay in nine studied populations of Zhabe horses; B) Linkage disequilibrium decay in Zhabe horses in comparison with Akhal-Teke, Caspian, Mongol, Arabian, and Thoroughbred horses; C) Neighbor net plot of pairwise FST between Zhabe horse populations and foreign breeds. Fig. 1 Section title: Results Educational score: 3.2446045875549316 Domain: biomedical Document type: Study Language: en Linkage disequilibrium (LD) analysis displayed higher values of R 2 coefficient in population 39 comparing to other Zhabe populations. Foreign breeds except Mongolian horses have shown much higher values; the highest R 2 coefficients and slower LD decay was in two Thoroughbred populations . Mongolian horses had closer LD values to Zhabe horses than to other foreign breeds. Section title: Results Educational score: 3.9245994091033936 Domain: biomedical Document type: Study Language: en Principal component analysis have shown a presence of outlying genotypes of Zhabe (populations 3 and 33) shifted towards Thoroughbred whereas most Zhabe samples have been combined into a single dense cluster. Principal component 1 (PC1) explaining 21.73 % of total variance contributes the most to discrimination between Zhabe horses and Thoroughbred, and PC2 with 8.27 % of explained variance separated Arabian horses. Whereas first two components place population 39 close to other Zhabe horses, PC3 has shown its significant deviation. Mongolian horses were placed close to Zhabe. Akhal-Teke and Caspian horses formed distinct cluster close to Zhabe. Fig. 2 Principal component analysis of Zhabe horses in comparison to five foreign breeds; A) Scatterplot of components 1 and 2; B) Scatterplot of components 2 and 3. Population codes according Table 1 . Fig. 2 Section title: Results Educational score: 4.002728462219238 Domain: biomedical Document type: Study Language: en ADMIXTURE analysis was performed for numbers of expected clusters K from 1 to 20. Cross-validation test have not revealed the best K value; although the error value was the lowest at K = 17, the absence of following inclining trend on the plot makes its selection doubtful . Fig. 3 A and B, demonstrates patterns for K= 2, 5, and 10 selected based on examination of diagrams as demonstrating most significant features of the obtained results. With K= 2 outlying genotypes persistent across all K configurations have been identified (shown orange in all diagrams). Comparative analysis with foreign breed allowed to attribute these individuals to inclusion of Thoroughbred into selection process. Population 39 was classified as a unique cluster dissimilar to either Zhabe or foreign breeds. Higher K patterns reveal within population heterogeneity of Zhabe breeds. Fig. 3 Results of ADMIXTURE analysis of Zhabe horses in comparison to five foreign breeds; A) Data on Zhabe horses, 43,422 SNP; B) Data on Zhabe horses combined with five foreign breeds (24,764 SNP); C) Plot of cross-validation error for data A (blue line) and B (red line). Population codes according Table 1 . Fig. 3 Section title: Results Educational score: 4.209163188934326 Domain: biomedical Document type: Study Language: en For genome wide association study, horse individuals identified as outlying genotypes by PCA and ADMIXTURE analysis were excluded from consideration. Total 823 animals were used for analysis, including 391 newly obtained genotypes and 432 genotypes from previously published data . The summary of phenotypic data used is shown in Table 3 . As a result, 126 SNPs had p- value adjusted by Monte-Carlo permutation test for association with body weight, and 99 SNPs for body size. All variants satisfying the selected significance threshold P < 0.001 were distributed occasionally, and no regions with high occurrence of significant SNPs were observed . After variant annotation and filtering of the results, total 57 SNPs have been retained, including 14 and 23 SNPs associated only with body size and weight, correspondingly, and 20 variants associated with both traits ( Table 4 ). Gene CFI (complement factor I) contained three revealed SNPs; for genes DLG2, KIT , and PRKG2 two variants have been identified; other genes contained single SNPs. Most annotated genes were have been involved into various regulatory processes and signal transmission. Table 3 Summary of phenotypic traits of Zhabe horse populations. Table 3 Population code Sex (percentage of mares in the sample) Age Weight HW OBL CC CBC mean sd mean sd mean sd mean sd mean sd mean sd 25 92.19 8.111 3.399 417.328 41.505 142.422 2.308 146.719 4.022 173.406 7.272 18.805 0.568 28 91.86 6.209 3.505 394.128 38.763 141.372 3.002 145.977 3.700 173.012 6.934 17.878 0.659 29 93.48 5.435 2.177 359.326 61.695 139.348 4.729 142.391 7.120 168.891 8.817 17.413 0.896 3 91.43 6.993 3.771 397.679 52.221 141.157 3.740 146.036 5.756 171.571 8.259 18.050 0.896 32 100.00 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 33 100.00 7.993 2.272 388.901 12.641 139.605 1.479 143.645 1.673 164.757 2.258 17.688 0.372 34 89.33 8.507 0.891 401.320 28.991 140.293 2.198 144.427 2.579 167.160 5.514 18.600 0.539 37 96.88 3.786 1.657 396.997 44.904 141.882 2.838 145.743 3.456 169.322 5.589 17.732 0.870 39 80.95 4.190 1.194 380.286 37.050 140.119 2.596 143.810 3.763 168.976 4.176 17.788 0.678 nd. - no data available. Fig. 4 Manhattan plots of P-values adjusted by the Monte-Carlo permutation test from genome-wide association analysis for body size (A) and weight (B) in Zhabe horses. Red line indicates significance threshold p= 0.001. Fig. 4 Table 4 Single nucleotide polymorhisms associated with horse weight and size annotated using VEP and DAVID tools. Table 4 Variant ID Location Gene P -value GO term – biological process Size Weight BIEC2_316765 15:68252917 ALK – 0.0008545 GO:0006468∼protein phosphorylation AX-103265166 2:55284182 BNIP3L 2.3 × 10 −5 1.80 × 10 −5 GO:0016239∼positive regulation of macroautophagy AX-104903715 4:93924679 BRAF 0.0007523 0.0004619 GO:0000165∼MAPK cascade BIEC2_946110 6:29507974 CACNA1C 0.0002397 – GO:0002520∼immune system development BIEC2_140284 11:13962473 CACNG4 – 3.181 × 10 −5 GO:0019226∼transmission of nerve impulse Affx-102683437 4:79003820 CADPS2 – 0.0001952 GO:0006887∼exocytosis AX-104609861 6:85773796 CCT2 – 0.0007669 GO:0006457∼protein folding BIEC2_508762 2:116459352 CFI – 0.0004362 GO:0006508∼proteolysis BIEC2_508766 2:116465026 CFI 0.0002012 0.0001068 GO:0006508∼proteolysis BIEC2_508769 2:116466442 CFI 0.0001972 0.0001277 GO:0006508∼proteolysis TBIEC2_331745 15:68417648 CLIP4 – 0.0002872 GO:0031122∼cytoplasmic microtubule organization Affx-101130275 4:60087882 CPVL 0.0009387 – GO:0006508∼proteolysis BIEC2_700308 27:2259858 CSGALNACT1 – 0.0004961 GO:0001958∼endochondral ossification BIEC2_725599 28:5564261 CSRP2 0.0003285 – GO:0045214∼sarcomere organization AX-103706694 1:120490380 CYP11A1 1 × 10 −6 1.00 × 10 −7 GO:0006700∼C21-steroid hormone biosynthetic process TBIEC2_498975 2:40275747 DISP3 0.0004661 – GO:0045665∼negative regulation of neuron differentiation AX-103721396 7:63690864 DLG2 0.0008281 1.00 × 10 −7 GO:0007268∼chemical synaptic transmission BIEC2_1004624 7:62981215 DLG2 0.0009895 – GO:0007268∼chemical synaptic transmission BIEC2_722382 27:39199656 DLGAP2 – 0.0002175 GO:0023052∼signaling AX-103583267 26:37333692 DSCAM – 0.0004288 GO:0007156∼homophilic cell adhesion via plasma membrane adhesion molecules Affx-103045508 2:27182213 EPB41 0.0008206 – GO:0007049∼cell cycle TBIEC2_889720 4:8502727 EPDR1 – 0.0004253 GO:0007160∼cell-matrix adhesion BIEC2_852365 3:115596394 EVC 0.0009599 – GO:0003416∼endochondral bone growth BIEC2_416905 18:63653165 FAM171B – 0.0009746 GO:0001525∼angiogenesis Affx-102845142 28:31765971 FBXO7 0.0003695 0.0001627 GO:0000422∼mitophagy BIEC2-472336 2:37787520 FHAD1 0.0003518 −0.0004809 – BIEC2_14481 1:32086433 FRAT1 5.366 × 10 −5 0.0006401 GO:0090263∼positive regulation of canonical Wnt signaling pathway Affx-102013817 17:62060245 GPC5 – 0.0004061 GO:0016477∼cell migration Affx-102676537 6:24907627 HDAC4 1.5 × 10 −5 3.00 × 10- 7 GO:0000122∼negative regulation of transcription from RNA polymerase II promoter BIEC2_14318 1:30611785 HPSE2 – 0.0002113 GO:0008283∼cell proliferation ITGA2B_19245752_GT 11:19245752 ITGA2B 1 × 10 −6 – GO:0001525∼angiogenesis KIT_79540741_W16 3:79540741 KIT 1 × 10 −6 1.00 × 10 −7 GO:0001541∼ovarian follicle development KIT_79580000_W7 3:79580000 KIT 1 × 10 −6 1.00 × 10 −7 GO:0001541∼ovarian follicle development BIEC2_320442 15:75799411 LDAH 0.0003885 – GO:0019915∼lipid storage Affx-101492057 10:19641430 NOSIP 6 × 10 −6 1.00 × 10 −7 – AX-104335949 5:40161837 NUP210L 1 × 10 −6 1.00 × 10 −7 – PAX3_11199140_SW4 6:11199140 PAX3 1 × 10 −6 1.00 × 10 −7 GO:0006355∼regulation of transcription AX-104963491 12:30951642 PITPNM1 2 × 10 −6 1 × 10 −7 GO:0015914∼phospholipid transport BIEC2_887173 5:2222786 PM20D1 0.0002478 – GO:0006520∼cellular amino acid metabolic process BIEC2_783022 3:57169047 PRKG2 – 0.0007432 GO:0006468∼protein phosphorylation BIEC2_783026 3:57171222 PRKG2 – 0.0003162 GO:0006468∼protein phosphorylation BIEC2_885537 4:107978404 PTPRN2 – 0.0009618 GO:0006470∼protein dephosphorylation BIEC2_591905 22:33100482 PTPRT 0.0006552 – GO:0006470∼protein dephosphorylation Affx-103079316 12:30867790 RAD9A – 3.127 × 10 −5 GO:0000076∼DNA replication checkpoint AX-104886193 25:31276468 RALGPS1 0.0001003 8.00 × 10 −7 GO:0007264∼small GTPase mediated signal transduction Affx-102836572 17:50354202 SLAIN1 0.0004986 1.00 × 10 −6 GO:0007020∼microtubule nucleation SLC36A1_25884457_Champagne 14:25884457 SLC36A1 1 × 10 −6 1.00 × 10 −7 GO:0015808∼L-alanine transport BIEC2_11418 1:24106986 SORCS1 0.0006665 – GO:0006892∼post-Golgi vesicle-mediated transport BIEC2_954544 6:50669754 SOX5 – 0.0009407 GO:0006357∼regulation of transcription from RNA polymerase II promoter BIEC2_1011868 7:87211576 SOX6 0.0006732 – GO:0006357∼regulation of transcription from RNA polymerase II promoter AX-104226721 9:36874369 SPIDR 1 × 10p 4.00 × 10 −7 GO:0000724∼double-strand break repair via homologous recombination AX-104855978 28:39009737 TEF 0.0007493 – GO:0006357∼regulation of transcription from RNA polymerase II promoter BIEC2_486399 2:69215803 TLL1 – 0.0006598 GO:0006508∼proteolysis BIEC2_1004550 7:62165511 TMEM126B – 0.0004348 GO:0032094∼response to food BIEC2_195234 12:24939247 TMEM138 – 6 × 10 −7 – AX-103728731 9:84365361 ZC3H3 0.0005481 2.00 × 10 −6 – Affx-101705964 10:25531136 ZNF787 0.0002468 – GO:0000122∼negative regulation of transcription from RNA polymerase II promoter Section title: Discussion Educational score: 4.207866668701172 Domain: biomedical Document type: Study Language: en The obtained results on genetic structure of Zhabe horses are in line with our previous findings . As we discussed in the mentioned paper, the specific conditions of traditional nomadic husbandry could affect genetic composition of Kazakh horses. Almost free grazing of horses on summer and winter pastures with low control of mating by the herders have been closer to the ways of existence of horses in the wild nature rather than horse breeds under strictly controllable stable conditions. Here we have examined 443 newly genotyped horse individuals in addition to the previously analyzed and reported horse samples (616) to have a deeper look on genetic structure of Zhabe horses in comparison with several foreign horse breeds, Akhal-Teke, Arabian, Caspian, Mongolian, and Thoroughbred (data by Ref. ). These particular breeds have been selected under specific considerations: Mongolian and Caspian horses have previously shown high genetic similarity to Kazakh horses ; Akhal-Teke breed was selected as another ancient breed with Central Asian origin; Arabian and Thoroughbred horses have been selected as reference well known breeds of stable maintenance and high breeding control; moreover, Thoroughbred horses are known to be involved to later breeding practice for improvement of Kazakh horses. Indeed, the presence of presumably hybrid genotypes between Zhabe and Thoroughbred horses was revealed by PCA and ADMIXTURE analysis. Population 3 (Kalka) which included the most of Thoroughbred hybrids was also shown closer to this breed by F ST analysis. In comparison, Zhabe horses display lower degree of LD comparing to foreign breeds. Such population genetics concepts as LD decay and ROH are considered as selection signatures in domestic animals . Comparing to foreign breeds, we have shown in our study that Zhabe horses indeed have these signatures less expressed. Lower LD parameters indicate higher individual variability within populations which is also illustrated by results of ADMIXTURE analysis. As Fig. 3 shows, besides individuals with identified admixture with Thoroughbred horses, higher K patterns demonstrate additional within populaion variability which is, however, is not associated with particluar populations. It was shown previously that such internal diversity within populations may be caused by the influence of groups of closely related horses, e.g. originated from the same productive sires . However, the lack of pedigree data on Kazakh horses due to low control of mating makes it impossible to evaluate such possible substructure in this study. Section title: Discussion Educational score: 3.7377190589904785 Domain: biomedical Document type: Study Language: en Population 39 representing unique white horses ‘Zhetysu Asyly’ posed a special interest. This lineage is a result of amateur selection and have not been an object of scientific evaluation to date. Although it was stated by the breeder (personal communication) that this breed have been established based on Zhabe and other Kazakh horses, no documents on the selection history are available. Our results reveal surprisingly high level of differentiation of population 39 comparing to Zhabe horses. Patterns of LD and F ST of these horses is more similar to foreign breeds, however PCA, F ST and ADMIXTURE analysis place this population as unique cluster. Considering strict artificial selection towards color, the observed differences should be considered selective signatures. If the declared assumption about their origin from Kazakh horses is correct, their genetic composition could be an example of rapid transformation of animal's genome under pressure of targeted selection. However, lack of reliable descriptive and genealogical data makes possible assumption speculative. Thus, so called ‘Zhetysy Asyly’ horses are a potential object for further deeper investigation. Section title: Discussion Educational score: 4.107645034790039 Domain: biomedical Document type: Study Language: en Previously, we have attempted GWAS to identify variants associated with body weight and size in Kazakh horses . The results of that work have demonstrated the absence of genomic regions strongly associated with the considered body traits. The variants with significant associations were distributed occasionally and did not form notable islands corresponding to genomic regions to increased association. Moreover, no SNPs associated with known major horse body size factors, genes LCORL , NCARG , and ZFAT , have been revealed. In the present study, we obtained similar results for selection of Zhabe horses. Interestingly, the loci identified here showed no correspondence to previously reported ones. Such a distribution of occasional SNPs could be a sign of the absence of strong selection signatures for body size and weight in Kazakh horses and, particularly, Zhabe horse type. Thus, these traits of Zhabe horses, as well as other Kazakh horse varieties, could be determined by environment to higher extent than genetic factors. Section title: Discussion Educational score: 4.067836284637451 Domain: biomedical Document type: Study Language: en Along with the previous findings, our results illustrate specific genetic properties of Kazakh horses. Traditional nomadic ways of horse breeding have lead to formation of unique landrace with low presence of selection signatures. We could suggest that this lack of dominating genetic factors developed by selection is the reason of high versatility and adaption of Kazakh horses to a range of conditions of Central Asia, from steppes to mountains. On the other hand, the genetic identity of Kazakh horses is vulnerable before targeted breeding practices and hybridization. As we have shown here , relatively recent hybridization events involving Thoroughbred horses have left distinctive signature allowing to identify source of admixture. Horses “Zhetysu Asyly” (population 39) resulted from targeted selection of Kazakh horses for white color demonstrate significant changes in their genetic composition from Zhabe horses and, considering low variability between breeds , Kazakh horses in general. Thus, Kazakh horses and, particularly, Zhabe type should be considered a unique legacy landrace requiring special protection. The obtained results provide genetic data on the Kazakh horses of Zhabe type which are traditional but yet understudied horse breed. To conserve their genetic composition and properties the breeding programs should be focused on the internal genetic resources of Kazakh horses rather than hybridization with distant breeds. Still, Kazakh horses may be a valuable genetic resource for developing new horse breeds due to their unique properties and adaption to environmental conditions. Section title: Conclusion Educational score: 3.922503709793091 Domain: biomedical Document type: Study Language: en The present work provides important insights into the genetic structure of Zhabe horses and other Kazakh horse populations. The analysis revealed that traditional nomadic breeding practices, characterized by minimal human interference, have shaped these horses into a unique landrace with low selection signatures and high genetic diversity. Population 39, the white horses known as ‘Zhetysu Asyly,’ displayed significant genetic differentiation, likely due to targeted breeding for coat color. The findings highlight the importance of conserving the genetic integrity of Kazakh horses, particularly Zhabe, to preserve their adaptability and historical significance, while recognizing their potential as a genetic resource for future breeding programs. Section title: CRediT authorship contribution statement Educational score: 0.9927184581756592 Domain: other Document type: Other Language: en Alexandr Pozharskiy: Writing – original draft, Visualization, Formal analysis, Data curation. Indira Beishova: Supervision, Resources, Project administration, Conceptualization. Askar Nametov: Writing – review & editing, Supervision. Alzhan Shamshidin: Resources. Tatyana Ulyanova: Methodology, Investigation. Alexandr Kovalchuk: Methodology, Investigation. Vadim Ulyanov: Investigation. Malika Shamekova: Resources, Investigation. Gulmira Bekova: Methodology, Investigation. Dilyara Gritsenko: Writing – review & editing, Supervision, Project administration, Conceptualization. Section title: Ethics statement Educational score: 1.0020151138305664 Domain: biomedical Document type: Other Language: en The study was conducted according to the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and approved by the Local Ethics Committee of Zhengir Khan West-Kazakhstan Agrarian Technical University . Section title: Data availability statement Educational score: 0.8878604769706726 Domain: other Document type: Other Language: en The raw data supporting the conclusions of this article will be made available by the authors on request. Section title: Funding Educational score: 1.1403753757476807 Domain: other Document type: Other Language: en This research was funded by the Ministry of Science and Higher Eduction of the Republic of Kazakhstan within the framework of the of the research project AP14870614 «Genetic marking of productive traits of the Kazakh horse of the Dzhabe type based on genome-wide coverage SNP genotyping» Section title: Declaration of competing interest Educational score: 0.9818206429481506 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Other | other | en | 0.999996 |
PMC11699314 | Section title: Introduction Educational score: 4.438709259033203 Domain: biomedical Document type: Study Language: en Soils are complex physicochemical matrices with a porous structure, a huge surface area, and an extremely variable supply of organic materials, nutrients, and water, providing a range of habitats for a multitude of organisms . They make up one of the most diverse habitats on Earth. Nowhere in nature are species so densely clustered as in soil communities . Microbial communities account for a large part of this biodiversity. Estimates of soil microbial diversity range from thousands to a million microbial species in just a few grams of soil . Soil microbial communities (Archaea, Bacteria, Fungi) play a crucial role in ecosystem functioning and resilience, particularly in biogeochemical cycles, organic matter mineralization, plant growth and productivity, soil structure maintenance, and soil pollutant reduction [ , , , , ]. Soil microbial communities are good indicators for monitoring soil quality and serve as early bioindicators of disturbances due to their essential role in soil functions and their high sensitivity to changes in soil conditions, combined with their short generation time . Recent advances in molecular biology have provided access to a large part of the soil microbial diversity, e.g., taxonomic richness or community composition . Moreover, several studies have reported that soil microorganisms serve as early and robust indicators of the impact of agricultural practices that threaten crop production sustainability, due to their small size, rapid generation time, and sensitivity to soil disturbances . Section title: Introduction Educational score: 3.4019205570220947 Domain: other Document type: Review Language: en Soils are fundamental resources for agricultural production and food security. As providers of more than 95 % of global food, they depend on intensive agricultural practices . Sustainable agriculture should satisfy global needs, i.e., not only food supply but also a range of other issues involving energy use, nutrient recycling, and the effects on adjacent ecosystems, including the impact on water bodies and climate change . Aiming for an agroecological transition, the biogas sector can represent a sustainable opportunity by developing (i) the production of renewable energies such as electricity, or biomethane, and (ii) the management of organic waste (OW) . Biogas is produced from the anaerobic digestion of organic matter. A wide variety of feedstock can be used (crop residues, animal manure, the organic fraction of municipal and industrial solid waste, or wastewater sludge) . This process converts biowaste into two economically useful products: renewable energy (biogas) and a fertilizer (digestate) . However, reports on the impacts of digestates on the soil microbiota are scarce, and rather contradictory, as reviewed by Karimi et al. . Section title: Introduction Educational score: 4.072287559509277 Domain: biomedical Document type: Study Language: en Most of the studies conducted to assess the impact of digestates on soil microbiota have been carried out under laboratory conditions often poorly linked to real-life agronomic conditions . For instance, some studies in short- ( ≤ 1 year) and mid- ( ≤ 5 years) term on field experiments reported positive or neutral effects after digestate application on soil organic carbon content (SOC) and some microbial parameters (e.g., microbial biomass and diversity) [ , , , , ]. Interestingly, Coelho et al. observed that microbial communities from different liquid digestates failed to establish in the soil after two years of repeated applications. However, studies conducted on long-term fertilization (≥5 years; organic and/or mineral fertilization) have reported more persistent impacts on soil characteristics , plant growth , and microbial diversity and activity [ , , ], suggesting that repeated digestate application over five years could have a lasting effect on soil microbial communities To our knowledge, no studies have assessed the long-term dynamics of soil microbial communities following repeated digestate applications. Therefore, no objective and robust conclusions can be drawn regarding the impact of digestate on soil microbial biodiversity, particularly in a real field context, with repeated applications over the long term . Section title: Introduction Educational score: 4.111779689788818 Domain: biomedical Document type: Study Language: en The field experiment “Effluents d'Elevage et Environnement” (EFELE; Rennes, France) was set up in 2012. It aims to comprehensively understand the long-term effects of repeated fertilization with different types of organic wastes (OW) on the soil quality and functioning. The experiment involved different fertilization practices, including mineral and organic inputs (cattle manure, pig slurry and pig slurry digestate). The present study aimed to assess the response of soil microbial communities (Archaea, Bacteria, Fungi) to 10 years of mineral and OW fertilization regimes by molecular DNA-based tools targeting the soil microbial biomass, soil microbial diversity, and soil microbial community structure. To our knowledge, no report had as yet employed an in-situ approach to monitor the dynamic effect of the repeated application of a digestate over a decade on soil microbial parameters. We hypothesized that (i) long-term historical and repeated digestate applications would strongly influence the edaphic soil properties, and in turn, affect the soil microbial parameters; and (ii) lasting modifications would only occur after several years of repeated applications. We assessed the long-term impact of historical and repeated fertilization practices on soil microbial communities, using digestate as an organic fertilizer. We also evaluated whether the digestate feedstock had a similar impact to that of the same undigested feedstock and other commonly used types of fertilizers. Section title: Experimental site, soil sampling strategy, and soil chemical analysis Educational score: 2.49613356590271 Domain: other Document type: Study Language: en The study was carried out at the EFELE experimental site (Le Rheu, France; 48°06′07″N, 1°47′44″W) of the SOERE-PRO 1 network, designed for long-term studies on the evolution of agrosystems after repeated applications of OW (see Refs. for a complete description). The soil was classified as a Luvisol-Redoxisol derived from aeolian silt deposited on schist material . The experiment was set up in 2012. The topsoil horizon (0–25 cm soil depth) had the following properties at the beginning of the trial: clay 14.2 %; silt 70.8 %; sand 15 %; total C 11.03 g kg −1 ; total N 1.14 g kg −1 ; C/N ratio 9.62; pH 6.10. The climate was mild temperate oceanic, with 711 mm year −1 mean annual rainfall and 11.2 °C mean annual temperature. Section title: Experimental site, soil sampling strategy, and soil chemical analysis Educational score: 4.100799560546875 Domain: biomedical Document type: Study Language: en The experiment was based on a winter wheat ( Triticum aestivum L.) and maize ( Zea mays L.) crop rotation, with a catch crop of white mustard ( Sinapis alba ) . The crop residues (including aboveground residues) were not returned to the soil. Before the start of the experiment in 2010 and 2011, maize was grown as a homogenization crop. The soil management activities performed from 2012 to 2022 are presented in Fig. 1 . The fertilization treatments included a mineral N fertilizer (MIN) in the form of ammonium nitrate (comprising 50 % nitrate and 50 % ammonium), a control (ON) that did not receive any organic or mineral nitrogen input (but fertilized with P and K), and three different OW: cattle manure (CM), pig slurry (PS), and the digestate obtained after anaerobic digestion of the same pig slurry used in the study (PS-DIG). The design of the trial was a complete randomized block where each treatment had 4 replicates, and each plot had a surface area of 109 m 2 (12.5 m × 8.7 m). MIN, PS, and DIG-PS were applied once a year in early spring (late March to mid-April), and CM every two years. The mean characteristics of the OW applied from 2012 to 2022 are given in Table 1 . The index of residual organic carbon (I ROC ) represents the proportion of organic matter that may be incorporated into the soil organic matter following OW application . The mean rate of MIN applied from 2012 to 2021 represented 110.9 kg N ha −1 yr −1 , 52.5 kg P 2 O 5 ha −1 yr −1 , and 78 kg K 2 O ha −1 yr −1 . The detailed amounts of OW and mineral fertilizer applied per year are presented in Table S 1 , Figure S 2 , and Figure S 3 . The application rates for the liquids OW (PS-DIG and PS) were determined according to their N fertilizer value, aligning with the regulations of the EU Nitrates Directive (91/676/EEC ). The CM inputs (solid OW) were reasoned based on the first limiting element, phosphorus, aiming not to exceed the threshold of an annual input of 100 kg P 2 O 5 ha −1 yr −1 . The application rate of MIN was calculated using the mineral N balance method recommended in France . Except for the ON plots, all plots, regardless of the treatments, showed similar yields throughout the years (18.65 t DM/ha for maize silage; 8.23 t DM/ha for wheat straw and 82.06 quintal/ha for wheat grain). Fig. 1 Agricultural practices at the EFELE from 2012 to 2021 on all treatments, including crop succession, fertilization, and soil management activities. The experiment was based on a winter wheat ( Triticum aestivum L.) and maize ( Zea mays L.) crop rotation, with a cover crop of white mustard ( Sinapis alba ). Wheat and maize were harvested, whereas mustard was mulched. Tillage was classified depending on soil depth (superficial <10 cm; deep >10 cm). Fertilization treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. “Others” management practices grouped: mechanical weeding, pesticide spreading, and liming. The liming rate was 370 kg ha −1 (granules containing 54.5 % CaO and 0.5 % MgO). Soil samples were collected in March every year from 2012 to 2022. Fig. 1 Table 1 Mean characteristics of the organic wastes (OW) applied in the EFELE plots from 2012 to 2021. Table 1 Treatment Applied quantity Dry matter Organic C Total N Organic N I ROC C/N pH (water) t FM ha −1 yr −1 % kg FM ha −1 yr −1 kg FM ha −1 yr −1 kg FM ha −1 yr −1 % Organic C CM 25a (0.0) 26.2a (3.6) 4692.5a (818.8) 313.3a (60.9) 248.1a (59.2) 58.8a (6.6) 15.2a (2.4) 9.4a (0.4) PS 31.4a (8.4) 7.2b (2.5) 844.5b (397.5) 160.6b (47.1) 41.6b (17.7) 49.3a (10.4) 5.2b (1.3) 8.5a (1.0) PS-DIG 32.9a (8.0) 5.8b (1.7) 645.3b (217.3) 159.5b (34.8) 36.8b (8.3) 54.7a (10) 4.0b (0.9) 9.0a (1.0) Fresh matter (FM). Values are means and letters denote the significant effect between treatments (p < 0.05) based on Tukey's HSD test p-value adjusted by the BH method. The standard error of the means is indicated in parentheses. CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Section title: Experimental site, soil sampling strategy, and soil chemical analysis Educational score: 4.1360673904418945 Domain: biomedical Document type: Study Language: en Soil samples were collected in early March (about 11 months after the latest fertilization treatment) every year from 2012 to 2022. The last soil sampling was in March 2022. Each sample was composed of 8 soil cores taken from the 0–25 cm horizon at random locations in each plot, and mixed and homogenized by 4 mm mesh sieving to remove above-ground plant material, other plant debris, roots, and stones. The sieved soil was freeze-dried and stored at −40 °C at the GenoSol soil conservatory (INRAE, Dijon, France). 2 DNA extraction and molecular analyses were conducted in 2022 on soil samples collected at the beginning of the trial in 2012, as well as 5 and 10 years later . This led to a total of 60 soil samples (5 treatments × 4 replicates × 3 years). A portion of the same soils sampled in 2012, 2016, and 2021 was air-dried for physicochemical analysis to determine soil particle size distribution, pH, SOC, soil total nitrogen, the soil C/N ratio, total phosphorus, and the cation exchange capacity (CEC). These analyses were performed at the INRAE Soil Laboratory Analysis. 3 The soil physicochemical data are given in Table 2 . Table 2 Soil physicochemical parameters of the EFELE plots according to the fertilization treatment and the sampling time. Table 2 Treatment Organic carbon Total N P 2 O 5 (Olsen) C:N pH (water) CEC g kg −1 g kg −1 g kg −1 cmol + kg −1 2012 CM 10.77a (0.574) 1.13a (0.05) 0.18a (0.02) 9.53a (0.05) 6.14a (0.05) 6.17a (0.20) MIN 11.45a (0.532) 1.19a (0.06) 0.18a (0.03) 9.61 ab (0.08) 5.97a (0.24) 6.10a (0.65) ON 11.16a (0.935) 1.15a (0.09) 0.19a (0.02) 9.72b (0.11) 6.14a (0.27) 6.36a (0.55) PS 10.95a (0.443) 1.13a (0.04) 0.19a (0.02) 9.62 ab (0.08) 6.16a (0.21) 6.44a (0.58) PS-DIG 10.51a (0.878) 1.10a (0.09) 0.17a (0.01) 9.57 ab (0.05) 5.99a (0.25) 5.71a (0.41) 2016 CM 11.12a (0.36) 1.19a (0.02) 0.14a (0.02) 9.37a (0.09) 6.16a (0.11) 6.63a (0.47) MIN 10.8a (0.53) 1.16a (0.06) 0.13a (0.02) 9.28a (0.03) 5.91a (0.20) 6.01a (0.63) ON 10.42a (0.84) 1.12a (0.07) 0.14a (0.02) 9.26a (0.14) 6.13a (0.19) 6.29a (0.48) PS 10.95a (0.64) 1.17a (0.06) 0.15a (0.02) 9.38a (0.08) 6.26a (0.14) 6.62a (0.50) PS-DIG 10.45a (0.65) 1.12a (0.07) 0.14a (0.01) 9.29a (0.02) 6.07a (0.16) 5.92a (0.26) 2021 CM 11.27b (0.39) 1.25b (0.05) 0.14a (0.01) 9.03a (0.09) 6.36a (0.14) 6.55a (0.28) MIN 9.89a (0.23) 1.11a (0.04) 0.14a (0.02) 8.89a (0.17) 5.85b (0.16) 5.80a (0.45) ON 9.61a (0.39) 1.08a (0.05) 0.17a (0.03) 8.93a (0.09) 6.17 ab (0.31) 5.67a (1.21) PS 10.14a (0.32) 1.15 ab (0.04) 0.15a (0.01) 8.83a (0.07) 6.14 ab (0.13) 6.38a (0.46) PS-DIG 9.79a (0.53) 1.09a (0.06) 0.14a (0.01) 8.94a (0.04) 6.06 ab (0.22) 5.82a (0.28) Data pertains to air-dried soil. Values are means and letters denote the significant effect of treatment for each sampling time (p < 0.05) based on Tukey's HSD test p-value adjusted by the BH method. The standard error of the means is indicated in parentheses. CM, cattle manure; PS, pig slurry; MIN, mineral fertilizer; ON, no fertilization; PS-DIG, pig slurry digestate. Section title: DNA extraction and purification Educational score: 4.120144844055176 Domain: biomedical Document type: Study Language: en Total DNA was extracted from 1 g (dry weight) of soil using a single procedure standardized by the GenoSol platform (INRAE, Dijon, France) 2 . This protocol is based on three main steps: (i) microbial cell lysis by physical and chemical action; (ii) deproteinization; and (iii) alcohol precipitation and washing of the extracted nucleic acids. The DNA concentrations of the crude extracts were determined by electrophoresis in 1 % agarose gel stained with ethidium bromide, using calf thymus DNA as a standard curve. Quantified crude DNA was used as an estimate of the soil molecular microbial biomass . In order to separate residual impurities – particularly humic substances – 100 μL of crude DNA was purified using a Nucleospin® Soil kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). The concentrations of purified DNA were finally measured using a Quantifluor staining kit (Promega, Madison, Wisconsin, USA), according to the manufacturer's instructions. Section title: High throughput sequencing of 16S and 18S rRNA gene sequences Educational score: 4.155482292175293 Domain: biomedical Document type: Study Language: en Prokaryotic (bacterial-archaeal) diversity was estimated from each DNA sample by metabarcoding of the 16S rRNA gene following the method described by . A 440-base fragment targeting the V3 to V4 regions was first amplified with the corresponding primers F479 (5′CAG CMG CYG CNG TAA NAC3′) and R888 (5′CCG YCA ATT CMT TTR AGT3′). Fungal diversity was estimated by metabarcoding of the 18S rRNA gene. A 350-base fragment targeting the V7 to V8 regions was first amplified with the corresponding primers FR1 (5′ANC CAT TCA ATC GGT ANT3′) and FF390 (5′CGA TAA CGA ACG AGA CCT3′) according to . Section title: High throughput sequencing of 16S and 18S rRNA gene sequences Educational score: 4.097355365753174 Domain: biomedical Document type: Study Language: en For each sample, PCR amplifications were performed with 5 ng of DNA in a total reaction mixture volume of 25 μL. The thermal profile of the prokaryotic PCR was as follow: initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension period at 72 °C for 7 min. The thermal profile of the fungal PCR was as follow: initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 1 min, and extension at 72 °C for 1 min, followed by a final extension period at 72 °C for 5 min. All PCR products were purified with an Agencourt® AMPure XP kit (Beckman Coulter, Brea, California, USA) and quantified with a Quantifluor staining kit (Promega, Madison, Wisconsin, USA). Section title: High throughput sequencing of 16S and 18S rRNA gene sequences Educational score: 4.187352180480957 Domain: biomedical Document type: Study Language: en A second PCR was run on the purified PCR products (7.5 ng of DNA for bacteria and archaea, 5 ng for fungi), using 10-base-pair multiplex identifiers (MIDs) added at the 5′ and 3’ ends of the primers for subsequent sample identification. The thermal profile of the second PCR for the preparation of bacterial and archaeal rDNA amplicon libraries was the same as previously described, but with only 7 cycles. The same procedure was followed for the preparation of fungal rDNA amplicon libraries (same thermal profile as in the first PCR, but only 7 cycles). Moreover, the denaturation step lasted 1 only min. All PCR products were purified using a MinElute PCR purification kit (Qiagen NV) and quantified with a Quantifluor staining kit (Promega, Madison, Wisconsin, USA). For all libraries, the samples were pooled at equimolar concentrations and purified using an Agencourt® AMPure XP kit (Beckman Coulter, Brea, California, USA) to remove excess nucleotides, salts, and enzymes. Finally, sequencing was carried out with a NovaSeq Illumina instrument (Illumina Inc. San Diego, California, USA) producing 250-bp paired reads. Section title: Bioinformatic analysis of 16S and 18S rRNA gene sequences Educational score: 4.234103679656982 Domain: biomedical Document type: Study Language: en Bioinformatic analyses were performed using the BIOCOM-PIPE pipeline . First, all 16S and 18S raw reads were sorted according to the MID sequences. Data preprocessing included initial trimming of raw reads using PRINSEQ and merging of paired-end reads using FLASH. The low-quality reads were discarded based on their minimum length, number of ambiguities (Ns), and primer sequences. Then, the reads were dereplicated to save computing time in the subsequent steps of the pipeline (i.e., clustering of strictly identical sequences). The dereplicated reads were aligned using Infernal alignment tool , and clustered into operational taxonomic units (OTUs) with a similarity threshold of 95 %. A filtering step was carried out to remove chimeras based on the quality of their taxonomic alignments . Finally, the retained reads were homogenized by random selection of 10,000 reads to compare the datasets efficiently and avoid biased community comparisons. As observed in previous studies, if sequencing depth is sufficient, such a homogenization step will produce robust results when describing patterns in α- and β-diversity . The retained high-quality reads were used for (i) the re-clustering step using ReClustOR to improve OTU consistency based on a reference OTU database taken from the RMQS project (French Soil Quality Monitoring Network) ; (ii) taxonomy-independent analyses to calculate diversity indexes by Hill numbers (richness, Shannon, Inverse Simpson) using the OTU dataset; and (iii) taxonomy-based analyses by similarity approaches against curated reference databases from SILVA r132 . The raw datasets are available in the EBI database system under project accession number PRJEB71034. Section title: Statistical analyses Educational score: 4.067465305328369 Domain: biomedical Document type: Study Language: en All statistical analyses were performed with R studio using the free statistics software R . OW characteristics, soil physicochemical parameters, soil molecular microbial biomass, alpha diversity index, and the relative abundance of soil prokaryotic and fungal phyla were processed by a two-way mixed analysis of variance (ANOVA). The model included the plot identity as a random factor to account for repeated measures within each plot, with ‘time’ as the within-subject factor and ‘treatment’ as the between-subject factor. A Tukey's HSD post hoc test with the Benjamini & Hochberg (BH) correction method was applied to adjust the p-values. Section title: Statistical analyses Educational score: 4.141612529754639 Domain: biomedical Document type: Study Language: en Alpha diversity was analyzed using Hill numbers generated by the vegan package . Hill numbers allow interpreting alpha diversity linearly by progressively considering the abundances of rare and dominant OTUs using the scaling parameter q (order of diversity) . Therefore, q = 0 represents richness, q = 1 corresponds to the exponential of Shannon entropy, weighing OTUs by their frequency without disproportionate favoring either rare or abundant ones, and q = 2 represents the inverse of Simpson index, where the abundant OTUs are overweighed . Similarities in the compositions of the prokaryotic and fungal communities among different fertilization treatments over time were evaluated by an analysis of similarities (ANOSIM) and an analysis of multivariate homogeneity of group dispersions (beta-dispersion), using 999 permutations and a significance threshold p < 0.05, based on the robust Aitchison dissimilarity distance . Section title: Statistical analyses Educational score: 4.135371208190918 Domain: biomedical Document type: Study Language: en To visualize the distribution patterns of microbial communities for each fertilization treatment over time, the non-metric multidimensional scaling (NMDS) approach was employed based on the robust Aitchison dissimilarity distance , using the metaMDS function from the vegan package . The significance of the differences between treatments and over time was assessed using a non-parametric permutational multivariate analysis of variance (PERMANOVA) based on the robust Aitchison dissimilarity distance . The relative effects of ‘time’, ‘treatment’, and ‘time × treatment’ were tested using the adonis2 function in the vegan package with 999 permutations, and a significance threshold p < 0.001; the “strata” option was used to constrain the permutations of samples within each time point. The abundance of soil prokaryotic and fungal phyla was subjected to a differential abundance analysis using the DESeq2 method to compare the cumulative effect of digestate to the other treatments . Section title: Statistical analyses Educational score: 4.0961079597473145 Domain: biomedical Document type: Study Language: en Finally, a variance partitioning approach was used to determine if the amounts of organic carbon and fertilizing elements (NPK) applied over 10 years could explain the variability observed in the dynamics of the soil molecular microbial biomass and the soil microbial community structure. For this purpose, the most parsimonious additive model was selected according to a forward selection procedure by minimizing the Akaike information criterion (AIC), using the rda and ordistep functions in the vegan package . Section title: OW and soil chemical characteristics Educational score: 4.051918983459473 Domain: biomedical Document type: Study Language: en Over a decade of repeated application of different organic wastes (OW), no statistically significant differences were observed between the physicochemical properties of undigested (PS) and digested (PS-DIG) organic matter ( p > 0.05, Table 1 ). In contrast, cattle manure (CM) exhibited significantly higher concentrations of carbon and nitrogen ( p < 0.05, Table 1 ). In 2012, before any treatment application, the physicochemical properties of the soil were similar in all plots ( p > 0.05, Table 2 ). Similarly, in 2016, no statistically significant differences were observed between treatments ( p > 0.05, Table 2 ). After a decade, in 2022, the soil chemical properties were lastingly modified, and the magnitude of the changes was treatment-dependent. Specifically, the soil organic carbon (SOC) content significantly decreased by an average of 7 % in the plots treated with PS and PS-DIG, and by 14 % in the MIN and ON plots ( p < 0.05, Table S 2). The only plots that maintained a stable SOC were those amended with CM ( p > 0.05, Table 2 and Table S 2). All treatments resulted in a significant decrease in the C/N ratio over time, by 7 % on average ( p < 0.05, Table S 2). Significant soil acidification was observed in the MIN plots ( p < 0.05, Table 2 and Table S 2), while the soil pH remained stable over time in the ON, PS, and PS-DIG plots ( p > 0.05, Table 2 and Table S 2). In contrast, the pH significantly increased in the CM plots ( p < 0.05, Table 2 and Table S 2). Section title: Effects of organic and inorganic fertilization on soil molecular microbial biomass Educational score: 4.23284387588501 Domain: biomedical Document type: Study Language: en A significant ‘year’ effect was observed on soil molecular microbial biomass regardless of the treatment . Overall, across all plots, soil molecular microbial biomass decreased by 32 % in 2017 compared to the beginning of the trial . In 2012, before any fertilizer application, a heterogeneity in soil molecular microbial biomass was observed, particularly between plots selected for mineral fertilization treatment and those designated to receive digestate . After ten years , significant differences were observed between plots amended with cattle manure (CM) and those unfertilized control plots (ON) , where the highest soil molecular microbial biomass was recorded when CM was repeatedly applied over a decade and lowest in ON plots . The redundancy analysis (RDA) model identified the parameters that explained variations in soil molecular microbial biomass over a decade ( Table 3 ). The temporal factor (‘year’) strongly influenced the model, explaining 43.3 % of the variance. In contrast, cumulative C organic inputs explained 4.2 % of the variation in soil molecular microbial biomass. Fig. 2 Soil molecular microbial biomass dynamics by year at the EFELE (experimental field site in Rennes, France) from 2012 to 2022. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Capital letters, differences among sampling time; lower-case letters, effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 2 Table 3 Effects of the cumulative inputs during the decade at the EFELE site on the soil molecular microbial biomass and microbial community structure. Table 3 Parameter Explanatory variables F P -value Variance explained (%) Soil molecular microbial biomass Year 23.09 0.001 43.3 C organic inputs 4.49 0.041 4.2 Residual – – 52.5 Structure of soil Prokaryotic community Year 8.14 0.001 21.3 C/N inputs 8.99 0.001 11.8 Residual – – 66.8 Structure of soil Fungal community Year 50.98 0.01 64.1 Residual – – 35.9 Variance partitioning approach, using Redundancy Analysis (RDA) to select the most parsimonious additive model, according to a forward selection procedure. F (Fisher's F-value), and p-value of Fisher's F from ANOVA test of RDA selected model. Scores from the NMDS ordination based on the robust Aitchison dissimilarity distance were employed for the variance partitioning approach of the soil microbial community structure. Section title: Effects on microbial diversity and community structure Educational score: 4.189914703369141 Domain: biomedical Document type: Study Language: en Following a decade of different fertilization regimes, significant differences in prokaryotic diversity indices were only observed in 2022 between digestate (PS-DIG) and pig slurry (PS) treated plots . Plots fertilized with digestate displayed higher values of richness (q = 0), OTUs frequency (q = 1), and effective number of dominant OTUs (q = 2) compared to PS plots . Regarding fungal community diversity indices, at the beginning of the experiment in 2012, significant differences in richness (q = 0) and OTUs frequency (q = 1) were observed, particularly among plots designated to receive pig slurry (PS) and digestate (PS-DIG) . Repeated digestate application over a decade induced a higher soil fungal effective number of dominant OTUs (q = 2) than in soils receiving slurry (PS) or cattle manure (CM) . Fig. 3 Soil prokaryotic community diversity dynamics by year at the EFELE (experimental field site in Rennes, France) based on Hill numbers. Hill numbers allow interpreting alpha diversity linearly by progressively considering the abundances (q-values) of rare and dominant OTUs. Therefore, ( A ) q0 represents richness, ( B ) q1 is the exponential of Shannon entropy, and ( C ) q2 is the inverse of Simpson index. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Lower-case letters: effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 3 Fig. 4 Soil fungal community diversity dynamics by year at the EFELE (experimental field site in Rennes, France) based on Hill numbers. Hill numbers allow interpreting alpha diversity linearly by progressively considering the abundances (q-values) of rare and dominant OTUs. Therefore, ( A ) q0 represents richness, ( B ) q1 is the exponential of Shannon entropy, and ( C ) q2 is the inverse of Simpson index. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Lower-case letters: effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 4 Section title: Effects on microbial diversity and community structure Educational score: 4.234692573547363 Domain: biomedical Document type: Study Language: en The analysis of similarities revealed significant differences in the microbial community structure (prokaryotic and fungal) among years and treatments (ANOSIM; p < 0.05, Table 4 ), although the ‘year’ effect seemed greater than the ‘treatment’ effect (ANOSIM; R statistic, Table 4 ). The analysis of multivariate homogeneity of group dispersions demonstrated that the dispersion of community structure differs significantly between years but not between treatments (beta-dispersion; Table 4 ). NMDS ordination confirmed these findings , which clearly highlighted distinct clustering by ‘year’ , indicating that the changes of soil microbial communities were more influenced by time than by fertilization type. PERMANOVA confirmed that both ‘year’ and ‘fertilization type’ had a significant effect on the prokaryotic and fungal community structure ( p < 0.05, Table 5 ). Pairwise comparisons revealed lasting modifications over time, with the largest differences between 2012 and 2022 . After 10 years, the microbial community structure in the plots receiving digestate (PS-DIG) differed significantly from those amended with cattle manure (both prokaryotic and fungal community structure) and pig slurry (fungal community structure) . In contrast, no significant differences were observed when comparing the microbial community structures between digestate fertilized plots and mineral fertilized (MIN) or unfertilized ones (ON) . Table 4 Analysis of similarities (ANOSIM), and analysis of multivariate homogeneity of group dispersions (beta-dispersion) depicting the differences in the prokaryotic and fungal community compositions between and within groups (‘treatment’ and ‘time’) based on the Robust-Aitchison distance matrices at the EFELE (experimental field site in Rennes, France), according to the treatment over time (999 permutations, significance threshold p < 0.05). Table 4 Prokaryotic community Fungal community ANOSIM Beta-dispersion ANOSIM Beta-dispersion R statistic P -value P -value R statistic P -value P -value Treatment 0.14 0.002 0.24 0.13 0.002 0.88 Year 0.29 0.001 0.04 0.60 0.001 0.01 R statistic: degree de separation between test groups ranging from −1 to 1; R = 0, not different; R = 1, completely different. Significance values were based on 999 permutations. Fig. 5 Non-metric multi-dimensional scaling (NMDS) ordination and PERMANOVA pairwise comparisons derived from robust Aitchison dissimilarity distances for ( A, B ) the prokaryotic community at the EFELE (experimental field site in Rennes, France) over 10 years of trial. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Big circles represent centroids, and little circles represent samples. (A) prokaryotic community structure colored by year, (B) prokaryotic community structure colored by treatment. Fig. 5 Fig. 6 Non-metric multi-dimensional scaling (NMDS) ordination and PERMANOVA pairwise comparisons derived from robust Aitchison dissimilarity distances for ( A, B ) the fungal community at the EFELE (experimental field site in Rennes, France) over 10 years of trial. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Big circles represent centroids, and little circles represent samples. (A) fungal community structure colored by year, and (B) fungal community structure colored by treatment. Fig. 6 Table 5 Permutational multivariate analysis of variance ( PERMANOVA) results depicting the differences in the prokaryotic and fungal community compositions based on the robust Aitchison distance matrices, at the EFELE (experimental field site in Rennes, France), according to the treatment over time (999 permutations, significance threshold p < 0.05). Table 5 Prokaryotic community Fungal community SS R 2 F P -value SS R 2 F P -value Treatment 0.44 0.11 1.78 0.001 0.69 0.07 2.88 0.001 Year 0.47 0.12 3.83 0.02 1.79 0.19 5.92 0.001 Treatment × Year 0.43 0.11 0.88 0.87 1.53 0.16 1.26 0.001 Residual 2.51 0.65 – – 5.44 0.57 – – Total 3.84 1 – – 9.46 1 – – SS (Sum of Squares), R 2 (Determination coefficient), F (Fisher's F-value), and p-value of Fisher's. Section title: Effects on microbial diversity and community structure Educational score: 4.11836051940918 Domain: biomedical Document type: Study Language: en The redundancy analysis (RDA) model identified the parameters that explain variations in the microbial community structure over a decade ( Table 3 ). The temporal factor (‘year’) had the strongest influence in both models. It represented 21.3 % and 64.1 % of the explained variance of the prokaryotic and fungal community structure, respectively ( Table 3 ). The C/N ratio of the applied product represented 11.8 % of the explained variance of the soil prokaryotic community structure. In contrast, none of the chemical properties of the treatments applied (cumulative organic carbon or NPK fertilizing elements) were able to explain the dynamics of variation in the structure of soil fungal communities ( Table 3 ). Section title: Effects on microbial diversity and community structure Educational score: 4.152534008026123 Domain: biomedical Document type: Study Language: en The temporal changes in the relative abundances of dominant soil microbial phyla over a decade following different fertilization regimes are illustrated in Fig. 7 , Fig. 8 . Overall, 13 prokaryotic and 6 fungal major phyla were identified across all treatments and years considered. The prokaryotic phyla were Acidobacteria, Actinobacteria-p, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes-p, Nitrospirae, Planctomycetes, Proteobacteria, Rokubacteria , Thaumarchaeota , and Verrucomicrobia . The fungal phyla were Ascomycota, Basidiomycota, Chytridiomycota, Cryptomycota, Mucoromycota , and Zoopagomycota . When comparing digestate to other OW, the repeated digestate application over a decade induced lower relative abundance in Thaumarchaeota but higher in Actinobacteria-p, Gemmatimonadetes-p, and Rokubacteria . For the fungal phyla, digestate favored Basidiomycota and Cryptomycota . The differential abundance analysis conducted using the DESeq2 method provided insights into the changes in the abundance of various microbial phyla in response to different fertilization treatments. Notably, when comparing the cumulative effect of a decade of annual digestate application to other treatments, fewer significant differences at the phylum level were observed between PS-DIG and MIN-treated plots, specifically in the Archaeal phyla Thaumarchaeota and Euryarchaeota , and the fungal phylum Blastocladiomycota . Fig. 7 Relative abundances of the major prokaryotic phyla by year at the EFELE (experimental field site in Rennes, France) from 2012 to 2022. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Relative taxonomic abundances below 5 % were grouped in “Others”. Lower-case letters, effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 7 Fig. 8 Relative abundances of the major fungal phyla by year at the EFELE (experimental field site in Rennes, France) from 2012 to 2022. Treatments: MIN, mineral fertilizer; ON, control that received no organic or mineral N input; three different OW: CM, cattle manure; PS, pig slurry; PS-DIG, pig slurry digestate. Lower-case letters, effect of the treatment for each sampling time. Different letters indicate significant differences ( p < 0.05) based on Tukey's HSD test; p -value adjusted by the BH method. Fig. 8 Section title: Discussion Educational score: 4.1246137619018555 Domain: biomedical Document type: Study Language: en Comparing to laboratory approaches, employing an in-situ methodology in real agronomical and pedological conditions provides a more comprehensive understanding of short- ( ≤ 1 year), mid- ( ≤ 5 years), and long- ( ≥ 6 years) term impact of digestates on soil microbiota. To our knowledge, no prior study has employed an in-situ approach to monitor the effects of repeated digestate applications over a decade. The overall objective of this study was to assess the long-term effects of repeated digestate applications as an organic fertilizer on soil microbial parameters, including molecular microbial biomass, diversity, structure, and composition. Furthermore, we aimed to evaluate how these effects relate to changes in edaphic soil properties, hypothesizing that significant modifications would emerge only after several years of repeated applications. Section title: Cumulative effects of fertilization treatment on soil edaphic properties Educational score: 4.221584320068359 Domain: biomedical Document type: Study Language: en As previously observed with a liquid digestate by Ref. , the repeated application of digestate did not induce a lasting increase of SOC content as commonly observed with classical OW such as farmyard manure or compost . The stabilization or increase of the SOC content was observed depending on the OW type and quantity applied. In the present study, CM plots resulted in a stable SOC content throughout the 10 years of the experiment, primarily attributed to the large quantity of organic carbon applied (total organic C (TOC) in the CM treatment: 28,155 kg ha _1 ), significantly higher than the other treatments. In contrast, a lower quantity of organic carbon was applied via digestate and pig slurry , explaining why the SOC content could not be maintained throughout the years. These findings align with the literature, emphasizing that increasing total C input is the primary driver for maintaining or rising SOC stocks . Moreover, as previously reported for organic farming systems, rising SOC levels is a gradual process closely related to biological soil quality and the quality of inputs materials . The treatments induced distinct dynamics of the total soil nitrogen content. These variations were attributed to differences in the proportions of total N applied via each treatment; specifically, the plots fertilized with digestate and mineral fertilizer recorded lower total soil N content at the end of the experiment. Indeed, over a decade of repeated fertilization, the cumulative total nitrogen supplied was lower in plots fertilized with digestate or mineral fertilizer compared to plots amended with organic products (cattle manure and pig slurry) . In line with prior studies, differences in carbon and nitrogen contents in the OW led to high variability in the C/N ratios across treatments . The C/N ratio of the OW is an indicator of their nitrogen mineralization capacity. When OW with C/N ratios below 20 are added to soils, net mineralization dominates . The C/N ratios below 10 favor rapid organic matter mineralization, while values above 10 result in slower mineralization ; microbial N immobilization becomes the dominant process with higher C/N ratios . An optimal C/N ratio in the range of 5–6 has been proposed for OW . In the present study, the C/N ratios of digestate ranged between 2.9 and 5.8, and for the other OW (pig slurry and cattle manure), the values ranged between 3.5 and 8.3 (PS) and 12.8 and 19 (CM), respectively. The lower C/N ratios of digestate may also explain why these inputs did not contribute to increases in the SOC content, favoring rapid mineralization of the organic matter and potentially resulting in different fates for nitrogen in the amended soils . Regarding soil pH, our study found no significant changes after 10 years of repeated digestate application, consistent with previous in-situ studies that also reported stable soil pH levels following repeated digestate inputs [ 51 , , , ]. Section title: Cumulative effect of digestate application on soil molecular microbial biomass Educational score: 4.2705183029174805 Domain: biomedical Document type: Study Language: en During the biogas production process, anaerobic microorganisms degrade most of the readily available carbon, leading to the subsequent production of methane and carbon dioxide . Consequently, a reduced amount of readily available carbon for soil microorganisms, and therefore a lower microbial biomass, can be expected when digested organic matter is used as an amendment compared to undigested organic matter . Contrary to these expectations, our findings highlighted that the soil molecular microbial biomass content was similar in plots treated with OW (digestate (PS-DIG), undigested organic matter (PS), and cattle manure (CM)), although their SOC content differed. As reported by Ref. , increases in SOC content were associated with improvements in soil biological activity and particularly in microbial biomass. Moreover, higher soil microbial biomass has been observed in plots amended with cattle manure . The results obtained in our study could be explained by a buffering effect of agricultural practices (e.g., crop rotation, the reasoning of the input doses, cover and catch crop) which exert a compensatory effect on soil molecular microbial biomass, not allowing for the observation of a more pronounced treatment-effect. Indeed, it is known that certain practices, such as the diversity of crop rotations or soil tillage , can impact soil microbial biomass. In addition, the variance partitioning approach identified the temporal parameter (‘year’) as the main factor explaining variations in soil molecular microbial biomass, with a significant and systematic decrease in biomass in all treatments in 2017. The winter of 2016/17 was drier and colder than usual . Typically, drainage seasons commenced between November and January, except for the winter of 2016/17, which started in February, lasted only one month, and had notably lower rainfall, resulting in reduced drainage volumes . This underscores the crucial role of soil temperature and moisture in driving seasonal changes in soil microbial communities. By influencing temperature and precipitation, climate can either mitigate or exacerbate the impact of land use on the soil microbial biomass . Moreover, the season of soil sampling may exert a greater influence on soil microbial communities than the application of digestate . Thus, in our study, land use and soil management practices probably buffered the effects of the treatments, whether organic or mineral, on the soil microbial biomass. These findings highlight the importance of fertilization and soil amendment, and the critical need for a long-term assessment of the potential impacts of any agricultural practice on soil microorganisms. Section title: The historical and cumulative fertilization regime drives the structure of the soil microbial communities Educational score: 4.140359878540039 Domain: biomedical Document type: Study Language: en As observed in previous studies, our results highlighted that repeated application of different fertilization treatments induced minor significant differences over time in alpha diversity indices (richness, the exponential of Shannon entropy, and the inverse of Simpson index) . Our findings highlighted that the long-term fertilization with digestate stimulated dominant OTUs more than other OW (pig slurry and cattle manure). Furthermore, our results revealed shifts and lasting modifications of the prokaryotic and fungal community structures in relation to time and the fertilization treatment, consistent with prior research, yearly organic and mineral fertilization treatments had a significant long-term impact on the soil microbial community structure . Field experiments have shown no significant short-term changes in the soil microbial community structure following digestate application . This indicates a different response of the microbial community structure to short- and long-term nutrient additions, whose effects take time before they become noticeable. Moreover, our results highlighted that (i) at least 5 years went by before lasting modifications of the soil microbial community structure were detected, and (ii) the distribution of the prokaryotic structure of the digestate fertilized plots was in between those of the mineral fertilizer and the other OW (pig slurry (PS) and cattle manure (CM)). Section title: The historical and cumulative fertilization regime drives the structure of the soil microbial communities Educational score: 4.136536598205566 Domain: biomedical Document type: Study Language: en The variance partitioning approach revealed that the temporal factor (‘year’) and, to a lesser extent, the cumulative C/N inputs best explained variations in the microbial community structure. As observed for the molecular microbial biomass, these findings underscored that the ‘year’ factor exerted a strong influence, likely reflecting environmental, climatic, and seasonal effects that could mask the impact of repeated applications of different fertilization treatments on soil microorganisms. Nevertheless, the nature of the inputs also plays a significant role in shaping these microbial communities. These findings align with previous studies which emphasize that both mineral and organic long-term fertilization can lead to changes in the soil microbial community composition , due to the stimulation of soil-borne microbes responding to the persistent modifications of the soil physicochemical properties resulting from the chemical composition of the applied OW . Section title: The historical and cumulative fertilization regime drives the structure of the soil microbial communities Educational score: 4.259023666381836 Domain: biomedical Document type: Study Language: en In our study, the variations of the soil microbial community composition were further reflected by the distinct proportions of the fungal and prokaryotic groups. The most abundant taxa were Proteobacteria (∼27 %) , Thaumarchaeota (∼25 %) , Bacteroidetes (∼19 %), and Actinobacteria-p (∼6.4 %). These are the major bacterial and archaeal taxa found in French soils . A decade of repeated digestate application resulted in a lower abundance of Thaumarchaeota and a higher abundance of Actinobacteria-p compared to the other OW (pig slurry and cattle manure). The phylum Thaumarchaeota comprises dominant archaea in the soil prokaryotic community, known for their pivotal role in soil ammonia oxidation by converting ammonia to nitrite and, subsequently, nitric oxide . Thus, a higher abundance of Thaumarchaeota could be expected after ammonia inputs via the digestate. However, contrary to these expectations, an antagonistic effect may be revealed as previously reported that oxygen availability might be suboptimal for soil-aerobic Thaumarchaeota ; therefore, our results could suggest a potential long-term consequence of the repeated application of an anaerobic digested product on the abundance of this phylum. However, to date, the impact of digestate application on the soil Thaumarchaeota phylum remains poorly documented. The phylum Actinobacteria is a Gram-positive, saprophytic, ubiquitous bacteria. They can produce a wide array of extracellular hydrolytic enzymes that degrade complex macromolecules that can break down animal and plant biomass. This makes them central organisms in carbon recycling . They are considered as oligotrophs because they dominate the decomposition of organic material when nutrients are limited . An increase in bacteria belonging to this phylum has already been observed after 42 days of digestate application . Furthermore, members of the Actinobacteria phylum have been reported as potassium-solubilizing and/or phosphate-solubilizing bacteria . The observed changes in the relative abundance of Actinobacteria-p may be linked to the cycling of phosphorus in the soil because the phosphorus content of the OW is one of the limiting elements to determine the application rates at the EFELE site. Section title: The historical and cumulative fertilization regime drives the structure of the soil microbial communities Educational score: 4.214145660400391 Domain: biomedical Document type: Study Language: en The most abundant fungal phyla in our in-situ approach were Ascomycota (∼43 %) , Basidiomycota (∼25 %) , Mucoromycota (∼16 %), and “Unknown Fungi” (∼8 %). These are worldwide dominant taxa in soil fungal communities . Ascomycota and Basidiomycota are major contributors to soil carbon cycling . Ascomycota members are generally defined as copiotrophic saprophytes. They are typically characterized as fast-growing, effective decomposers of labile carbon and are abundant dwellers of soils with high C availability . By contrast, Basidiomycota members are defined as oligotrophic saprophytes and symbiotrophs . Basidiomycota are assumed to possess improved metabolic capacities over Ascomycota for decomposing more recalcitrant soil C compounds . In 2022, the higher abundances of Ascomycota in plots amended with traditional organic products (PS and CM) may be attributed to organic carbon availability within these treatments. In contrast, the higher relative abundance of Basidiomycota in plots receiving digestate or mineral fertilizer may be explained by the reduced amount of readily available carbon for soil microorganisms when digested organic matter is used as an amendment, having a similar effect to a mineral fertilizer . Section title: Conclusions Educational score: 4.17380428314209 Domain: biomedical Document type: Study Language: en This study provides the first field-based analysis of the dynamic effects of repeated digestate applications over a decade, assessing whether the impacts of digestate differ from those of its undigested feedstock and other commonly used fertilizers. Our findings provided new insights regarding the long-term effects of one type of digestate on soil microorganisms. After a decade of repeated applications of different OW (cattle manure, pig slurry and digestate of pig slurry) or mineral fertilizer, lasting changes were observed in the soil's physicochemical properties and microbial parameters across all treatments. These changes were primarily explained by the effect of time and to a lesser extent by the quality of the applied product. Our results highlight that the response of soil microbial communities to repeated digestate application is similar to that of mineral fertilizer but strongly influenced and buffered by other agricultural practices and environmental and climatic conditions. Hence, when assessing the impact of digestate on soil microbial communities, associated agricultural practices should be considered. Further research is needed to better understand the cumulative effects of repeated digestate applications on soil microbial communities, particularly across diverse agro-pedological contexts. This includes not only soil type but also interactions with agricultural practices, such as fertilization history, additive fertilization (additional nutrient inputs), tillage, and plant cover (e.g., crop type, cover crops, rotation, and plant diversity). Section title: CRediT authorship contribution statement Educational score: 0.9368098378181458 Domain: other Document type: Other Language: en Daniela Mora-Salguero: Writing – original draft, Methodology, Formal analysis. Lionel Ranjard: Writing – review & editing. Thierry Morvan: Writing – review & editing, Methodology. Samuel Dequiedt: Writing – review & editing, Formal analysis, Data curation. Vincent Jean-Baptiste: Writing – review & editing. Sophie Sadet-Bourgeteau: Writing – review & editing, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization. Section title: Funding Educational score: 0.9437368512153625 Domain: other Document type: Other Language: en This project was funded by Institut Agro Dijon. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Section title: Declaration of competing interest Educational score: 0.9803839325904846 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper. | Study | biomedical | en | 0.999999 |
PMC11699321 | Section title: Introduction Educational score: 4.045415878295898 Domain: biomedical Document type: Study Language: en An increased focus has been placed on the production of polymer composites reinforced with natural fibres over the past few decades. This is primarily due to concerns surrounding sustainability and degradability, as evidenced by various scholarly sources . The use of natural fibers like Fique fibers are being proposed for creating composites that are lightweight and less expensive than traditional synthetic fibers, including carbon fiber, Kevlar, and fiber glass [ , , , ]. Furthermore, reinforced polymer composites made from natural fibers may be an eco-friendly option. The mechanical and physical characteristics of pure cellulose fibers are influenced by their source and characteristics, including but not limited to diameter, length, and specific gravity. These properties play a pivotal role in identifying the potential applications of these fibers. The use of natural fibers, including sugar palm fiber, OPEFB fiber, kenaf fiber, flax fiber, pineapple leaf fiber, banana fiber, and jute in polymer matrix composites (PMCs) present numerous benefits, such as recyclability, reduced density, biodegradability, accessibility, enhanced mechanical and physical characteristics, and favorable thermal steadiness of the resultant materials [ , , , ]. The use of natural fibers, including but not limited to kenaf, jute, and hemp, as reinforcement for polymer composites has resulted in exceptional thermal properties that are comparable to those of synthetic fibers. This has led to an expansion of their potential applications in various fields, including aerospace and construction industries, as evidenced by studies [ , , , , ]. Before their implementation in structural applications, it is necessary to use testing methodologies to examine the composite structure and assess its performance under cyclic loading and thermal conditions. Section title: Introduction Educational score: 4.191376686096191 Domain: biomedical Document type: Study Language: en The categorization of natural fiber can be delineated into two primary classifications based on its source: inorganic and organic. Typically, inorganic natural fibers consist of mineral fibers, including wollastonite, fibrous brucite, and asbestos. Various plant species, including grass, bamboo, wheat, and bagasse, can produce fibers from their stalks. The constituents of plant fibers primarily comprise three fundamental elements, namely cellulose, hemicelluloses, and lignin. The fibers in question are commonly known as lignocellulosic materials, because cellulose constitutes the primary chemical, alongside varying quantities of lignin and hemicellulose . Mechanical characteristics of fibers are attributed to the hydrogen bonds present between macromolecules, as well as the orientation of crystalline cellulose fibrils within the cell wall. Lignin exhibits strong adherence to hemicellulose and forms cross-links with polysaccharides, thereby occupying the interstitial spaces within the cell wall matrix composed of pectin, cellulose, and hemicellulose constituents, thereby conferring mechanical stability to the cell wall . Section title: Introduction Educational score: 4.268484115600586 Domain: biomedical Document type: Study Language: en The primary constraints associated with natural fibers pertain to their hydrophilic characteristics, which can augment moisture absorption and undermine the bonding interplay between the fibers and polymer matrices [ , , , ]. The drawbacks associated with natural fibers, such as inadequate interfacial bonding and the manifestation of hydrophilic properties, can be overcome by using diverse chemical processing that modify the compatibility of the natural fibers with the matrix. The application of chemical processing results in the formation of natural fibers that are fully cured and cross-linked. These fibers serve to reinforce the polymeric matrix, thereby leading to enhanced mechanical strength and thermal stability. This has been documented in previous studies . Improved compatibility among the natural fibers and polymer binder might be achieved through the efficient removal of surface contaminations and hydroxyl group constituents . This phenomenon facilitates a robust chemical adhesion at the interfaces of two phases, resulting in a well-established network. This process has the potential to enhance the utility of natural fibers in various domains, including marine and automobile sectors, by augmenting their resistance to wear, tear, and creep [ , , , ]. The augmentation of these characteristics has the potential to enhance the longevity of the material, particularly when subjected to severe circumstances, such as elevated temperatures, higher levels of humidity, and acidic environment [ , , , ]. Section title: Introduction Educational score: 4.224978446960449 Domain: biomedical Document type: Study Language: en The investigation of thermal behavior under diverse conditions is a crucial aspect to be considered for the design and development of hybrid composites for multiple applications. The nonlinearity of thermal property fluctuations in hybrid composites presents a challenge in determining and forecasting thermal conductivity [ , , ]. The incorporation of filler particles of silicon carbide into the pristine epoxy polymer composites results in an enhancement of thermal conductivity, which is directly proportional to the percentage of volume fraction. The thermal conductivity of silicon carbide in isolation is notably higher than that of hybrid composites. However, when combined with epoxy resin, silicon carbide exhibits reduced thermal conductivity due to the relatively diminutive size of the filler particles, limiting their efficacy in producing conductive pathways is limited . Consequently, insufficient cooling may lead to the malfunction of electronic devices. The operational temperature range limit is a critical factor that determines the resilience and recital of electronic devices. Precise management of thermal conditions is a crucial factor in determining the high performance of semiconductors . The efficacy of using high-performance computing systems is depends on the selection of filler materials with superior thermal conductivity. The use of hybrid nano-fillers in the modification of polymer matrices has opened up extensive avenues for research, enabling the attainment of superior thermal and mechanical properties applicable across various fields. Section title: Introduction Educational score: 4.166794776916504 Domain: biomedical Document type: Study Language: en According to previous research, various nanoparticle fillers are frequently used to enhance the thermal conductivity and electrical resistivity of polymers . Owing to the inherently low thermal conductivity of polymers, various fillers including mica, zinc oxide, glass fiber, boron nitride, aluminum nitride, silicon carbides, and alumina are incorporated . The enhancement of thermal resistance can be observed in silica hybrid composites, which can be attributed to the robust interfacial bonding between silica particles and epoxy resin, as reported in Ref. . The incorporation of graphene nanoplates (GNPs) at a concentration of 1 % by weight resulted in a notable improvement in thermal conductivity. This effect can be attributed to the uniform distribution of GNPs within the epoxy polymer matrix as well as the establishment of internal bonding. Some studies involved a comparative analysis of the experimental thermal conductivity values obtained for polymer composites filled with particulate matter, with the mathematical methods, namely the rule of mixture thermal conductivity model, parallel thermal conductivity model, and Maxwell thermal conductivity model . To enhance the thermal conductivity and thermal stability of polymer composites, fillers including graphene, CNT, copper mesh and powder, can be incorporated into carbon fiber–reinforced polymer (CFRP) to create hybrid polymer composites . Section title: Introduction Educational score: 4.1346611976623535 Domain: biomedical Document type: Review Language: en The use of hybrid fillers including boron nitride, SiC, and aluminum nitride in polymer composites has been observed to be a more efficacious approach in reducing the CTE . A few experiments were conducted by adding SiC nanoparticles into glass fiber-based epoxy composites to evaluate their thermal stability. The results of thermal experiments indicated that composites with 5 % and 20 % SiC contents exhibited relatively high thermal conductivities . Thermogravimetric analysis (TGA) was conducted on composites comprising polyester resin, glass fiber, and jute fiber. The results indicated that the composite demonstrated a lower weight loss with increasing temperature . In a TGA comparison study of hybrid polymer composites, carbon fiber composite, and glass fiber composite, it was found that the thermal stability of the hybrid composites was superior . The TGA of the composite material consisting of PMMA toughened glass and epoxy revealed a greater degree of mass loss, whereas the incorporation of SiC helped to maintain its temperature stability . The findings suggest that the incorporation of GNPs, graphene oxide (GO), reduced graphene oxide (rGO), and multi-walled carbon nanotubes (MWCNTs) results in an increase in thermal conductivity . The impact of filler materials on the glass transition temperature of polymers is significant. Certain composites undergo testing for structural stability at low temperatures, as indicated in Ref. . Hence, from all the above discussions it could be understood that that the incorporation of natural or inorganic nanoparticles exhibited better thermal characteristics when compared with the composites without nanoparticles. Accordingly, the focus of the current review article was to articulate the significance of nanoparticle addition to the natural fiber–based polymer composites in improving the thermal characteristics. Section title: Sisal fiber composites Educational score: 4.232688903808594 Domain: biomedical Document type: Study Language: en Natural fiber polymer composites could be analyzed for their disintegration by temperature rise and weight loss in relation to temperature, including residue material beyond maximum heating, using TGA. The lignocellulosic fiber combined polymer matrix that makes up natural fiber composites. The rate at which these materials degrade in heat depends on the reinforcement and matrix used, with each component degrading at distinct temperatures. When something degrades at a certain temperature, it loses mass in accordance with that temperature. The process of reducing weight in fiber composites can be broken down into three distinct phases: first, because of the evaporation of water; second, owing to the breakdown of the fiber reinforcement; and third, because of de-polymerization in the binder itself. Recently, the hybridization process has enhanced the thermal resistance of fiber composites. Among the hybridization influences in the thermal resistance of sisal composites are the following. Researchers evaluated the thermal properties exhibited by the jute or sisal hybrid composites. As a result of the initial drying process, the composites lost approximately 5 wt%. The deterioration of binder and fiber caused 75 % of the composite's mass loss when the temperature began to rise. The composites completely degraded when subjected to high temperatures. Maximum thermal stability was observed in a composite made up of 50 % jute and 50 % sisal fiber. Additionally, alkaline treatment was found to be advantageous in improving the composites' thermal stability . Section title: Sisal fiber composites Educational score: 3.9654154777526855 Domain: biomedical Document type: Study Language: en Gupta obtained comparable findings from a different study that investigated identical jute or sisal epoxy composites with varying fiber weight fractions . Similarly, the temperature resistance of sisal material was improved by glass fiber incorporation. This increase can be attributed in large part to the fact that glass fiber has more thermal resistance than sisal fiber. The TGA revealed that the sisal or glass fiber composites generated significantly more charred residue than any of the other composites developed . There was some variation in thermal characteristics between sisal with glass hybrid composites made using an extrusion process and those made using an injection molding technique . The research findings point to the importance of fiber dispersion in the matrix for improving the thermal resistance of composites. The influence of particle reinforcement on the thermal characteristics of sisal polyester composite was reported by Venkatram et al. . Comparison research was conducted after adding 3 wt% nanoclay to an existing sisal polyester composite. Section title: Sisal fiber composites Educational score: 4.288424491882324 Domain: biomedical Document type: Study Language: en The composite without nanoclays was less stable at high temperatures than those reinforced with nanoclay. At 290 °C, the sisal composites lost 82 % of their weight whereas the hybrid composite lost only 79 % of its weight . Charcoal units added to sisal-based composites exerted a positive influence on the deterioration and temperature resistance of the hybrid composite . High degradation temperatures are required to break down the charcoal particles. For this reason, the hybrid mixture with 8, 6, in addition, 4 wt% charcoal retained 30%–40 % of weight regardless of its decomposition at 450 °C. After being heated to 750 °C, the mixture of composite with 8 wt% charcoal retained nearly 38 % of the composite remnant. The thermal characteristics of sisal with SiC-blended composites were reported to be negatively impacted by Teja et al. . The thermal degradation of composites made with 30 wt% sisal fiber and either 5 or 10 wt% SiC particles increased, but the composites still exhibited high thermal stability compared to the pure 30 wt% sisal composites . Sisal, banana plus coir fiber were used to reinforce unique hybrid composites developed by Sumesh et al. . Nanoparticle incorporation improved the thermal resistance of the hybrid composites. Particle reinforcement increased the deterioration temperature of these hybrid composites from 50 % to 70 %. Though particle reinforcement was enhanced by the incorporation of 5 % nanoparticles, mechanical and thermal properties were degraded as an outcome of a greater aggregation of particles . Pappu et al. found that a hybrid composite of polylactic acid (PLA) and sisal and hemp fibers had the same thermal characteristics as pure PLA material. The TGA revealed no change in the thermal properties of the composite after natural fiber was added to the PLA matrix. The hybrid material lost weight in two distinct phases. At temperatures of 300 °C, weight loss began and at 410 °C, it accelerated significantly. At temperatures above 15 °C, the composite began to degrade rapidly and completely, as revealed by Samal et al. . In 2009, Jarukumjorn and Suppakarn explored the rise in heat distortion temperature (HDT) of sisal with glass hybrid PP composites as a function of fiber weight percentage. Adding more reinforcement in the form of sisal fibers raised the HDT. The stiffness of these composites was improved by the addition of the sisal fiber reinforcement, which might account for the observed increases in HDT. The thermal characteristics of a composite made of 10 % sisal fiber with 20 % glass fiber was found to be superior . Section title: Sisal fiber composites Educational score: 4.138964653015137 Domain: biomedical Document type: Study Language: en Govindan and Srinivasan observed identical findings from their study of sisal with basalt hybrid PLA composite. At 250 °C, hybrid composites made of PP and sisal fiber degraded at a lower rate than composites made of sisal fiber alone. Nimanpure et al. reported on the thermal resistance of kenaf with sisal hybrid composites. Thermal degradation was different between composites of different weight percentages. High thermal stability was observed in the hybrid composite made up of 20 % sisal fiber and 20 % kenaf fiber. At 300 °C, this composite lost 10 % of its weight. By improving the fiber–matrix interface, chemical processing of fiber enhanced the thermal stability of the composites. Meenakshi and Krishnamoorthy found that the deterioration temperature of epoxy composites reinforced with sisal, flax, and glass fiber layers was between 306 and 315 °C. Desai et al. showed that carbon epoxy composites benefit from reinforcement with silane-treated sisal fiber. Further, it was reported that the enhanced bonding prevents the free radicals formed during degradation initiation from further spreading. Sisal fiber's degradation temperature is changed when it is reinforced in a hybrid composite because of the removal of organic material during treatment . Maleic anhydride-grafted polypropylene (MAPP) was found to improve the thermal stability of thermoplastic hybrid composites . MAPP was shown to improve the thermal stability of a hybrid polypropylene composite material with coir fiber and sisal fiber combined yarn reinforcement . Asaithambi et al. investigated the thermal characteristics of a banana with sisal fiber–reinforced PLA hybrid composite that had been chemically treated with benzoyl peroxide. The composites' first deterioration along with the final deterioration temperature were slowed by the chemically treated fiber reinforcement. Section title: Jute fiber composites Educational score: 4.021757125854492 Domain: biomedical Document type: Study Language: en Jute fibers are the common reinforcements in polymer matrices and are compatible with almost all other natural fibers and fillers when it comes to hybridization. Composites maintain the 40 % jute fabric weight incorporating the 3 wt% of into a polyester with jute composite significantly improve their tensile, flexural, and impact properties, as shown in Fig. 1 . Fourier-transform infrared spectroscopy (FTIR) spectroscopy confirmed the presence of OH connection among ATH, ZHO, with jute fabric, which are responsible for the enhanced mechanical characteristics. After performing a scanning electron microscopy (SEM) examination of the broken surfaces, it was determined that the synergy of the nanoparticles led to enhanced interfacial bonding. The TGA additionally demonstrated that the thermal resistance of the composites improved. Endothermic disintegration regarding ATH and ZHO fine particles as a char layer along with water molecules over combustion significantly improved the flame retardancy for the prepared composites, as evidenced by cone calorimetry along with horizontal igniting tests. By working together, ATH and ZHO particles improve mechanical, thermal, and fireproof qualities of polyester with jute composites . Fig. 1 Hydroxide-based nanoparticles addition in jute/polyester composites . Fig. 1 Section title: Jute fiber composites Educational score: 4.183988094329834 Domain: biomedical Document type: Study Language: en The environmentally beneficial natural fiber–reinforced polylactide composite (NFPC) has been the subject of some attempts to fortify it, but conventional methods reduce its ductility. To simultaneously increase the NFPC's strength and toughness, the authors of the present research synthesized rigid–soft core-shell nanoparticles. Nano-silica along with poly (butyl acrylate) rubber was chosen for their molecular properties, which made them ideal candidates for the core and shell roles, respectively. Crystalline, thermal, as well as mechanical characteristics of NFPC were studied in the presence of core-shell nanoparticles. The findings demonstrated that the core-shell nano-fillers can aid in the formation of a deeper crystalline grain within the PLA matrix and an increase in the material's thermal stability. A bonus is that the elongation at the break of NFPC was not compromised while the nanoparticle's rigid–soft core-shell construction increased the material's strength and stiffness. Furthermore, the resilience enhancement techniques and synergistic effect of core-shell nanoparticles have been demonstrated through field-emission SEM. Toughness is enhanced due to the core-shell filler inducing micro-cracks, shear bands, and matrix fibrillation . Section title: Flax fiber composites Educational score: 4.173618793487549 Domain: biomedical Document type: Study Language: en Natural fibers are increasingly used in the manufacture of polymer composites, even though these substances have become highly combustible. In this research, titanium dioxide, polydopamine, and diammonium phosphate were added to flax fiber composites made with a polyurethane-based shape memory polymer matrix (PUSMPM) to increase their fire resistance. Polydopamine, a shape memory polymer matrix reinforced using titanium dioxide, was applied to flax fibers that had been processed using diammonium phosphate. The defensive char layer of polydopamine can be seen in SEM images within the generated composites both before and following combustion. Owing to this coating, the composite material burned with much less of a loss of mass than it would have without it. PDA-coated TiO 2 -doped flax fiber composite showed substantial enhancement over uncoated flax fiber composite, with a limiting oxygen index value of 24 % compared to the noncoated flax fiber composite's 20 %. The flame propagation rate was reduced by 81 down to 21 mm/min during the horizontal burning experiment. Using TGA, along with differential thermal analysis, we looked at how polydopamine and titanium dioxide affected fibers and the PUSMPM. Titanium dioxide, polydopamine, and diammonium phosphate were shown in this investigation to significantly increase the combustibility of natural fiber composites made from a PUSMPM . Section title: Poplar fiber composites Educational score: 4.21895694732666 Domain: biomedical Document type: Study Language: en Impact of nanoscale iron oxide (Fe 2 O 3 ) particles on medium-density fiberboard (MDF) physical and chemical characteristics was studied. In this investigation, experimented with untreated poplar fibers along with a variety of nano iron oxide loadings (0.5, 1.5, and 2.5 wt%). Before being embedded into natural fibers, Fe 2 O 3 nanoparticles were mixed into urea formaldehyde resin using a high-vacuum mechanical stirrer. Dry mat layers were created by winding untreated poplar fibers onto metal frames. Twenty distinct composite specimens were created. Thickness swelling, retention of water, moisture content, and density were measured for each composite specimen using industry standards EN-322, EN-317, EN-323, and ASTM D570. Results showed that using iron oxide nanoparticles that were uniformly dispersed greatly enhanced thickness swelling ( T s ). At the highest loading, 3 wt%, water absorption (WA) increased by as much as 49.2 % and 34.5 %, respectively. This study used SEM to examine the nanoparticles' microstructure and determine whether or not iron oxide nanoparticles demonstrate favorable interactions with urea formaldehyde along with poplar wood fibers. A differential scanning calorimetry (DSC) and a TGA were performed to look into the effects of Fe 2 O 3 nanoparticles on heat and mass transfer. Fe 2 O 3 improved the resin's curing temperature as well as thermal stability. To regulate the dissemination of Fe 2 O 3 a one-way analysis of variance was created. As a result, the incorporation of iron oxide nanoparticles into an epoxy polymer results in a more rigid matrix, which in turn increases nano-MDF's potential for exhibiting improved physical characteristics . Table 1 explains the types of nanoparticles reinforced with natural fiber-based composites. Table 1 Types of nanoparticles in natural fiber–reinforced composites. Table 1 Nanoparticle Concentration (wt.%) Natural fiber Reinforcement Matrix Ref. Nanoclay 0, 1, 3, 5 Sisal Polypropylene 0, 2, 4 Bagasse High-density polyethylene (HDPE) 1–4 Bagasse Polypropylene 0, 1, 3, 5 Sisal Epoxy 0, 2, 4 Bagasse Polyethylene 0, 1, 3, 5 Bamboo High-density polyethylene (HDPE) + malleated polyethylene (MAPE) Bacterial cellulose Surface treated Sisal Acrylated epoxidized soybean oil (AESO) Hemp Poly ( l -lactic acid) + cellulose acetate butyrate (CAB) Sisal Acrylated epoxidized soybean oil (AESO) MWCNTs 0.1, 0.5, 1 Palm oil Epoxy Oil palm CNTs 0, 0.5, 1-2 Flax Epoxy Graphene 0.01–0.05 Palm oil Epoxy Nanoclay 0, 5, 8, 10 Flax Epoxidized soybean oil (ESO) Titanium dioxide, TiO 2 0–4 Jute Epoxy Zinc oxide, ZnO 0–4 Jute Epoxy Silica, SiO 2 0, 1.5, 3 Flax Polypropylene (PP) 0, 5 Sisal Polyester Zirconium oxide, ZrO 2 Grafted Flax Poly (lactic acid) Graphene – Bagasse Polypropylene 0–5 Kenaf Alumina, Al 2 O 3 0–3 Flax/PLA Epoxy MWCNTs 0–0.6 Ramie Epoxy Titanium dioxide, TiO 2 0–7 Flax Epoxy Oil palm CNTs 0.15 Bamboo Epoxy Silica, SiO 2 0, 2, 5 Bagasse High-density polyethylene (HDPE) Nanoclay 0–1.5 Hemp Unsaturated polyester (UP) + epoxidized methyl soyate (EMS) Titanium dioxide, TiO 2 0, 2 Bagasse Ethylene co-vinyl acetate (EVA) Nanoclay 1, 2.5, 5, 10 Hemp Poly (lactic acid) Cenosphere – Bamboo Epoxy Bacterial cellulose Surface treated Sisal Poly ( l -lactic acid) + cellulose acetate butyrate (CAB) Oil palm CNTs – Kenaf Epoxy 0, 0.5, 1–4 Hemp Graphene 0.3, 1, 3 Jute Epoxy Alumina, Al 2 O 3 0, 5, 10, 15 (μm) Jute Epoxy 10 (μm) Coir Titanium dioxide, TiO 2 Grafted Flax Poly (lactic acid) Exfoliated graphite, xGnp 0, 1, 3, 5 Kenaf Poly (lactic acid) Section title: Thermal characteristics of nanoparticle-based hybrid composites Educational score: 4.210137367248535 Domain: biomedical Document type: Study Language: en Graphite is composed of numerous layers of carbon atoms piled on top of each other. Graphene is a monolayer of graphite that can be mechanically exfoliated. The material exhibits notable characteristics such as exceptional strength, low mass, and excellent resistance to corrosion. In comparison to metals, it also has superior electrical and thermal conductivities. Additionally, its remarkable sensitivity renders it suitable for use in nanosensor technology. The advantageous characteristics of graphene, such as its lack of metallic contaminants and two-dimensional structure, make it a valuable material in the field of nanofabrication. Graphene can be synthesized using various techniques, such as mechanical exfoliation, intercalation, chemical synthesis, and burning of agricultural waste materials. This substance finds utility in a diverse range of applications. Graphene exhibits superior thermal conductivity compared to copper, enabling more effective heat dissipation from miniature electronic devices such as computer chips. Numerous industries anticipate the use of materials or nanocomposites that possess distinctive features. The integration of graphene into polymers has been found to considerably enhance the attributes of composites, including electrical conductivity, mechanical strength, thermal stability, and fire resistance . It effectively prevents the initiation and propagation of cracks when subjected to periodic loading. The results of the fatigue studies indicate that after 800 cycles, the hybrid composite material consisting of bamboo, glass fibers, and nanoparticles exhibits a load capacity that is 22.5 % greater than that of all other composite mixtures. The fatigue life of hybrid composites exhibits an upward trend when the filler dimension decreases. Section title: Thermal characteristics of nanoparticle-based hybrid composites Educational score: 4.136844158172607 Domain: biomedical Document type: Study Language: en The thermal resistance of the hybrid bamboo/glass FRP composite with nanoparticles was enhanced, as indicated by the findings of TGA/DTG studies . The investigation examines the features of the micro- and nanoparticles of hydrophobic graphene oxide (HO) and HO Janus (HOJ) by the use of several analytical techniques, including field-emission scanning electron microscopy, TGA, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, and contact and centrifuge angle tests. Various micro- and nanocomposite samples of PS/PMMA were fabricated using solution mixing techniques and subsequently evaluated through thermal conduction experiments. Fig. 2 depicts the optical microscopic (OM) images of the PMMA/PS blends containing HOJ nanoparticles. The findings of the study indicate that the incorporation of HO micro- and nanoparticles in either phase of the PS/PMMA blend resulted in a slight increase in the conduction coefficient (K). However, it was observed that the combined presence of these particles in both phases had a more substantial effect on K. In contrast, while evaluating the outcomes of the blend specimens with and without the incorporation of HOJ micro- and nanoparticle, a significant enhancement in the heat conductivity of the polymer with polymer interface was seen as a result of the inclusion of Janus particles. Furthermore, it was observed that the thermal resistance of the samples exhibited a reduction as their thermal conductivity increased. This trend was particularly pronounced in the samples that contained both HO and HOJ micro- and nanoparticles . Fig. 2 OM images depicting the presence of HOJ nanoparticles in PMMA/PS polymer blends . Fig. 2 Section title: Thermal behavior of nanocomposites Educational score: 4.176374912261963 Domain: biomedical Document type: Study Language: en Nanoparticle-based polymer composites are potential candidates for various engineering applications apart from natural-fiber and hybrid composites. Polymer composites are normally reinforced with organic or inorganic nanoparticles and were assessed for their physical and thermal properties in most cases. Cellulose-based nanocomposites and carbon or graphene-based nanocomposites were analyzed by numerous researchers for their thermal and electrical properties [ , , , ]. Bioactive polymeric nanocomposites are materials that are considered essential and have gained significant interest due to their wide range of applications within the human organism. A complex of poly (hydroxyethyl methacrylate) (pHEMA) was fabricated using hydroxyethyl methacrylate (HEMA) as the primary material with the aid of a twin-screw extruder. The nanocomposites based on pHEMA were fabricated by incorporating titanium oxide nanoparticles (1 wt % TiO 2 ) and graphene oxide (0.1 wt% GO) and their thermomechanical properties were investigated. The PT and PTG nanocomposites exhibited notable alterations in microstructural behavior, resulting in enhanced thermomechanical characteristics as compared to pure pHEMA. This study investigated the suitability of pure pHEMA as well as PT and PTG nanocomposites for implantation in dental implications . Section title: Thermal behavior of nanocomposites Educational score: 4.1342854499816895 Domain: biomedical Document type: Study Language: en A few experimental works used betel nut husk fiber (BNHF) derived nanocellulose reinforced in polyvinyl alcohol (PVA) matrix with various weight fractions and their thermal behaviour was evaluated using TGA and DSC techniques. It was understood from the results that the addition of nanocellulose to PVA matrix enhanced thermal stability of the nanocomposites due to the establishment of strong hydrogen bonding between the composite constituents . In some other studies, vinyl laurate micro fibrillated cellulose (VL-MFC) were reinforced in PLA matrix and their rheological and thermal properties were evaluated. It was observed from the results that the thermal stability of the PLA matrix was increased due to the addition of VL-MFC while the storage and loss modulus of the VL-MFC nanocomposites increased with the addition of the micro cellulose. These changes were supported by the better interfacial adhesion between the nanoparticles and the matrix and due to the liquid-to-solid transition in the composites when subjected to change in temperatures . From the above discussions, it could be understood that the thermal stability of the polymer composites were improved by the incorporation off the nanoparticles. Section title: Thermal behavior of nanocomposites Educational score: 4.163469314575195 Domain: biomedical Document type: Study Language: en The composite polymer electrolytes are specifically designed to be compatible with the 3D printing process known as fused deposition modelling. The composite materials used in this study were composed of altered poly (ethylene glycol) (PEG), lithium bis-(trifluoromethylsulfonyl) imide (LiTFSI), and nanofillers based on SiO 2 . The end group modification of PEG was effectively carried out, resulting in the production of telechelic PEG that incorporates either ureidopyrimidone (UPy) or barbiturate moieties. These modified PEG molecules possess the ability to form supramolecular networks through hydrogen bonding interactions, thereby providing self-healing properties to the electrolyte system. Silica NPs were used as a filler to modify the mechanical properties of the electrolyte to facilitate its 3D-printing capability. It is anticipated that the surface modification of the NPs using an ionic liquid (IL) or hydrophobic alkyl chains will result in an enhanced dispersion of the NPs within the polymer matrix. The rheological properties of composites containing varying concentrations of nanoparticles (in increment of 5 %, from 5 % to 15 %) and LiTFSI salt were examined to acquire an in-depth understanding of their suitability for 3D printing. Additionally, the ionic conductivity of these composites was assessed using broadband dielectric spectroscopy. The successful 3D printing of the composite electrolyte, PEG with other constituents in combination with 15 % NP-IL, demonstrates its potential as a viable option for printable composite electrolytes. TGA experiment was performed using the Netzsch TG 209 F3 instrument. Samples weighing between 5 and 10 mg were carefully positioned into alumina crucibles. These crucibles were then subjected to controlled heating in an environment free of reactive gases, with a heating rate of 10 K −1 . Section title: Thermal behavior of nanocomposites Educational score: 4.2231245040893555 Domain: biomedical Document type: Study Language: en DSC measurements were conducted using a Netzsch DSC 204 F1 instrument. Before measurement, the samples were subjected to an air-drying process in a vacuum environment at 80 °C. Subsequently, the dried samples were carefully positioned in aluminum pans and measurements were carried out in the presence of a nitrogen atmosphere. The thermal history of the samples was eliminated with the application of heat at 100 °C. The cooling process was conducted until a temperature of −20 °C was reached, with a cooling rate. Subsequently, heating curves were recorded up to a temperature of 170 °C, using a heating rate. The samples underwent a drying process at 80 °C under vacuum conditions for 24 h. Subsequently, measurements of shear rate vs viscosity were conducted at various temperatures. The silica nanopowder, which incorporated alkyl groups (referred to as NP-alk), demonstrated the stretching vibration of the alkene of C–H within the range of approximately 3000 cm −1 . The findings from the thermogravimetric study are illustrated in Fig. 3 . The nanoparticles that underwent modification exhibited a reduction in weight when exposed to elevated temperatures, which can be attributed to the breakdown of the organic modifiers present on their surfaces. This outcome serves as evidence of the effective functionalization of the nanoparticles. The Ludox SM30 nanoparticles functionalized with ionic liquid groups (referred to as NP-IL) exhibited thermal stability up to 350 °C, consistent with previous findings reported in the literature for nanoparticles functionalized with ILs . In contrast, the thermal stability of NP-alk was found to be considerably lower, reaching only up to 250 °C. The estimation of the size of the nanoparticles (with a range of 10–120 nm) was conducted using dynamic light scattering and TEM measurements. Furthermore, proton nuclear magnetic resonance ( 1 H NMR) and solid-state cross-polarization/magic angle spinning silicon-29 nuclear magnetic resonance (CP/MAS 29Si-NMR) techniques were also used to characterize NPs . Fig. 3 Thermal degradation curves of nanocomposites with silica nanocomposites with surface modification done using ionic liquids (blue line) and Alkyl compounds (black line) . Fig. 3 Section title: Summary and conclusions Educational score: 3.8645243644714355 Domain: biomedical Document type: Study Language: en Natural fiber composites have become the first-choice materials in various engineering applications due to their economic feasibility, technicality and environmental friendliness. Though many research works were undertaken for developing novel composite materials using natural fibers, various technical and economic constraints retarded the full-scale implementation of natural fiber composites in different commercial applications. A high level of priority must be given to the thermal ability of the natural fiber composites during their processing and the retention of their inherent properties even after exposure to thermal and other heat-related environments. In this context, the thermal analysis of the composite materials paved way for the development of newer and novel composite materials if the selection process of the materials is rightly optimized. Various approaches were analyzed in the previously published literature to improve the thermal properties of the natural fiber–reinforced polymer composites. Some of the commonly adopted techniques were tailoring the fiber and matrix type, addition of organic and inorganic fillers specifically in nanoscale, orientation, chemical modification, and loading of the natural fibers. In most of the studies, it was pointed out that the addition of nanoparticles resulted in a better enhancement of the thermal properties of the composites. Section title: Summary and conclusions Educational score: 4.082978248596191 Domain: biomedical Document type: Study Language: en Some of the commonly analyzed thermal behavior includes the mass loss of the nanoparticles reinforced composites when heated, the temperature of complete degradation of the composites, their glass transition temperature, and the viscoelastic behavior of the composites. The addition of nanoparticles facilitated the composites to attain better thermal properties. Specifically, the viscoelastic properties of the natural fiber–reinforced polymer composites could be readily improved by the addition of nanoparticles, which promote good interfacial adhesion between the matrix and the reinforcements and improve the energy dispersion. The addition of nanoparticles also governed the thermal degradation of the polymer composites by enhancing the molecular mass of the composites, increasing the cross-linkages, and increasing tacticity, which in turn affects the glass transition properties of the composites. Additionally, the hybridization of the natural fiber composites with the nanoparticles improved the crystallization kinetics, heat of fusion, heat capacity, melting behavior, and liquid crystal transition temperature, which enhance the thermal characteristics of the composites. From all the above studies, it could be concluded that the nanoparticles incorporated in natural fiber composites exhibit better thermal behavior and are highly suitable as renewable materials for various high-temperature applications. Section title: CRediT authorship contribution statement Educational score: 0.8098545670509338 Domain: other Document type: Other Language: en D. Balaji: Writing – original draft, Supervision, Data curation, Conceptualization. P. Sathish Kumar: Writing – review & editing, Writing – original draft, Supervision, Data curation, Conceptualization. V. Bhuvaneshwari: Writing – original draft, Supervision, Data curation, Conceptualization. L. Rajeshkumar: Writing – original draft, Supervision, Data curation, Conceptualization. Manoj Kumar Singh: Writing – review & editing, Supervision, Data curation, Conceptualization. M.R. Sanjay: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Suchart Siengchin: Writing – review & editing, Supervision, Conceptualization. Section title: Consent to participate Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. Section title: Future scope Educational score: 4.002086639404297 Domain: biomedical Document type: Study Language: en The trend of using nanoparticles for enhancing the thermal properties of the natural fiber–reinforced polymer nanocomposites was analyzed using a patent database due to its frequently updated nature. This analysis provides a path for researchers in this domain to take direction on decision making of their research work. This also paves a pathway to critically assess the application point-of-view of the material through the identification of the innovations using this material . It could be understood from many of the earlier research works that natural fiber composites with nanoparticles as hybrid reinforcements possess better thermal behaviour and thermal stability for various engineering applications. Since the thermal behaviour of the nanoparticle-reinforced natural fiber composites were found to be better, they can capture a significant market share in electrical and sports applications. To further enhance the potential of the nanoparticle-reinforced natural fiber composites, the weathering behaviour of the composites specifically in outdoor environments must be assessed in detail. Exposure of the composites to sunlight, moisture and UV light can be monitored and their behaviour can be evaluated under such conditions to enhance the applications spectrum of the composites . Section title: Future scope Educational score: 1.8166850805282593 Domain: other Document type: Study Language: en Table 2 reveals impact of natural fiber with nanoparticles searched in English all categories, with keywords “natural fiber reinforced composites” and “nanoparticles”. Eight keywords were searched. The researchers in this domain can focus on the International Patent Classification (IPC) categories C04B, B29C, C08J, and C08L. In Over the past decade, there has been slow but steady progress. The recent inclusion of advanced materials suggests the potential for novel nanoparticles to introduce newer application. Furthermore, only two countries have contributed to this technology, indicating substantial opportunities for growth and development in the future . Table 2 Patent landscape analysis . Table 2 S. No. Countries Count IPC Count Year Count 1 United States of America 3 C04B 3 2014 1 2 Patent Cooperation Treaty 3 B29C 2 2015 1 3 India 2 C08J 2 2016 0 4 C08L 2 2017 1 5 A61F 1 2018 0 6 A61L 1 2019 0 7 B01D 1 2020 1 8 B01J 1 2021 1 9 B28B 1 2022 1 10 B29D 1 2023 0 Section title: Availability of data and materials Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. Section title: Ethics approval Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. Section title: Consent for publication Educational score: 1.0029362440109253 Domain: other Document type: Other Language: en This manuscript does not contain any individual person's data in any form. Section title: Funding Educational score: 1.0266131162643433 Domain: other Document type: Other Language: fr Not applicable. Section title: Declaration of competing interest Educational score: 0.9956112504005432 Domain: other Document type: Other Language: en The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:The author(s) of this paper, L. Rajeshkumar and M. R. Sanjay, works as an Associate Editor at Heliyon Materials Science. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Study | biomedical | en | 0.999997 |
PMC11699328 | Section title: Introduction Educational score: 3.278853416442871 Domain: other Document type: Study Language: en The use of unconventional oil and gas production technologies is increasing due to the continuous increase in energy demand . Accordingly, the exploitation of unconventional reservoirs through hydraulic fracturing is an inevitable choice in the industry . Hydraulic fracturing (HF) operations inject large and highly pressurized volumes of fracturing fluid into tight shale formations to fracture the rock and allow the extraction of trapped oil and gas through the created flow pathways . The most widely used type of fracturing fluid is slick-water , which is a water-based fluid that contains a fraction of added chemicals (e.g., friction reducers) and proppants . Flowback and produced water returning to the surface during production consist of the fraction of the injected fracturing fluid that is recovered and the brine, respectively . Reported fracturing fluid flow-back recovery values vary significantly across different formations, reflecting that a substantial volume of fluid typically remains in the subsurface . For example, recovery rates for the Marcellus shale are reported at 7.8 % , for the Qusaiba formation at 9.6 % , for the Montney formation at 36 % , and for the Haynesville formation at 5 % . These examples highlight the generally low flow-back recovery rates across formations, which contributes to notable fluid retention in the subsurface. Section title: Introduction Educational score: 3.8874728679656982 Domain: biomedical Document type: Study Language: en Meanwhile, environmentalists are constantly warning on the potential risks associated with the migration of the remaining fraction of this fluid (>70 %) which imbibed into the shale formation. Particularly, there are concerns on possible groundwater contamination because the additives present in the fracturing fluid might find their way to water aquifers . Toxicity information available about fracturing fluid chemicals show that being exposed to fracturing fluid poses serious health hazards on humans [ , , , ]. Public pressure concerning this issue is constantly intensifying because toxicity data is limited and disclosures of fracturing fluid are questionable. Previously, voluntary disclosures were minimal , and mandatory disclosure regulations used to have shortcomings concerning the chemicals' identification requirements, imposed penalties in case of insufficient reporting, and lenient timelines of reporting . A recent study by Bonetti et al. showed that revisiting and tightening disclosure mandates led to a decrease in the quantity of toxic chemicals used. Assessing the environmental impact of fracturing fluid, monitoring its fate, and tracking the diffusion of the fluid's constituents post injection are necessary to eliminate possible hazards. Section title: Introduction Educational score: 2.3254055976867676 Domain: other Document type: Study Language: en Unfortunately, the fate of the injected fluid in the subsurface is not clearly understood [ , ]. So, investigating the fate of the un-recovered fraction of the fluid requires a thorough understanding of the fluid transport in the subsurface post injection. The literature highlights the issues of the un-recovered fracturing fluid by addressing three main topics: (i) Upward fluid migration into drinking water sources [ , , , , , , , ], (ii) fluid retention in complex fractured systems created due to hydraulic fracturing [ 5 , , , , ], and (iii) the effect of operational parameters (e.g. shut-in period) on fluid recovery [ 21 , , , , , , ]. Section title: Introduction Educational score: 1.2037664651870728 Domain: other Document type: Review Language: en Accordingly, as this review paper demonstrates, the key parameters controlling the flow-back in hydraulic fracturing are primarily related to formation and operational properties (e.g., permeable pathway properties, shut-in time, and fracture network complexity and properties). Section title: Introduction Educational score: 1.8365483283996582 Domain: other Document type: Study Language: en Furthermore, instances where induced seismicity was correlated with hydraulic fracturing are presented and provide further evidence that fracturing fluids are diffusing and migrating to permeable underground formations. Fluid diffusion has been identified as the primary trigger behind recorded earthquakes in western Canada [ , , , ] and the Sichuan Basin in China . Hence, low flow back recovery may be attributed to underground fluid diffusion in addition to fluid imbibition into the matrix, retention within the fracture systems, and potential upward migration. Section title: Introduction Educational score: 3.165210247039795 Domain: other Document type: Study Language: en Additionally, although hydraulic fracturing is widely recognized for its use in the oil and gas industry, its application has expanded into sustainability-focused projects such as carbon capture and storage (CCS) and enhanced geothermal systems (EGS). In these areas, hydraulic fracturing is used to improve permeability, create heat reservoirs in EGS, and enhance CO₂ injectivity in storage reservoirs. Although the types and concentrations of chemicals used in fracturing fluids for CCS and EGS are typically simpler and lower than those in oil and gas applications, there remain critical environmental risks—such as induced seismicity, fluid migration, and CO₂ leakage—that are important to consider. These shared concerns highlight the broader relevance of hydraulic fracturing fate studies, as they illuminate fluid retention and migration behaviors that could affect subsurface and surface water resources across applications. By evaluating hydraulic fracturing's impacts in CCS and EGS alongside traditional oil and gas contexts, we gain a more comprehensive understanding of how this technology can support, rather than undermine, sustainability goals. Addressing these risks proactively is essential to ensuring that hydraulic fracturing contributes positively to energy transition efforts and environmental protection measures [ , , , , , , , ]. Section title: Introduction Educational score: 2.536770820617676 Domain: other Document type: Review Language: en This review is essential for addressing a significant gap in understanding the environmental impacts of fracturing fluid and has two primary objectives: first, to compile data on the composition of fracking fluid and the associated hazards which highlight the environmental concerns rising from the uncertainty surrounding the fate of the fracturing fluid; and second, to establish correlations between the environmental and geologic observations, such as water contamination, induced seismicity, subsurface complexity and the recovery rate. Section title: Methodology Educational score: 3.336200475692749 Domain: biomedical Document type: Review Language: en Peer reviewed articles from scientific journals, Environmental Protection Agency (EPA) reports, and conferences papers were analyzed and discussed in order to describe the fate, transport and recovery of fracturing fluids, in addition to their environmental and health impacts. The 100+ papers referenced in this review included both numerical simulations and case studies. Accordingly, the articles referenced are classified into three categories representing the main areas of the study, which are (1) environmental impact of HF fluid and associated risks; (2) fate of HF fluid; and (3) induced seismicity triggered by HF fluid diffusion and its correlation to flow back. Section title: Methodology Educational score: 4.080531597137451 Domain: biomedical Document type: Study Language: en First, HF fluid composition and toxicity are illustrated to emphasize the criticalness of surface and subsurface risks associated with the transport and migration of HF fluid. Next, the fate of HF fluid was examined due to the subsurface threats posed by its migration, particularly the retention of a significant fraction of HF fluid in the subsurface post-production. This analysis involved exploring the potential reasons behind the low flowback recovery values and establishing correlations between HF fluid recovery and the various factors affecting it. Finally, a correlation between induced seismicity and load recovery is demonstrated by highlighting the spatial and temporal link between seismic activity and hydraulic fracturing operations in which HF fluid migrates to critically stressed faults. Moreover, this research identifies the limitations and gaps in the literature, which are related to the scarcity of data. Disclosure data about the composition of the fracturing fluid is limited , and subsurface monitoring data utilized to characterize fluid migration are rare in comparison to the large number of hydraulically fractured wells . Such limitations impede groundwater contamination investigations and restrict the publishing of relevant studies, leading to shortcomings in irrefutable documentation of drinking water contamination by fracturing fluid . Section title: Methodology Educational score: 1.6815751791000366 Domain: biomedical Document type: Other Language: en Certainly, as this is a controversial topic in the industry, the papers were reviewed from a scientific point of view without any bias. Furthermore, the studies were selected from various sources and geographic locations to avoid biases and ensure objectivity in the analysis. Section title: Fracturing fluid composition Educational score: 2.4042341709136963 Domain: other Document type: Other Language: en Fracturing fluid composition, required for the success of the fracturing operations, consists of a base fluid (e.g. water, acid, oil), proppants, and chemical additives such as gelling agents, crosslinkers, corrosion inhibitors, and friction reducers. Since the introduction of hydraulic fracturing, there has been a continuous development in fracturing fluid composition, shifting from a hydrocarbon to an aqueous basis . Among the various base fluids and their corresponding fluid types (see Table 1 ), the fluid composition selection is based on the formation targeted and fracturing job results anticipated [ , , , ]. Table 1 Fluid types employed for the different types of fracturing fluid base . Table 1 Base Fluid Fluid Type Water Linear gel Crosslinked gel Slickwater Viscoelastic surfactant gel Oil Linear gel Crosslinked gel Emulsion Acid Linear gel Crosslinked gel Emulsion Alcohol Methanol and water mixture 100 % Methanol Foam Water-based Acid-based Alcohol-based CO 2 -based Emulsion Water-oil emulsion CO 2 -methanol Section title: Fracturing fluid composition Educational score: 1.4028738737106323 Domain: other Document type: Other Language: en Recent literature indicates that the most commonly used fracturing fluid is water-based, with a typical description as detailed in Fig. 1 [ 2 , 14 , 56 , , , , , , ]. However, specific regions, such as California, often employ alternative fluids like acid-based fracturing fluids due to unique geological and resource characteristics, highlighting how local conditions can influence fracturing fluid selection. Fig. 1 Water-based fracturing fluid composition (reproduced from Ref. ). Fig. 1 Section title: Fracturing fluid composition Educational score: 1.4742635488510132 Domain: other Document type: Other Language: en Operators state that only a small percentage of the fluid is occupied by chemicals and about 99 % is occupied by the water and proppants. Nevertheless, this does not negate the need to counterattack the negative consequences resulting from the usage of fracturing fluid and the fact that hydraulic fracturing treatments require large volumes of fluid and thus a large volume of chemicals is used regardless of their percentage in the composition . Accordingly, regulatory authorities demand operators to provide a disclosure of the chemical additives present in the hydraulic fracturing fluid . For example, this data is available for many states in the USA and for Alberta and British Columbia, which are the provinces with the highest fracturing activity in Canada . Section title: Toxicity of additives Educational score: 2.4723548889160156 Domain: biomedical Document type: Other Language: en Chemical additives in the fracturing fluid pose a threat on the environment and human health . Environmentalists are especially alert regarding the issue of threats posed on groundwater's integrity due to possible contamination by fracturing fluid chemicals, which are labeled as “chemicals of concern”, due to their toxicity (e.g., mammalian, aquatic) and human health related hazards (i.e., effects on nervous system, immune system, respiratory system, reproductive system, and sensory organs) [ 15 , 54 , , , ]. Nevertheless, severity of these effects varies and is controlled by degree and way of exposure to these chemicals. Section title: Toxicity of additives Educational score: 4.116811752319336 Domain: biomedical Document type: Study Language: en In studies dedicated to evaluating toxicity of additives present in hydraulic fracturing fluids, authors usually follow the same approach; compiling lists of disclosed chemicals, then scanning databases for toxicological data, and possible health effects. Key findings from the studies provide different pieces of information about the type of toxicity associated with the chemicals, toxicity ratings, hazard rankings, and associated health effects. Table 2 shows some of the interesting findings presented in the literature. Furthermore, Kahrilas et al. reviewed the toxicity of biocides used in fracturing fluids, highlighting their potential as serious irritants to sensory organs and their considerable acute toxicity to aquatic life. Data from their study are summarized and illustrated in Fig. 2 to show the types of chronic toxicity associated with certain biocides and their degradation products used in fracturing fluid formulations. Table 2 Summary of toxicity data availability for hydraulic fracturing fluid chemicals as reported in each study, including the types of data presented and the biological systems affected. Table 2 Number of chemicals studied Toxicity data availability Toxicity data available/most affected systems Reference 1076 10 % Chronic oral toxicity values 36 22 % Chronic oral toxicity values Note: Additives reported in fracturing fluids in at least 10 % of wells in the U.S. 600 58.8 % • >75 % affect sensory organs, respiratory and gastrointestinal systems • 52 % affect nervous system, immune and cardiovascular systems, and kidneys • 37 % affect endocrine system • 25 % cause cancer 81 69 % • 16 % low/moderate toxicity • 31 % lacked toxicity data • 53 % considered as non-toxic 168 N/A 14 % associated with reproductive and developmental toxicity 320 60 % Chronic and/or acute hazard screening values 924 21 % Reproductive and developmental toxicity reported 28 82 % Mammalian and aquatic acute toxicity measurements Note: Additives used in more than 10 % of fracturing treatments in California 20 45 % Mammalian and aquatic acute toxicity measurements Fig. 2 Toxicological data of biocides used in hydraulic fracturing operations . Fig. 2 Section title: Toxicity of additives Educational score: 2.3790907859802246 Domain: other Document type: Study Language: en Despite extensive data collection and studies demonstrating the toxicity of fracturing fluid constituents, comprehensive toxicity assessment remains challenging. In studies examining these chemicals, toxicity data is often incomplete, with many chemicals lacking toxicity information . Furthermore, as Caldwell et al. notes, the information disclosed by industries on the chemicals used is still not detailed enough, and this lack of transparency continues to hinder scientists' ability to assess potential chemical interactions in the subsurface and the resulting changes in toxicity. Section title: Associated risks Educational score: 1.5050126314163208 Domain: other Document type: Other Language: en From an environmental perspective, risks associated with the use of fracturing fluid are numerous. A significant portion of the literature addresses the risk of water bodies contamination, which can be the result of aboveground incidents (e.g., spillage of the fracturing fluid during transport) or the infiltration of the fluid into aquifers through migration into subsurface layers [ 17 , 18 , 53 , 66 , , , , , , , , , ]. Section title: Associated risks Educational score: 1.674914002418518 Domain: other Document type: Other Language: en One of the pre-requisites required to assess environmental hazards, as per Shah et al. , is tracking the motion of the hydraulic fracturing fluid. Similar to Kreipl & Kreipl's classification, they categorized possible risks into two groups: surface risks and downhole risks. Section title: Surface risks Educational score: 2.6714894771575928 Domain: biomedical Document type: Study Language: en Risks classified as surface risks primarily include fracturing fluid spills that occur during transport and leakage incidents from storage pits . Mrdjen & Lee emphasized the adverse human health effects due to possible contamination of surface water bodies, often utilized as sources of drinking water, by chemicals used in hydraulic fracturing operations . This important issue has not been addressed considerably in the literature. Fracturing fluid and flow-back and/or produced water spills and leakage can occur both prior to and during injection (e.g., spills during the transport of fracking water and additives to the site for mixing, and leakage from storage tanks) and throughout production (e.g., accidental spills of produced water on the soils or illegal deliberate releases by some operators) . Section title: Surface risks Educational score: 3.112053155899048 Domain: other Document type: Study Language: en Numerous studies have compiled and analyzed spill records [ 8 , 54 , 71 , 75 , 76 , 80 , , , ]. Scientists are able to categorize spills linked to hydraulic fracturing by type of material, pathways, sources, causes, and volume spilled . For example, the EPA report examined data of more than 36,000 spills recorded over a period of 6–7 years in 11 states in the U.S. and found that the most prevalent material types that are spilled are flow-back and produced water (49 % of the total material spilled), the source of these spills are mainly from storage (46 % of the total source type of spills), and the cause is mainly due to human error (33 % of the time) and equipment failures (27 % of the time). A detailed description is synthesized in Fig. 3 . Fig. 3 Percent distribution of material related to hydraulic fracturing that are spilled, their source and cause. These values were calculated using the number of spills per material, source, and cause types relative to the total number of spills with data synthesized from the U.S. EPA . Fig. 3 Section title: Surface risks Educational score: 1.512567162513733 Domain: other Document type: Study Language: en As a demonstration of spill data analysis in states with significant unconventional oil and gas activity, spill rates calculated by Maloney et al. in Colorado, New Mexico, North Dakota, and Pennsylvania are presented in Table 3 . Differences in spill rates across states may reflect variability in reporting requirements, which differ in terms of volume thresholds, spill location specifications, and methods of reporting (written or verbal). Table 3 Spill rates calculated from spill data collected between 2005 and 2014 across selected U.S. states . Table 3 Location Spill rate (%) Data timeframe Data source State of Colorado 7.1 % 2005–2014∗ a Colorado Oil and Gas Conservation Commission New Mexico 12.1 % New Mexico Oil Conservation Division North Dakota 40.7 % Oilfield Environmental Incident reports Pennsylvania 17.6 % Notice of violations (NOV) database a For Colorado, the data timeframe is from January 1, 2005, to December 31, 2013, due to a significant change in spill reporting starting in 2014. While for the other states, the timeframe is from January 1, 2005, to December 31, 2014. Section title: Surface risks Educational score: 1.6799653768539429 Domain: other Document type: Study Language: en To further analyze state-specific trends, we highlight annual variations in reported spill numbers as documented by Patterson et al. . Fluctuations within each state, along with an overall upward trend in reported spills, suggest that tighter reporting requirements have increased reported spill counts . Notably, North Dakota experienced a marked rise in reported spills post-2010, the year which marks the transition from verbal to written reporting obligations in the state . As for the State of Colorado, regulatory changes in 2008 and 2013, which included lowering the minimum reportable spill volume to 1 barrel in 2013 likely contributed to the consistent rise in number of reported spills. Fig. 4 Annual number of reported spills from 2005 to 2014 across four states . Fig. 4 Section title: Surface risks Educational score: 3.496288537979126 Domain: biomedical Document type: Other Language: en It must be highlighted that although the spill rates usually reported might be misinterpreted as inconsequential, the volume per spill ranges from small values like 1 Gal to critically high values reaching 1 MGal , thereby amplifying exposure risks. Estimating fracturing fluid spill rates serves as a basis for spill assessment. Spills of all material types, even if non-toxic, must be incorporated into spill probability assessments, since a spill during transport due to a wreck incident has the same likelihood whether the fluid is fracturing fluid or any other chemical; if the content is not safe it will pose a serious environmental hazard. Another important factor in assessment and mitigation practices is monitoring how changes in reporting requirements impact the spill rates recorded, where stricter mandates are leading to an improvement in accuracy, details, and number of reported spills. Section title: Downhole risks Educational score: 1.327595591545105 Domain: other Document type: Other Language: en Groundwater is an invaluable and versatile resource; in countries like the United States, reliance on groundwater for purposes such as public use, irrigation, livestock, and human consumption is significantly higher than on surface water sources, mainly because it is less susceptible to pollution compared to surface water . So, it is vital to prevent groundwater contamination. Section title: Downhole risks Educational score: 1.3994501829147339 Domain: other Document type: Study Language: en Nevertheless, the downhole risks associated with hydraulic fracturing present a distinct threat to groundwater quality, as they primarily relate to the possible upward fracturing fluid migration towards drinking water aquifers . A considerable amount of the literature has discussed the potential pathways, and it was found that, mainly, pathways can be created as a consequence of poor well integrity (e.g., poor cementing) and/or undesired hydraulic communication in the subsurface [ 2 , 18 , 53 , 66 , 78 , 79 , , , ]. Section title: Downhole risks Educational score: 1.1634010076522827 Domain: other Document type: Other Language: en Researchers identified the most common pathways: 1. Defective casing installments and/or poor cementing jobs. 2. Intersection between created hydraulic fractures and aquifer. 3. Intersection between created hydraulic fractures and pre-existing fractures or faults. 4. Intersection between created hydraulic fractures and adjacent wells (e.g., abandoned wells, offset wells). Section title: Downhole risks Educational score: 2.9334447383880615 Domain: other Document type: Study Language: en Although hydraulic fracturing design and operation plans should be prepared to avoid undesirable fluid leakage, well design and planning has a level of uncertainty. Faulty cementing, leaky casing connections, casing corrosion, and cement sheath degradation are contributors that jeopardize well integrity and promote fluid loss . For example, shallow leaky casings near the aquifers, and annular spaces created by cement sheath failure form channels and conduits for the fracturing fluid to infiltrate into unfavorable zones . Also, initiated fractures might extend out of the designated interval and provoke unanticipated communication, especially in the presence of unmapped faults . Furthermore, pathways generated due to communication between hydraulic fractures and wells are attributable to well communication/interference. One of the most important types of this interference is fracture communication, known as frac-hits, and it is highly controlled by well spacing, where tighter spacing leads to a higher degree of interference among wells . Simply put, groundwater contamination is possible through these pathways if two conditions are met: presence of a fluid and a driving force for fluid flow . Section title: Downhole risks Educational score: 1.9389792680740356 Domain: other Document type: Other Language: en Despite the fact that groundwater contamination by hydraulic fracturing additives has rarely been irrefutably documented in the academic literature and such events are considered to be of relatively low occurrence, there have been reported contamination incidents of drinking water sources by the fracturing fluid and the health implications are alarming. Notable examples of water contamination linked to hydraulic fracturing operations are. A. Jackson County, West Virginia in 1982: fracturing fluid gel was detected possibly due to a nearby gas wells that could serve as conduits for fluid flow . B. West Divide Creek in Garfield County, Colorado in 2004: BTEX (benzene, toluene, ethylbenzene and xylene) was detected possibly due to natural fractures and faults and poor wellbore integrity (i.e., improper cementing job) that could serve as conduits for fluid flow . C. Pavillion in Fremont County, Wyoming in 2008: Fracturing fluid components possibly due to the short distance between the aquifer and the well . Section title: Downhole risks Educational score: 1.6834217309951782 Domain: other Document type: Other Language: en On another note, even though methane detection in drinking water is out of the scope of this paper, the literature is rich with documented cases, so it is worth pointing out that the phenomena controlling gas migration is not entirely specific to gases and could also apply to hydraulic fracturing fluid migration , which adds to the plausibility of aquifer contamination incidents. Section title: Downhole risks Educational score: 3.845120668411255 Domain: biomedical Document type: Study Language: en Digiulio & Jackson assessed the effect of fracturing operations on subsurface drinking water sources. The motivation behind this work was based on an EPA investigation done in 2008 in response to complaints in the Pavillion Field about changes in taste and odor of drinking water from domestic wells. To evaluate potential upward migration of fracturing fluid additives, the EPA drilled a couple of monitoring wells near the production wells (with a separating distance of 200–300 m) and sampled them. The two wells “MW01” and “MW02” were screened at intervals of 233–239 m and 296–302 m below ground surface, respectively. In the Pavillion Field, the maximum depth of groundwater use was 322 m (at the time the study was conducted) and some stimulation activities (acid stimulation/hydraulic fracturing) in the area occurred at depths as shallow as 213 and 322 m below ground surface. In the sampling results of the monitoring wells, several constituents of the fracturing fluid and/or their corresponding degradation products were detected, in considerable concentrations. Organic compounds that were detected include naphthalene, alkylbenzenes, diethylene glycol, octylphenol, and trimethylbenzene. These results provide extra credibility to the suggested scenario of the infiltration of fracturing fluid chemical additives into the drinking water sources. Section title: Downhole risks Educational score: 1.7802824974060059 Domain: other Document type: Study Language: en It was possible for Digiulio & Jackson , via examination of previous incidents in the Pavillion field (not enough data is provided by the authors to document previous contamination incidents), to derive that the transport of additives of the fracturing fluid in the Pavillion field was possible due to the occurrence of the following phenomena: (i) casing failure facilitating seepage of the fluid, (ii) fracturing fluid injection into drinking water sources or near them, (iii) fluid leak-off via fractures, and (iv) lithological heterogeneity in the subsurface and the existence of permeable layers allowing fluid migration . Section title: Downhole risks Educational score: 4.02252197265625 Domain: biomedical Document type: Study Language: en Based on facts and discussions analyzed in this section, it must be noted that even when the hydraulic fracturing fluid does not or takes several years to migrate towards the underground drinking water sources, it remains toxic posing a risk to human health, especially through the other existing exposure pathways. Human exposure to fracturing fluid chemicals primarily results from accidental spills and operational accidents. However, these chemicals are not exclusive to fracturing fluids; many are also components of other operational fluids or are, for example, hydrocarbon derivatives . This overlap complicates the attribution of contamination solely to hydraulic fracturing, especially since detected chemicals in environmental samples sometimes consist of complex mixtures of organic compounds, like petroleum distillates . Additionally, there exists a lack of robust pre-injection water quality checks in some regions, which complicates the ability to always demonstrate that the injection caused the contamination. So, to accurately pinpoint and limit contamination directly related to fracturing fluid injection, concentration of additives should not exceed the limits established by regulatory acts involved with monitoring the quality of drinking water quality . In addition, not all the operators disclose fracturing fluid constituents they use, and the toxicity-related information is somewhat limited . This obstructs investigations examining environmental hazards linked to the use of fracturing fluid. Section title: Downhole risks Educational score: 2.2622392177581787 Domain: other Document type: Study Language: en The recognition and examination of environmental risks and health hazards resulting, during and/or after fracturing operations, from the usage of fracturing fluids takes several years . As a result, long time periods must be allocated to track and simulate fracturing fluid transport in the subsurface . Furthermore, low hydraulic fracturing fluid flow-back recovery values that are usually reported [ , , ] prove that a relatively considerable fraction of the fluid is retained in the subsurface with the possibility of infiltrating into underground drinking water sources (e.g. Pavillion field). Consequently, the following section is dedicated to investigating fate of fracturing fluid and phenomena/parameters influencing its flow-back. Section title: Fate of fracturing fluids Educational score: 1.5159653425216675 Domain: other Document type: Other Language: en Flow-back recovery of the fracturing fluid, termed as “load recovery”, is the fraction of the fracturing fluid that is recovered at the surface after the commencement of production. It is given in percentages and the first three months of production usually contribute to the main portion of the load recovery value . According to reported values, load recovery can be relatively low and ranges between 10 and 50 % . Fig. 5 Load recovery values and ranges for shales (where accurate data was available, load recovery values were constrained in a range) (data obtained from Refs. [ 62 , , , , ]). Fig. 5 Section title: Fate of fracturing fluids Educational score: 1.7550592422485352 Domain: other Document type: Other Language: en The low recovery values are noteworthy since contaminants can migrate to the subsurface after production, and several factors control the fluid's migration and, consequently, its fate. This section discusses some of the key phenomena and points out the subsurface complexities that lead to impediments in studying fluid flow and assessing possible environmental impacts. Section title: Fluid migration Educational score: 4.150233268737793 Domain: biomedical Document type: Study Language: en Discrepancy in flow-back rates and ambiguity surrounding the fate of the fracturing fluid have garnered research interest due to the uncertainty in the causes and the risk of aquifer contamination , which is strongly dictated by fluid migration and directly attributed to low flow-back ratios. The associated downhole risks have been previously addressed in sub-section 3.3.2 . Low recovery values are the consequence of hydraulic communication resulting from the intersection between induced hydraulic fractures and permeable pathways previously existing in the formation, like faults and abandoned wells. There is a large number of published studies that investigated the upward flow of HF fluid and that gave, indirectly, insights regarding the fate of HF fluid that is not produced back [ 6 , , , , , , , 97 , 98 ]. For instance, Birdsell et al. conducted a study where they simulated hydraulic fracturing fluid migration post injection into a Marcellus shale reservoir and the results showed that, over a period of 20 years, 68 % of the injected fracturing fluid was extracted by the production well, 0.1 % infiltrated into the overlying aquifer, and the remaining diffused elsewhere in the subsurface. The parameters that are positively correlated to, and control, the speed of fluid migration (presented from the most influential to least influential) are: 1) the proximity of a permeable pathway to HF, 2) permeable pathway permeability, 3) injection pressure, 4) permeable pathway width, 5) overburden heterogeneity, and 6) overburden permeability. As an illustration, the speed of fluid migration is higher in the case of higher proximity between HF region and the fault (i.e. shorter distance), when compared to cases of lower proximity . Section title: Correlations impacting load recovery Educational score: 3.384477376937866 Domain: other Document type: Study Language: en Correlations existing between load recovery and certain parameters (e.g. fracture network complexity, and shut-in period) have become a target of studies discussing fracturing fluid recovery, as illustrated in Table 4 which shows correlations derived specifically from numerical simulation studies. Negative correlation means that an increase in the ‘parameter studied’ yields a decrease in the load recovery, whereas a positive correlation means that an increase in the ‘parameter studied’ yields an increase in the load recovery. Table 4 Correlations between formation and operational parameters and load recovery. Negative correlation means that an increase in the ‘parameter studied’ yields a decrease in the load recovery, whereas a positive correlation means that an increase in the ‘parameter studied’ yields an increase in the load recovery. Table 4 Parameter studied Relation with load recovery References Shut-in period Negatively correlated Natural fracture density Negatively correlated Positively correlated Fracture closure Negatively correlated Positively correlated Rock wettability Hydrophobicity: positively correlated Hydraulic fracture permeability Positively correlated Reservoir pressure Positively correlated Geochemical interactions Negatively correlated Fracture network complexity Negatively correlated Section title: Correlations impacting load recovery Educational score: 2.19744873046875 Domain: other Document type: Study Language: en Some of the correlations presented in Table 4 are expected. For example, a variety of studies have confirmed that extended shut-in periods and increased fracture network complexity promote hydraulic fracturing fluid imbibition into surrounding layers and fluid retention, respectively, leading to a reduction in load recovery. However, other parameters, such as natural fracture density, and fracture closure, exhibit intriguing correlations to load recovery. Section title: Effect of fracture network complexity Educational score: 2.136340379714966 Domain: other Document type: Study Language: en Literature findings support microseismic monitoring observations that demonstrate the development of complex fracture systems as a consequence of hydraulic fracturing . Fracture network complexity in these systems arises from the existing natural fractures that are reactivated, and hydraulic fractures that are induced, and thus generates a broad spectrum of fracture dimensions. This complexity has its adverse effects on both fracturing fluid and hydrocarbon recovery rates . Section title: Effect of fracture network complexity Educational score: 4.0728912353515625 Domain: biomedical Document type: Study Language: en Complex fracture networks developed through multi-stage hydraulic fracturing in tight reservoirs can contribute to low flow-back percentages due to fluid retention in the fracture network and fluid leak-off from the fracture to the matrix . Fluid leakage phenomenon, known as “fluid loss”, is defined as the leakage of the fracturing fluid via the main fracture into the formation. Various numerical models dedicated to fluid leak-off simulation have been developed [ , , , , 105 ], and controlling properties were identified. Fluid leak-off is positively correlated to net fracture pressure, fracture length, natural fracture permeability and density, reservoir temperature and brine salinity. Conversely, reservoir pressure and fracturing fluid viscosity are negatively correlated with fluid leak-off . Section title: Effect of fracture network complexity Educational score: 1.931368350982666 Domain: other Document type: Study Language: en Table 5 shows the load recovery values as a function of the fracture complexity index, as obtained by Ghanbari & Dehghanpour and Liao et al. in their respective simulation studies. Table 5 Load recovery results of two numerical studies as a function of fracture complexity. Table 5 Section title: Effect of fracture network complexity Educational score: 3.9841864109039307 Domain: biomedical Document type: Study Language: en Using flow-back data from 18 wells in the Horn River Basin, Ghanbari & Dehghanpour categorized wells based on flow-back performance and gas production, linking higher fracture complexity to lower water recovery and higher gas production. To backup the basis of their categorization, Ghanbari & Dehghanpour developed a numerical model of a flow-back process. To account for complexity in the model, a fracture complexity index (FCI) controlled by the volume of natural and hydraulic fractures was introduced and showed that increased fracture complexity restricts load recovery while promoting gas production. Liao et al. employed the same FCI equation developed by Ghanbari & Dehghanpour and derived similar conclusions. As anticipated, their results revealed that as the fracture network complexity increases, fracturing fluid load recovery declines. To illustrate, the load recovery decreased from 90 % to 43.9 % as the fracture complexity index increased from 0 to 0.75. However, despite demonstrating the same trend in the relation between fracture complexity and fluid recovery, there is a substantial disparity in load recovery values, with an order of magnitude difference. This discrepancy can be attributed to variations in values of model parameters, such as matrix permeability (0.01 mD in Liao et al.’s study vs. 0.001 mD in Ghanbari & Dehghanpour's study) and hydraulic fracture permeability . Section title: Effect of fracture network complexity Educational score: 3.939854860305786 Domain: biomedical Document type: Study Language: en Effect of fracture network complexity on fluids' flow and recovery can be explained as follows: During injection, the hydraulic fracturing fluid and proppants both fill the created hydraulic fracture while only the fluid flows into the natural fractures. Thus, the hydraulic fracture's aperture becomes greater than the natural fracture's aperture and the production of fluids from the hydraulic fracture becomes easier when compared to that from natural fractures. However, the higher the complexity of the fracture system created, the wider the contact surface area that is between the matrix and fractures. Hence, imbibition amplifies leading to an increase in the fraction of the water that gets imbibed into the shale matrix during shut-in, and displaces accumulated gas in fractures, which in-turn gets produced at higher ease by the production well. This leads to constrained load recovery and enhanced gas production during early production, and vice versa . Section title: Effect of natural fracture density Educational score: 3.071718454360962 Domain: other Document type: Study Language: en Natural fracture density is one of the fracture network characteristics that determines the degree of complexity. The findings for the numerical simulations by Liu et al. contradict other models' that proposed that the increased contact area due to an increase in natural fracture density results in the retention of higher volumes of water in the subsurface. Liu et al.’s results may be considered as a demonstration of the counter-effect of an increased contact area, which could yield a boost in hydraulic communication and hence in water recovery. The apparent contradiction might be due to the different scales and input of these models. For example, natural fracture permeability in Liao et al.’s study is three orders of magnitude higher than the value used in Liu et al.’s study. Section title: Effect of fracture closure Educational score: 4.08705997467041 Domain: biomedical Document type: Study Language: en Another noteworthy feature is the fracture closure. Fracture closure hinders the flow of a fraction of the fracturing fluid from the fractures back to the well, thereby increasing retained volume . While some studies support that accounting for fracture compressibility variations leads to lower load recovery , the study done by Seales et al. shows that fracture closure mechanisms might actually “squeeze” the water out of the saturated fractures and enhance early flow-back. This divergence in findings could stem from differing modeling assumptions. In the studies by Liu et al. and Liao et al. , fracture permeability varied with factors like fracture closure and pressure changes, whereas Seales et al.’s model assumed it to be constant. Section title: Effect of fracture closure Educational score: 3.452648162841797 Domain: other Document type: Study Language: en In conclusion, while fracture network complexity generally leads to lower fluid recovery and higher hydrocarbon production, the specific outcomes depend on a range of factors, including fracture closure properties and the interactions between natural and induced fractures. These complexities highlight the challenges in accurately predicting well performance and fluid flow-back. Additionally, in complex geological settings with local heterogeneities, in-situ stress fields, spatial inconsistency in rock mass properties, and pre-existing bedding planes and fault systems, hydraulic fracturing operations become more challenging. These factors, coupled with the complexities in fracture network characterization, highlight the difficulties in accurately predicting flow-back and hydrocarbon recovery . Section title: Induced seismicity and load recovery correlation Educational score: 3.5903098583221436 Domain: other Document type: Study Language: en In studies examining seismicity referred to hydraulic fracturing activities, the primary focus is often on seismic events arising from wastewater injection . This process involves the disposal of flowback water, which consists of the original fracturing fluid (i.e. water and chemical additives) along with naturally present constituents from the formation, into designated wells . This emphasis is due to the fact that earthquakes triggered by wastewater injection generally exhibit higher magnitudes than those directly induced by hydraulic fracturing, with recorded events reaching magnitudes of 5.6 , 5.8 , and even 5.9 . These elevated magnitudes are typically ascribed to the substantial volumes of injected wastewater and the resultant pore pressure increases, which can stimulate seismic activity . Nonetheless, induced earthquakes recorded in areas near hydraulic fracturing sites can be attributed to hydraulic fracturing processes [ , , , , 44 , 45 , 87 , , , , , , , , ], especially under specific hydrogeological conditions . A spatial and temporal link has been developed in regions like the Western Canada Sedimentary Basin (WCSB) [ , , ] and the Sichuan Basin, China . This association is supported by data from well monitoring and the compilation of operational data and seismic activity into catalogs . An example of such data is the one compiled by Verdon & Rodriguez-Pradilla (see Table 6 ), showcasing seismic activity related to fracturing in 12 major shale plays in the Western Canada Sedimentary Basin (WCSB) and the United States using data from regulators (i.e., Alberta Energy Regulator and the British Columbia Oil and Gas Commission) and FracFocus ( www.fracfocus.org ), respectively. Table 6 Hydraulic fracturing-induced seismic activity in 12 major shale plays (adapted from ). Table 6 Play Number of wells Total HF volume (x 10 6 m 3 ) Number of associated earthquakes Largest event magnitude Number of associated wells Bakken 15,375 288 1 3.3 3 Barnett 6427 45 2 2.9 3 Duvernay 1946 44 569 4.1 145 Eagle Ford 15,825 407 389 3.7 731 Fayetteville 2863 28 421 3.9 336 Haynesville 3650 208 18 3.8 9 Horn River 344 14 30 3.6 25 Marcellus 11,530 456 2 2.8 2 Montney 8281 80 388 4.4 603 Niobrara 17,314 367 9 2.6 45 Utica 3146 157 37 3.8 24 Woodford 4424 198 794 3.6 584 Section title: Induced seismicity and load recovery correlation Educational score: 1.7103002071380615 Domain: other Document type: Study Language: en Although no formula can be derived to link number of wells or any other parameter to the magnitude or occurrence of seismic events, this kind of data is highly useful for studying the relationship between hydraulic fracturing and induced seismicity. Verdon & Rodriguez-Pradilla specifically acknowledge that not all earthquakes are caused by hydraulic fracturing, as some may result from natural causes or other industrial activities. To improve data accuracy and ensure that the majority of the listed events are induced by hydraulic fracturing, they applied the following criteria: (i) Used a 10 km distance from the operation site. (ii) Counted seismic events with magnitude ≥2.0 from the beginning of operations to 10 days after they concluded. (iii) Compared seismic activity during operations to the baseline activity observed in the area 6 months before and 6 months after the operational period. Section title: Induced seismicity and load recovery correlation Educational score: 3.738049268722534 Domain: other Document type: Study Language: en One of the fundamental processes responsible for fluid induced seismicity is determined to be fluid diffusion, succeeding fluid injection. This diffusion is enabled due to the hydraulic communication established between hydraulic fractures and faults, allowing the fracturing fluid to reach existing faults. Hence, pre-existing faults are reactivated due to pore pressure increase during or after hydraulic fracturing that is enough to trigger earthquakes . Fluid pressurization and injection have become key research attractions due to their role in fault reactivation . He & Li used the data presented in Table 7 and conducted a simulation study that demonstrated that amplified fluid injection increases the probability of fault reactivation. As per the data presented by Wu et al. in their review paper (see Table 7 ), we can highlight that fluid injection in any unconventional energy project has the potential to induce seismicity, which broadens the scope of research concerned with the injection and fate of the fracturing fluid. Table 7 Data showing the link between flow back and HF and induced seismicity. Table 7 Reference Presented Data Treatment stage Flow-back after the stage Seismicity associated with the stage II No flow-back First seismic event III Aggressive flow-back Weak seismicity IV No flow-back Significant seismicity V Aggressive flow-back Weak seismicity Overall flow-back 20 % Location Reason Significant recorded magnitude Pohang, South Korea Geothermal reservoir stimulation MW = 5.5 Sichuan, China Shale gas production MW = 4.7 Oklahoma, United States Wastewater disposal MW > 4.0 Data Value Five earthquakes recorded during fracturing MW > 4.0 Magnitude of largest recorded event MW = 4.7 Distance separating the source of seismicity and injection point 2–3 km Magnitude of largest recorded event MW = 3.9 Time of occurrence of largest recorded event Two weeks after fracturing completion Distance separating clusters from fractured wells 2 km Flow-back 7 % Period between start of HF and first recorded seismic event 5 days Period between start of HF and largest recorded seismic event 3 months Magnitude of largest recorded event MW = 2.2 Flow-back 40 % Section title: Induced seismicity and load recovery correlation Educational score: 3.9482967853546143 Domain: other Document type: Study Language: en According to Schultz et al. , the injected fluid volume is the dominating operational parameter that is directly positively correlated to both the number of seismic events recorded and their magnitude. Although this behavior is expected, a fluid injection over a long period also boosts fluid seepage into surrounding formation , which in turn affects flow-back recovery. In their report done to study induced seismicity in the Bowland Shale in the UK, De Pater & Baisch represented a plausible relation between fracturing flow-back and induced seismicity. Table 7 illustrates the degree of seismicity associated with each stage of hydraulic fracturing as a function of the well flow-back. They suggested that the pattern of seismicity shown is the result of flow-back routines, where strong seismicity is associated with low fluid flow-back volumes, and the fact that only about 20 % of the injected fluid was recovered in this operation supports their observations. Hence, they recommended aggressive fluid flow-back and reduced injected volumes as possible seismicity mitigation practices. Likewise, Bao & Eaton compiled data of a four-month period for six hydraulic fracturing well pads in the Duvernay formation, and the most interesting data are presented in Table 7 . Moreover, Tan et al. used available injection data from two well pads, named “N5” and “N7”, in Sichuan Basin, as a basis for their study. Data derived from pad N5 shows the time period separating the onset of fracturing activities and seismic events occurrence ( Table 7 ), and the flow-back ratio. Values synthesized in Table 7 prove the suggested link between hydraulic fracturing and induced seismicity as per the discussion above, and provide evidence that the earthquake distributions are spatially concentrated nearby hydraulic fracturing well pads and follow chronologically the timing of stimulation activities by few days or weeks. Section title: Induced seismicity and load recovery correlation Educational score: 3.6928632259368896 Domain: other Document type: Study Language: en Although conclusions discussed in this section might be considered redundant, they act as support to the hypothesis that connects induced seismicity to low fracturing fluid flow-back recovery since authors present fluid diffusion as one of the major phenomena contributing to induced seismicity in the proximity of hydraulic fracturing locations. For instance, Tan et al. and Bao & Eaton reported load recovery values in cases where fluid diffusion was believed to be the main trigger behind seismic events (i.e., earthquakes), and these values show that more than 50 % and 90 %, respectively, of the injected fluid remained trapped in the subsurface in these cases. Nevertheless, it is worth highlighting that the magnitude of the events is a result of a combination of factors including the injection pressure, cumulative injected volumes, position of created fractures with respect to existing faults, and hydraulic communication between the fractures and these faults which is also discussed in Yehya et al. . Section title: Emerging technologies in fracturing fluids Educational score: 2.031400442123413 Domain: other Document type: Other Language: en Recent advancements in hydraulic fracturing technology aim to develop environmentally friendly fluids to reduce toxicity and environmental impact [ , , ], while also addressing technical challenges associated with conventional fracturing fluids . This shift involves the transition from traditional fluids, such as water- and oil-based systems, to unconventional alternatives like viscoelastic surfactant (VES) fluids, liquefied petroleum gas (LPG)-based fluids, and CO₂-based fluids . These new formulations aim to minimize issues like permeability reduction, pore clogging, and formation damage caused by residues left by polymer-based fracturing fluids . Section title: Emerging technologies in fracturing fluids Educational score: 2.53000545501709 Domain: biomedical Document type: Other Language: en In particular, viscoelastic surfactant (VES) fluids are gaining attention for their capacity to act as clean fluids, leaving minimal residue and providing enhanced environmental performance . VES fluids address common limitations of polymer-based fluids by eliminating insoluble residues, reducing friction, and limiting formation damage . Section title: Emerging technologies in fracturing fluids Educational score: 3.7856314182281494 Domain: biomedical Document type: Study Language: en To further improve environmental safety and performance, researchers are exploring the use of alternative additives in these fluids. These include biodegradable and green surfactants, nanocomposite friction reducers, hydrogel additives, and amino acid-based surfactants, each offering enhanced biodegradability and lower ecological impact . By incorporating such components, fracturing fluids are engineered to deliver higher lubricity, increased temperature resistance, and better proppant transport, thus supporting efficient hydraulic fracturing with reduced environmental risks . Section title: Emerging technologies in fracturing fluids Educational score: 1.1873822212219238 Domain: other Document type: Other Language: en Overall, these innovations towards green alternatives for fracturing fluid reflect a growing emphasis on sustainability in hydraulic fracturing, balancing improved operational performance with reduced environmental footprint. However, parallel to this progress, equal efforts should be dedicated to evaluating and addressing potential environmental risks associated with these emerging alternatives. Section title: Conclusions and outlook Educational score: 1.4342925548553467 Domain: other Document type: Other Language: en The public, in general, and environmentalists, in specific, are consistently raising concerns regarding risks associated with the rapid expansion of hydraulic fracturing treatments worldwide. Hydraulic fracturing fluid poses environmental risks that can be classified into surface risks (e.g. spills, felt seismic events), and subsurface risks (e.g. groundwater contamination). Also, more than half of the injected volume of fracturing fluid is not produced back to the surface, and the higher the percentage of fracturing fluid that remains in the subsurface, the higher are the risks associated with it. Section title: Conclusions and outlook Educational score: 3.5812461376190186 Domain: other Document type: Study Language: en In this work, the main objective is not to discuss the risks related to all the stages of a hydraulic fracturing job, but to discuss the environmental impact of the fracturing fluid. First, we discuss the environmental and health risks associated with HF fluid and its implications on drinking water and highlight reported cases of contamination. Then, we inspect available field data on flow-back recovery rates and load recoveries of the HF fluid, and summarize the parameters and hydrogeological conditions controlling the values of recovery. Hence, we show how HF fluid recovery correlates with the fluid's upward migration through permeable pathways, retention in fracture networks, leak-off into the formation, and induced seismicity. Section title: Conclusions and outlook Educational score: 4.130630970001221 Domain: biomedical Document type: Study Language: en Main conclusions derived from the literature can be summarized as follows: 1. The high number of toxic chemicals added to the HF fluid and the limited disclosure of toxicity data highlight the importance of addressing the surface and subsurface risks of HF fluid. 2. Load recovery is correlated to fracture properties (e.g. natural fracture density) and operational parameters such as shut-in period. Most evidently long shut-in period leads to low hydraulic fluid recovery values. 3. Fluid migration in the subsurface via a permeable pathway, which contributes to low recovery and aquifer contamination, is mostly sensitive to properties of the existing permeable pathway. For instance, a strong positive correlation exists between speed of fluid migration and permeability of the pathway. 4. Fluid leak-off from the fracture to the matrix is considered to be a strong contributor to the low load recovery values due to the retention of the HF fluid. 5. Higher fracture network complexity boosts oil/gas recovery and curbs water recovery. 6. Field evidence support the hypothesis suggesting a negative correlation between induced seismicity in the vicinity of fracturing operations and load recovery; which is explained by fluid diffusion into fractured regions. Section title: Conclusions and outlook Educational score: 1.847448468208313 Domain: other Document type: Other Language: en Environmental impact of the HF fluid content and fate (e.g., migration) of the HF fluid are interrelated and must be evaluated simultaneously to assess related risks, and therefore be able to make decisions regarding hydraulic fracturing operations’ design. Accordingly, when assessing environmental risks, it is essential to examine the underlying causes of low recovery rates and the parameters and phenomena that influence fluid transport and recovery. Recent literature stresses on the need for a comprehensive investigation into the causes of increased seismicity proximal to hydraulic fracturing sites, as hydraulic fracturing is employed not only in shale development but also in renewable energy projects. Section title: Conclusions and outlook Educational score: 3.98534893989563 Domain: biomedical Document type: Study Language: en Further research is essential to specifically address the environmental impacts of fracturing fluids as complex chemical mixtures, independent of the hydraulic fracturing processes. Although existing studies provide insight into potential environmental risks, the majority concentrate on the operational aspects of hydraulic fracturing rather than the toxicity and long-term environmental effects of the fluid's chemical constituents. Comprehensive risk assessment requires more extensive chemical analysis of fracturing fluids, as well as toxicological and epidemiological data that quantify exposure risks to humans and animals. Also, detailed exposure assessments are necessary to understand the pathways through which these chemicals affect biological systems. This will assist in the development of appropriate safety precautions and disclosure mandates that reduce the harm. Section title: Conclusions and outlook Educational score: 1.3184553384780884 Domain: other Document type: Other Language: en Furthermore, implications on groundwater contamination might take tens of years to be detected, so, serious efforts should be put in place to gain more access to data needed to develop risk mitigation plans that take into consideration the timescales associated with the conditions and mechanisms of fluid migration in the subsurface. Section title: Conclusions and outlook Educational score: 1.2143447399139404 Domain: other Document type: Other Language: en Finally, while innovative approaches to create more sustainable fracturing fluid alternatives are promising, equal attention must be dedicated to a thorough evaluation of their environmental impacts to ensure they meet both safety and sustainability standards while achieving the desired fracturing outcomes. Section title: CRediT authorship contribution statement Educational score: 1.0028690099716187 Domain: other Document type: Other Language: en Sara Makki: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Elsa Maalouf: Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. Alissar Yehya: Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. Section title: Data availability statement Educational score: 0.8947381377220154 Domain: other Document type: Other Language: en The data that support the findings of this study are available on request from the corresponding author. Section title: Funding statement Educational score: 0.960715115070343 Domain: other Document type: Other Language: en This research is funded by the University Research Board at the 10.13039/100007688 American University of Beirut, Awards # 104392 and # 104260 . Section title: Declaration of competing interest Educational score: 0.9818207025527954 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Study | other | en | 0.999998 |
PMC11699343 | Section title: Introduction Educational score: 4.626765251159668 Domain: biomedical Document type: Review Language: en Flavor is a critical quality attribute in food and is a primary driver for consumer acceptance and food purchasing decisions. The development of flavor in food is a complex process, involving numerous chemical molecules, including volatiles, non-volatiles and high-molecular weight compounds . The sensation of flavor results from simultaneous stimulation of our chemical senses, mainly odor and taste, triggered by these chemicals originally present in raw food material or generated during food processing and storage. Traditional flavor research focused on identifying key odor-active and taste-active chemicals in food, which are less than a few hundred compounds . Advances in analytical techniques and artificial intelligence (AI), which can handle large datasets, have led recent flavor research toward a more systemic assessment of flavor-related chemicals. This includes the measurement of compounds that are tasteless and odorless but impact flavor perception (e.g., flavor enhancers) and compounds that interact with other molecules to modify flavor profile . Studies on flavor precursors and flavor synthetic pathways have also increasingly been introduced in fresh fruits and vegetables, as well as processed food products . With these trends, a new omics subdiscipline named ‘flavoromics’ was born in 2008 and has addressed various challenges in food flavor research . Flavoromics combines analytical chemistry, sensory evaluation, and data science to comprehensively understand the relationships between chemical compositions and flavor traits in food. To characterize a wide range of compounds, including unknown molecules that might affect flavor formation and regulation, flavoromics often employs untargeted chemical analysis using analytical techniques such as gas chromatography–ion mobility spectrometry (GC–IMS), gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–high-resolution mass spectrometry (LC–HRMS) . A similar term, sensomics, uses a combination of sensory evaluation and instrumental analysis, like flavoromics, while sensomics concentrates on the identification and quantification of aroma-active compounds at the molecular level, which is assisted by experiments such as aroma extract dilution analysis (AEDA) and aroma recombination and omission . Multi-omics, the integration of more than one omics approach (e.g., metabolomics with transcriptomics), is another emerging trend to understand in-depth biochemical mechanisms and regulatory processes behind the flavor phenotypes of food across different biological layers . Section title: Introduction Educational score: 1.363083839416504 Domain: biomedical Document type: Other Language: en In this special issue, a total of 71 articles regarding the modern flavor chemistry of food were collected between June 2023 and April 2024. Section title: Flavor chemistry in raw agricultural commodities Educational score: 4.16636848449707 Domain: biomedical Document type: Study Language: en Sugars and organic acids are significant chemical components in fresh fruits, contributing to their balanced sweetness and sourness. The total soluble solids to titratable acidity (TSS/TA) ratio is commonly used to assess the flavor quality and ripeness of fruits . However, efforts have been made to measure individual sugars and organic acids for a better understanding of fruit flavor, because each compound may have a different taste activity value (TAV), and their contents and ratios vary among fruits. Sugars, acids, and their proportions and distribution were examined in a cherry species ( Prunus pseudocerasus ) by high-performance liquid chromatography–ultraviolet/refractive index detection (HPLC–UV/RID) . With the support of sensory evaluation and the TSS/TA ratio, key indicators of cherry flavor, including glucose, fructose, and maltose were identified, and new cherry grading criteria were proposed. Liu, Song, et al. explored alterations in flavor-related compounds in yellow and white-fleshed loquats ( Eriobotrya japonica ) using HPLC–UV/RID and LC–MS-based metabolomics approach. Differentially accumulated metabolites, including sugars and organic acids (e.g., malic acid), were found in loquats of different colors and at different development stages. Section title: Flavor chemistry in raw agricultural commodities Educational score: 4.193579196929932 Domain: biomedical Document type: Study Language: en Interestingly, one collected article used a targeted metabolomics strategy, and reported the identification of ethyl vanillin, a ‘synthetic’ vanilla flavoring compound, in strawberry ( Fragaria × ananassa ) . It was the first time that ethyl vanillin had been observed in natural food. The presence of ethyl vanillin in strawberry was thoroughly confirmed by multiple analytical techniques including GC–MS/MS, LC–HRMS and LC–MS/MS with the use of isotope-labeled ethyl vanillin and a global metabolite library (NIST). The same research group published another article regarding the discovery of a novel bitter-masking compound in allspice ( Pimenta dioica ) using sensory-guided isolation . The structures of the isolated molecules were determined by nuclear magnetic resonance (NMR) and LC–HRMS, and one molecule exhibited promising bitter masking activity against quinine, as determined by sensory evaluation. Molecular docking analysis indicated that the compound could act as an antagonist of one of the bitter receptors, TAS2R14. Section title: Flavor chemistry in raw agricultural commodities Educational score: 4.088637828826904 Domain: biomedical Document type: Study Language: en In addition to the above topics, the flavor qualities of buckwheat ( Fagopyrum spp.) , pear ( Pyrus spp.) , wampee ( Clausena lansium ) , vanilla beans ( Vanilla planifolia ) and pepper seeds ( Capsicum annuum ) were assessed by characterizing flavor-related compounds using gas chromatography with flame ionization detection (GC–FID), GC–MS, GC–IMS, HPLC–UV/RID and/or LC–MS/MS, followed by multivariate statistical analysis. One study employed widely targeted metabolomics and annotated nearly 1000 metabolites in pear . The relationship between metabolites and sensory attributes was uncovered through statistical correlation network analysis, and key sensory-related metabolites in pear were identified based on gene significance and module memberships. Another study examined flavor precursors and downstream aroma volatiles in pepper seeds, demonstrating that certain fatty acids and amino acids were highly correlated with the formation of pepper aroma . Section title: Flavor chemistry in processed foods Educational score: 1.8909423351287842 Domain: biomedical Document type: Other Language: en Fifty-five out of the 71 articles in this special issue studied flavor chemistry in processed food products, including marinated and stewed beef , cooked pork , sausages , chicken soup , porridge , plant-based meat , sauerkraut , coffee , yogurt , tea , wine , soda , a fruit by-product , and processed oil . Section title: Flavor chemistry in processed foods Educational score: 4.101799964904785 Domain: biomedical Document type: Study Language: en For solid processed foods, key odorants associated with the warmed-over flavor of stewed beef after refrigeration and reheating were characterized using a sensomics approach, which combined sensory evaluation with GC–IMS/MS analysis . Shao et al. explored the effect of different starter cultures on the microbial communities and flavor compounds of fermented sausages using 16S rRNA gene sequencing and chemical analysis, demonstrating that a mixed starter enhanced the flavor of fermented sausages by generating a high level of umami taste-related compounds. With growing attention on plant-based foods, one study utilized a soybean by-product as an additive for the production of plant-based patties and assessed the quality of the patties via physicochemical measurements and GC–MS analysis . The developed patties showed increased water-holding capacity, improved texture profiles and lower levels of undesirable flavor volatiles (e.g., benzaldehyde, nonanal, 2-heptanone) compared to control patties without the soybean by-product. Luo et al. introduced a novel fermented food using a pineapple by-product and whey protein. The effect of fermentation on the product quality was clarified by measuring changes in carbohydrate and amino acid profiles, organoleptic properties, and the microbial community of the product. Section title: Flavor chemistry in processed foods Educational score: 4.119634628295898 Domain: biomedical Document type: Study Language: en For liquid processed foods, the influence of roasting conditions on the physicochemical properties and flavor quality of coffee ( Coffea arabica ) was assessed by analyzing caffeine, chlorogenic acid, total flavonoid content, antioxidant capacity, flavor, and flavor compounds using various instruments such as HPLC–UV, electronic tongue, electronic nose, and GC–MS-olfactometry (GC–MS-O) . Ouyang et al. employed untargeted volatile analysis using two-dimensional gas chromatography-olfactometry–quadrupole-time-of-flight mass spectrometry (GC × GC-O–QTOF-MS) to exhaustively characterize the odorants of black tea from different provinces in China. A total of 190 volatiles were identified in the tea, and among them, 23 compounds were confirmed as key odorants contributing to the distinct aromas of black tea in different regions. In another study, the aroma profiles of Italian wines were determined by GC–IMS during spontaneous and inoculated fermentation processes, demonstrating that spontaneous fermentation effectively enhanced the aroma characteristics of wine, with increased levels of corresponding volatiles, showing its aging potential . Barba et al. examined the effect of sugar alcohols on the release of aroma compounds in soda beverages using GC–MS and NMR, which may help guide the use of sugar alcohols and aroma volatiles in the formulation of sugar-free or reduced-sugar beverages. Additionally, a new sample preparation technique, called dispersive liquid-liquid microextraction, was introduced and applied to the determination of toxic α-dicarbonyl compounds, such as glyoxal and methylglyoxal, in sesame oil, which has potential for use in the quality and safety control of oil . Section title: Application of multi-omics in food flavor Educational score: 4.078056335449219 Domain: biomedical Document type: Review Language: en Several studies in this issue utilized multi-omics strategies to support and confirm their hypotheses across different biological layers in food products, such as tea , cheese and fermented foods . An integrated metabolomics-transcriptomics approach was employed in oolong tea research, revealing dynamic changes in metabolites and flavor formation during tea manufacturing process . Differentially expressed metabolites and genes were associated, and central metabolic pathways and network (sugar, amino acid and flavonoid metabolism) altered during different tea processing steps were identified. Another study examined variations in metabolite profiles and bacterial communities in Liupao tea during tea processing using a joint metabolomic and metagenomic analysis . The two omics datasets were correlated, and the results indicated that metabolic changes in Liupao tea were not primarily derived from bacterial activities, but rather from other processing conditions, such as high temperature and humidity. A combination of metabolomics and metagenomics was also used in yak cheese , and fermented dairy and vegetable products to elucidate the fundamental mechanisms behind the flavor formation of these products in different contexts. Section title: Concluding remarks Educational score: 3.516810178756714 Domain: biomedical Document type: Other Language: en Chemicals in food are the end products of cellular signaling processes in raw foods or products generated during processing and storage in processed foods. These chemicals directly reflect food phenotypes, such as flavor, which is linked to food quality, safety and sensory properties. This special issue presents recent advances in the flavor chemistry of food and introduces the most up-to-date technologies and approaches for food flavor research. However, challenges remain in the field, as flavor is a complex trait influenced by numerous compounds, their levels, compositions, interactions and reactions. Future advancements in analytical techniques, along with the application of AI technologies such as machine learning and deep learning algorithms, are expected to enhance the measurement and assessment of food flavor from large and complex datasets across various research topics. Finally, the guest editors sincerely thank the authors, reviewers, editors, editorial office, and all those who contributed to this special issue. Section title: Declaration of competing interest Educational score: 0.9818206429481506 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Review | biomedical | en | 0.999994 |
PMC11699348 | Section title: Introduction Educational score: 4.4384565353393555 Domain: biomedical Document type: Study Language: en Low back pain (LBP) presents a remarkably prevalent affliction and has been the leading cause of years lived with disability (YLDs) . In 2020, an estimated 619 million people worldwide were affected by low back pain, and the number is estimated to be 843 million (759–933) by 2050 . Various elements including vertebrae, sacroiliac joint, intervertebral disc, neurovascular structure, and soft tissue can alone or jointly result in low back pain . Intervertebral disc degeneration (IVDD), which contributes to spinal canal stenosis, spondylolisthesis, and deformity, has been identified as one of the major causes of LBP . Notably, annual economic costs related to IVDD exceed US$100 billion in the United States (see Table 1 , Table 2 , Table 3 , Table 4 , Table 5 ). Table 1 Applications of chondroitin sulfate-based biomaterial in IVDD regeneration and repair. Table 1 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Hyaluronic acid (HA), tetra poly (ethylene glycol) with maleimide end groups (Teran-PEG-MAL), and chondroitin sulfate (CS) Ex situ solution penetration In vitro, in vivo (rat) Injectable anti-inflammatory hydrogel, with Diels-Alder reaction-mediated sequential crosslinking enhanced mechanical property and pH-responsive release function Chondroitin sulfate, poly (ethylene glycol)- b -poly (trimethylene carbonate)-acrylate (PEG- b -PTMC-Ac) Bovine NPCs Ex situ solution penetration In vitro In situ forming, mechanically resilient hydrogel, with high NPCs viability and ECM production. poly (N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS), alginate microparticles Growth differentiation factor 6 (GDF-6) Solution mix Ex vivo, in vitro In situ forming hydrogel, enhanced initial injectability, bioadhesiveness, and mechanical performance. Decellularised extracellular matrix of bovine coccygeal discs functionalized with chondroitin sulfate Solution mix In vitro Injectable, self-assembled biomimetic hydrogel, boosting sGAG production and preserving rounded cell morphology of nasal chondrocytes Table 2 Applications of hyaluronic acid-based biomaterial in IVDD regeneration and repair. Table 2 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Hyaluronic acid-1,4-butanediol diglycidyl ether (HA-BDDE), HA-poly (N-isopropylacrylamide) (HA-pNIPAM) NP cells In situ penetrating In vitro, ex vivo (bovine IVD) Support high cell viability, facilitate ECM production, and withstand confined cyclic axial compression. Type Ⅰ collagen, tyramine-substituted hyaluronic acid BM-MSCs Solution mix In vitro Promote NP ECM biopolymers, increase hydration degree, and ensure cell adhesion and survival. Fibrin, collagen, and hyaluronic acid Solution mix In vitro Maintain cell viability, and increase the sGAG to collagen ratio. Hyaluronic acid, zirconium oxide Nanoparticles addition into solution In vivo (goat) and ex vivo (rabbit) Real-time visualization and monitoring, support disc height and promote anabolism of IVD Polyethylene glycol, hyaluronic acid, PPS Mesenchymal precursor cells Ex situ penetrating In vitro Injectable, enzymatically-crosslinked hydrogel, enhance MPCs differentiation to NP-like tissue. HA and poly (N-isopropylacrylamide) (pNIPAM) Stromal cell-derived factor 1 (SDF-1) Direct amidation reaction Ex vivo (bovine IVD) Increase both the number of recruited cells and their migration distance within the intervertebral disc (IVD) tissue. Collagen, HA, and gelatin microspheres Transforming growth factorβ3 (TGF-β3) Ex situ penetration In vitro and in vivo (mouse) possess superior viscoelastic properties and injectability, support MSC and NC differentiation HA, butanediol diglycidyl ether In situ crosslinking In vitro and in vivo (rabbit) Injectable, restore the disc hydration and function Aldehyde hyaluronic acid, poly (amidoamine) siRNA Solution mix In vitro and in vivo (rat) Transport siSTING and silence STING signaling pathway in NPC, alleviate IVD inflammation and degeneration Methacrylated hyaluronic acid microspheres circSTC2 silencing gene vectors Microfluidics In vitro and in vivo (rat) Silence circSTC2 expression in NPCs to maintain the ECM metabolism balance in the nutrient-restricted microenvironment and hinder IVDD Oxidized and methacrylated glycosaminoglycan, fibrin, and poly (ethylene glycol) diacrylate (PEGDA) In situ solution penetration In vitro and ex vivo (bovine) Injectable two-part hydrogel with high adhesion strength, low cytotoxicity, and mechanical compliance Thiolated HA and PEG vinylsulfone Ex situ penetration In vitro HA with lower molecular weight is advantageous for maximizing cell viability and sGAG synthesis. Hyaluronic acid, collagen In situ crosslinking Ex vivo (rat) Restore disc hydration and demonstrate superior instantaneous and equilibrium moduli Table 3 Applications of cellulose-based biomaterial in IVDD regeneration and repair. Table 3 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Carboxymethylcellulose Esterification of hydroxyl groups In vitro Injectable, photocrosslinked, and water-soluble hydrogel, with NP cells viable and mechanical integrity Carboxymethylcellulose (CMC) and methylcellulose (MC) Esterification of hydroxyl groups In vitro, ex vivo (bovine caudal IVDs), in vivo (rat) Thermoresponsive, tunable, biocompatible hydrogel with long-term structurally stable Carboxymethylcellulose Human mesenchymal stem cells (hMSCs) Esterification of hydroxyl groups In vitro Photocrosslinked and biocompatible hydrogel, NP ECM elaboration, improved mechanical properties [ , , ] Chitosan carboxymethyl cellulose Adipose-derived stem cells (ASCs) Ex situ solution penetration In vivo (ovine), in vitro Long-term stabilization of injectable composite hydrogel in degenerated IVDs in a large animal model Poly (ethylene glycol) dimethacrylate and nano-fibrillated cellulose Ex situ solution penetration Ex vivo (bovine IVDs) In situ injection and photopolymerization, restore IVD height and maintain after 0.5 million loading cycles Methacrylated gellan-gum reinforced with cellulose nanocrystals Solution mix In vitro Injectable composite hydrogel with improved scaffold stiffness that augments matrix entanglement Cellulose-alginate double network hydrogel-based annulus fibrosus and cellulose hydrogel-based nucleus pulposus Growth differentiation factor-5, SKP peptide (MSC homing peptide) and RGD peptide (cell adhesion peptide) In situ addition In vitro and in vivo (rats) Recruit and induce MSCs differentiation to NP-like phenotype, has high compressibility, shape memory property and comparable mechanical strength Cellulose nanofiber-based AF and type Ⅱ collagen-based NP Rat NPCs and AFCs Bioprinted bacterial cellulose by the fermentation of Acetobacter xylinum on a micropatterned template In vitro and in vivo (rats) Mimic native IVD and manifest distinguished structural and functional performance Table 4 Applications of alginate-based biomaterial in IVDD regeneration and repair. Table 4 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Alginate, collagen-cryogel, hyaluronic acid Froze and lyophilize alginate and collagen, crosslinked with EDC/NHS In vivo (rat) Ameliorate mechanical allodynia, preserve IVD hydration, and maintain the structure integrity of the disc. Alginate In situ crosslink by calcium carbonate and glucono-δ-lactone In vitro, ex vivo (bovine caudal IVDs) Reestablish function by reducing height loss during long-term cyclic loading and remains contained within the disc with no leakage at the injection site Ultra-purified alginate BMSCs, bone marrow aspirate concentrate (BMAC) In situ gelling In vitro, in vivo (rabbits) Hinder IVD degeneration in histological grade and promote the repair of IVD defects. Alginate beads Human NPCs, Wharton's jelly MSC-derived extracellular vesicles Solution mix In vitro Preserve cell viability, promote ECM deposition while inhibiting inflammation. Alginate, poly (ethylene glycol) diacrylate (PEGDA) microcryogels Mesenchymal stem cells (MSCs) Ex vivo solution penetration In vitro, in vivo (dog), ex vivo (canine IVD) Injectable hydrogel with enhanced elasticity, alleviate IVDD in an ex vivo organ culture model and an in vivo canine model. Alginate Syndecan-binding peptides (AG73), integrin-binding peptides (cyclic RGD) Strain Promoted Azide Alkyne Cycloaddition (SPAAC) click chemistry, maleimide-thiol click chemistry, solution mix. In vitro cRGD and AG73 loaded alginate hydrogel, contribute to higher cell viability, adhesion, biosynthetic property, and NPC phenotype expression. Alginate, poly (N-isopropylacrylamide) (PNIPAAm), silicate ceramics NPCs Solution mix, in situ crosslinking In vitro, in vivo (rat) Thermosensitive, immunoregulatory, promote matrix synthesis. Mesoporous bioactive glasses, sodium alginate melatonin Solution mix In vitro, in vivo (rat) Possess favorable physical and mechanical properties, and alleviate IL-1β triggered oxidative stress and inflammation. Ultra-purified alginate Human IVD cells Solution dilution In vitro, in vivo (sheep and rabbit) Reduced cytotoxicity, favorable biomechanical property, and anabolism enhanced phenotype. Ultra-purified alginate Rabbit BMSCs Solution dilution In vitro, in vivo (rabbit) Upregulate ECM and phenotypic marker, augment IVD regeneration. Ultra-purified alginate Rapidly expanding clones of human MSCs Solution mixture In vivo (rat) Suppress inflammatory cytokine production, reduce nociceptive behavior of rats. Sodium alginate, gelatin Antagomir-204–3p Solution mix In vitro, in vivo ( Improve IVD mechanical property, sustain hydration, and preserve structural integrity. Oxidized alginate microbeads, fibrin RGD peptides, AFCs Genipin-crosslinking, ex situ solution penetration In vitro, ex vivo (bovine IVD) Preserve high cell viability, inhibit AFCs apoptosis, reduce herniation risk in bovine caudal IVD organ for a long-term. Alginate IL-1 receptor antagonist (IL-1Ra), soluble tumor necrosis factor receptor-1 (sTNFR1) Cell encapsulation in solution In vitro, ex vivo (human cadaveric IVD) Inhibit IL-1β and TNF expression, promote proteoglycan and collagen production Alginate, poly (ε-caprolactone) microfibers AFCs and NPCs Ex situ fabrication In vitro, in vivo (rat) Enhance ECM deposition and mechanical properties. Table 5 Applications of chitosan-based biomaterial in IVDD regeneration and repair. Table 5 Material Biological factor, agent or cell Preparation method Type of study Function and advantage Ref Chitosan-glycerophophate NP cells Solution mix In vitro Thermosensitive and biocompatible hydrogel that retains proteoglycan of NP cells Chitosan-glycerophosphate Mesenchyme stem cells Solution mix In vitro Temperature-sensitive hydrogel, capable of inducing proteoglycans and collagens production of MSCs Chitosan-glycerophosphate (SHC0.075BGP0.1) NP cells Solution mix In vitro, ex vivo (human cadaveric IVDs) Similar mechanical properties to the human NP tissue, induction of highest GAG N -hexanoyl glycol chitosan N -hexanoylation reaction of glycol chitosan In vitro, in vivo (rat), ex vivo (pig) Thermos-sensitive injectable hydrogel with no cytotoxicity and no adverse effects in a rat model, maintaining the stability of defective porcine IVD for 28 days. Chitosan and poly-(γ-glutamic acid nanoparticles diclofenac Ex situ solution penetration In vitro, in vivo (rat) Ch/Df/γ-PGA NPs inhibits pro-inflammatory mediator production and reverse ECM synthesis. [ , , ] Methacrylate chitosan and aldehyde polyethylene glycol Solution mix, Schiff base reaction In vitro, in vivo (rat) Rapidly in situ forming injectable hydrogel with low cytotoxicity and enhanced mechanical strength. Chitosan, gelatine Link N Solution mix In vitro Improved mechanical properties in compression and pro-anabolism function Chitosan, poly (l-lactic acid) Human umbilical cord mesenchymal stem cells (hUCMSCs) Solution mix In vivo (rabbit) Beneficial for cell adhesion and proliferation, enhance bone connection between adjacent vertebrae and promote AF tissue regeneration Chitosan with dissolved perfluorotributylamine (core), polycaprolactone (shell) Annulus fibrosus stem cells (AFSCs) Coaxial electrospinning, solution mix In vitro, in vivo Persistent oxygen release, promoting proliferation, migration and ECM production of AFSCs. Chitosan, poly (butylene succinate-co-terephthalate) (PBST), poly (ether ether ketone) (PEEK) IVD cells In vitro fabrication In vitro, in vivo (pig) Support IVD cell growth, possess a good compressive stress and elastic moduli. Chitosan, poly (butylene succinate-co-terephthalate) copolyester AF and NP cells In vitro fabrication In vitro, in vivo (rabbit) Maintain the biological function of IVD cells and retain the height of intervertebral Section title: Introduction Educational score: 4.237462043762207 Domain: biomedical Document type: Review Language: en IVDD is a complex and multifactorial process that involves biochemical, structural, and mechanical changes in the disc tissue. The pathological process includes (a) progressive cell senescence and cell death , (b) cell dysfunction and declined anabolic production of extracellular matrix (ECM) , (c) inflammatory cells and cytokines, and increased catabolic activity . The loss of hydration and structural integrity accelerates the biomechanical property to fail and results in pain and disability. Furthermore, there exist complex interactions between the degenerated disc, adjacent vertebral endplates, and the paraspinal muscles. Changes in the endplates can alter nutrient transport to the disc, exacerbating degeneration, while the paraspinal musculature may undergo compensatory changes, further influencing spinal biomechanics and pain perception . Understanding these interconnected factors is critical for developing holistic therapeutic strategies. Section title: Introduction Educational score: 4.053830146789551 Domain: biomedical Document type: Review Language: en Although progress in understanding the molecular basis of IVDD has been made, the current treatments for IVDD, either conservative or operative options, are limited. Non-surgical treatments including immobilization, analgesic drugs, and other physical therapy, mainly aim to alleviate the pain . In severe cases, invasive surgical treatments like interbody fusion or arthroplasty are necessary to provide mechanical structural support and stability . Although pain control can be beneficial for patients, the long-term prognosis of anti-inflammatory drug use is not optimal. Additionally, the risk of adjacent segment degeneration (ASD) exists with invasive surgical procedures . Essentially, conservative treatments or surgical options can not address the underlying etiology or facilitate the regeneration of the degenerated disc tissue. In comparison, polysaccharide-based biomaterials possess the potential to alleviate the inflammatory microenvironment , halt cell senescence and apoptosis , improve biomechanical instability , and prevent IVD from degeneration. This is beneficial for addressing IVDD from the etiological source. Section title: Introduction Educational score: 4.4468584060668945 Domain: biomedical Document type: Review Language: en Since traditional treatments could not reverse degenerative progress, restore tissue homeostasis, and reconstruct the mechanical stability of the spine. New strategies aimed to counteract intervertebral disc degeneration are urgent. Many attempts have been made in the following fields. (a) anti-inflammatory strategies to regulate the inflammatory microenvironment ; (b) vascularization and innervation growth inhibition ; (c) anti-catabolic strategies ; (d) pro-anabolic strategies ; (e) endogenous and exogenous cell supplementation . The main purposes of ingenious therapeutics are to alleviate inflammation in IVD which contributes to discogenic pain and degenerative symptoms and to remodel anabolic/catabolic balance in the nucleus pulposus (NP). Among these regenerative approaches, emerging materials such as synthetic hydrogels, decellularized tissue matrices, peptide-based scaffolds, and stimuli-responsive smart materials have shown promise in supporting disc cell viability, mimicking the disc's extracellular matrix, and enhancing mechanical resilience [ , , , ]. Advanced cellular approaches, including stem cell therapies, gene editing, and endogenous or exogenous biological factor recruitment are also under investigation to directly repair or regenerate damaged tissue [ 8 , , , ]. Against this backdrop, polysaccharide-based biomaterials stand out due to their biocompatibility, tunable mechanical properties, and ability to interact with native disc cells and matrix components. Section title: Introduction Educational score: 4.062017440795898 Domain: biomedical Document type: Study Language: en In situ IVD injection has become the paradigm for drug delivery in that systematical administration can barely reach the disc due to avascularity and immune privilege. However, puncture itself is an invasive procedure that may impair annulus fibrosus (AF) and therefore facilitate IVDD and the simply-delivered drugs are susceptible to transient clearance, limited absorption by nucleus pulposus cells (NPCs), and off-target side effects . In such a situation, biomaterial delivery systems based on tissue engineering hold promise in IVD repair and regeneration. The bioinspired delivery platform can not only bear the mechanical pressure of the IVD but also improve drug efficacy due to their excellent biocompatibility, biodegradability, accurate tissue targeting, and sustained release , of which polysaccharide-based materials are ideal candidates . Section title: Anatomy and physiology of intervertebral disc Educational score: 4.165579319000244 Domain: biomedical Document type: Study Language: en The intervertebral disc, a fibrocartilaginous joint between adjacent vertebrae, consists of three main components: the gelatinous NP as the core, the annulus fibrosus surrounding the periphery, and the cartilage endplates (CEP) sandwiching aforesaid two structures . The NP contains two types of cells, NPCs and notochordal cells, the latter of which only exist in the embryonic and fetal stages. Its extracellular matrix (ECM) is defined by a network of irregularly organized type Ⅱ collagen embedded in amorphous proteoglycans . The AF is made up of 15–25 concentric layers of type I collagen fibers, with inner and outer regions, primarily responsible for resisting tensile forces . The upper and lower CEPs, through which the main source of nutrition is diffused and metabolites are excreted, interface the vertebral body and the disc . Fig. 1 (A) A schematic view of the human IVD (Adapted from Ref. ). (B and C) Representative H&E staining and representative T2-weighted MRI scans of disc samples from normal, moderately degenerated and severely degenerated groups. Fig. 1 Section title: Anatomy and physiology of intervertebral disc Educational score: 4.763520240783691 Domain: biomedical Document type: Study Language: en Proteoglycans are proteins with glycosaminoglycan chains covalently attached. The glycosaminoglycan chains endow aggrecan osmotic properties to swell and withstand compressive pressure, to which the remarkable chondroitin sulfate (CS) and keratin sulfate (KS) chains are ascribed . Of note, as a key constituent of ECM (65 % in NP and 20 % in AF), aggrecan is essential in endowing IVD mechanical property to resist axial compression in that negatively charged glycosaminoglycans strongly attract water molecules and bind growth factors and cell adhesion molecules . Recent advances have demonstrated that aggrecan is critical for the preservation of appropriate stiffening of the ECM by providing biomechanical cues during IVD development . In addition, aggrecan restrains endothelial cell adhesion and cell migration, therefore helping maintain the avascular condition as well as inhibiting nerve growth . In particular, chondroitin sulfate (CS), as one kind of glycosaminoglycan, contributes to hydration, cell survival, and the non-innervated state of IVD . With growth and development, glycosaminoglycan chains undergo several changes including that the CS shortens while the KS elongates, accompanied by sulfation site transferring from the 4-carbon to the 6-carbon of N-acetyl galactosamine . Section title: Pathophysiology and comprised biomechanism of IVDD Educational score: 5.018293380737305 Domain: biomedical Document type: Study Language: en IVDD defines the crucial pathological basis of degenerative disc disease (DDD), triggered by multiple factors including genetics, environmental factors, and mechanical factors . Cytokines, for example, IL-1β and TNF-α, aggravate the degenerative process and promote cellular senescence, autophagy, and apoptosis. The inflammatory cascade mediates catabolism through upregulating extracellular matrix enzymes such as MMP3 and ADAMTS production and restrains anabolism by inhibiting proteoglycan expression [ , , ]. The reduction of proteoglycans is the most prominent biochemical variation and is recognized as a hallmark of IVDD . With proteoglycan and type-Ⅱ collagen collapsing, the strength-bearing capacity of IVD and biomechanical integrity was impaired, leading to AF tear and subsequent NP exposure to the circulatory system and immune system . In the meantime, evoked inflammatory cytokines, IL-6, prostaglandin E2 (PGE2), and pro-innervating factors nerve growth factor (NGF) stimulate nerve ingrowth, leading to symptomatic pain . Upregulated vascular endothelium growth factor (VEGF) and chemokines, CCL1, 3 and 13, CXCL10 by NPCs provoke vascularization and immune cells infiltration, which accumulates the inflammatory cytokines including IL-1β and TNF-α, aggravating catabolism and ECM loss . In addition, CEP calcification evolves with aging, breaking the supply/demand balance. The nutrient diffused inclines while demand rises due to increased infiltrated immune cells, which accelerates the disorder of energy metabolism . This imbalance restrains the nutrients for NPCs and annulus fibrosus cells (AFCs) and further affects cell viability. Furthermore, a deteriorating extracellular environment characterized by significant acidification with metabolite cumulating induces further cell senescence and apoptosis and aggravates degradative enzyme production . Fig. 2 Schematic representation of biomechanical causes, biological factors and microenvironment changes in the process of IVDD. Multifactorial changes are involved in IVDD, including cellular changes, anabolism/catabolism imbalance, inflammatory response, mitochondrial dysfunction, calcification and endplate changes, and altered biomechanics. Loss of notochordal cell marks the beginning of the degeneration process, followed by nucleus pulposus cell decrease. Reduced matrix production and aggravated matrix degradation define phenotypic changes, leading to disc dehydration and loss of height. Pro-inflammatory cytokines upregulation and immune cell infiltration exacerbate disc microenvironment. Neovascularization and nerve infiltration are associated with discogenic pain. Mitochondrial dysfunction including accumulated ROS, impaired mtRNA, and altered mitochondrial dynamics contributes to and perpetuates disc degeneration. Endplate calcification and subchondral sclerosis limit nutrient supply to disc. IL-1β: Interleukin 1β, IL-6 and IL-17: Interleukin 6 and 17, TNF-α: Tumor necrosis factor α, PGE2: Prostaglandin E2, MMPs: Metalloproteinases, TIMPs: Tissue inhibitors of metalloproteinases, BMP: Bone morphogenetic protein, TGF-β: Transforming growth factor β, NGF: Nerve growth factor, ADAMTS: A disintegrin and metalloproteinase with thrombospondin motifs, VEGF: Vascular endothelium growth factor, ROS: Reactive oxygen species, mtRNA: mitochondrial RNA. Fig. 2 Section title: Pathophysiology and comprised biomechanism of IVDD Educational score: 4.351002216339111 Domain: biomedical Document type: Study Language: en During IVDD, proteoglycan in NP undergoes a significant decrease and leads to the ratio of NP/AF proteoglycan dropping, which indicates the importance of high proteoglycan concentration in healthy IVD . Hyaluronic acid, as another major component of proteoglycans with aggrecan monomers linked, imbibes water and subsequently renders IVD mechanical strength. In addition, hyaluronan demonstrates an anti-inflammatory property by inhibiting tetratricopeptide repeats 3 (IFIT3) and insulin-like growth factor binding protein-3 (IGFBP3) and a matrix remodeling action by improving anabolism of extracellular matrix and reducing ADAMTS4 . Although the content of aggrecan declines in NP during aging, there is a compensating rise of aggrecan concentration in the inner AF . Furthermore, as MMPs and aggrecanases cleave aggrecan, whether fragmented aggrecan is harmful to disc needs to be elucidated. Section title: Pathophysiology and comprised biomechanism of IVDD Educational score: 4.192824840545654 Domain: biomedical Document type: Study Language: en Mitochondrial dysfunction is associated with the development of IVDD through affecting a series of changes including mitochondrial fusion, autophagy, and cell apoptosis . Interestingly, hyaluronic acid can preserve mitochondrial function via activating mitophagy in NPCs, thus attenuating IVDD . Conversely, free hydroxyl radicals from hydrogen peroxide and superoxide are detrimental for HA to depolymerization . Section title: Overview of polysaccharides Educational score: 4.337090492248535 Domain: biomedical Document type: Review Language: en Polysaccharides are made up of monosaccharides and connected by O-glycosidic bonds. Widespread in nature, a variety of sources of polysaccharides includes algae (agarose), plants (starch, pectin), microorganisms (xanthan gum, gellan gum) and animals (chondroitin, heparin, hyaluronan) . With regard to their chemical structure, the various functional groups of different polysaccharides lay the foundation for physicochemical modifiability, so as to endow more characteristics and functionalities . In addition to the similarities between polysaccharides and the extracellular matrix (ECM), polysaccharides exert essential biological roles in molecular recognition, cell adhesion, and signal transduction . Therefore, considering their impressive merits including biocompatibility, biodegradability, hydrophilicity, nontoxicity, bioactivity, and water retention capacity, polysaccharides have garnered significant attention for diverse biomedical applications including drug delivery, tissue repair, as well as targeted therapy . Several common forms of polysaccharide-based biomaterials include hydrogels (e.g. hyaluronic acid, chondroitin sulfate, and alginate), scaffolds (e.g. chitosan and cellulose), nanoparticles (e.g. chitosan, hyaluronic acid, and alginate), microspheres (e.g. alginate and chitosan), nanofibers (chitosan, collagen, and silk fibroin), and nanocrystals (e.g. cellulose). These forms of polysaccharide-based materials provide a versatile toolkit for addressing different aspects of IVDD. Section title: Chemical properties Educational score: 3.927008867263794 Domain: biomedical Document type: Other Language: en As a polymeric carbohydrate, a polysaccharide is composed of more than 10 monosaccharides, among which the same monosaccharides constitute homopolysaccharides while different monosaccharides constitute heteropolysaccharides. According to the glycosidic linkage type and electrical charges, the polysaccharides can be classified into straight or branched chains and polyanion, electroneutral, or polycation polysaccharides respectively . Section title: Chemical properties Educational score: 3.598773717880249 Domain: biomedical Document type: Other Language: en Polysaccharides that derive from natural sources have their own drawbacks including microorganism pollution, batch variation, different hydroxide content, and unstable mechanical strength . However, the chemical modifications minimize these limitations and impart versatile functionalization to polysaccharides. There are diverse approaches including carboxymethylation, oxidation, sulfation, and polymer graft to achieve this transformation. Section title: Crosslinking mechanisms of polysaccharides Educational score: 4.074172496795654 Domain: biomedical Document type: Study Language: en Hydrated polysaccharides in an aqueous environment define the process of production of the hydrogel. As such, one of the most extensive usages of polysaccharides in tissue engineering and biomedical applications is hydrogel establishment. The mechanism whereby polysaccharides form network structure is mainly divided into physical crosslinking that is defined by ionic interaction, of which alginate represents the paradigm and chemical crosslinking that includes radical polymerization (e.g. dextran), aldehyde (e.g. amine contained polysaccharides), addition reaction, and condensation reaction. Section title: Biomedical applications of various polysaccharide-based materials in IVDD Educational score: 3.996187925338745 Domain: biomedical Document type: Review Language: en Mammalian polysaccharides including hyaluronic acid, keratan sulfate, and chondroitin sulfate naturally exist in vivo and constitute the ECM of the intervertebral disc. Glycosaminoglycan chains covalently attach to core protein and compose proteoglycans, contributing to structure integrity and microenvironmental homeostasis of intervertebral discs. In the following section, the fabrication and application of polysaccharide-based materials and biological properties in intervertebral disc repair and regeneration will be discussed. Section title: Chondroitin sulfate Educational score: 4.741799354553223 Domain: biomedical Document type: Review Language: en Widely distributed in vertebrates, invertebrates, and bacteria, chondroitin sulfate is characterized as a linear polysaccharide composed of repeated disaccharides of N-acetyl galactosamine and glucuronic acid. In regard to different sulfated residues at carbon positions, CS is classified into CS-A (chondroitin-4-sulfate), CS-C (chondroitin-6-sulfate), CS-D (chondroitin-2,6-sulfate) and CS-E (chondroitin-4,6-sulfate) . There has been reports that CS significantly inhibited NF-κB activation and nuclear translocation, along with the production of inflammation cytokines and inducible nitric oxide synthase . As a prominent constituent of the ECM, apart from anti-inflammatory, anti-oxidant, anti-coagulating, and immunomodulatory functions, CS serves as a signaling molecule that can influence cell signal transmission and promotes the formation of collagen and proteoglycans . Moreover, CS has been reported to promote ECM production via the integrin-WNT5A pathway and notochordal cells-derived CS has been observed to inhibit hyperinnervation and peripheral nociception . CS is now extensively utilized in biomedical applications because of its renowned compatibility with biological systems, biodegradability, and hydrophilicity . Section title: Applications of chondroitin sulfate-based materials in NP regeneration Educational score: 4.171813011169434 Domain: biomedical Document type: Study Language: en Based on the anti-inflammatory properties of CS, Luo et al. engineered a versatile hydrogel system (HA/CS). This hydrogel is injectable and remarkably self-crosslinked post-injection through boronate ester bonds. Further enhancement of mechanical strength through sequential coupling through the Diels-Alder reaction to simulate the human NP. CS can degrade under physiological stimuli such as hyaluronidase enzyme, reactive oxygen species, and pH change, thus enabling site-specific release. As such, this hydrogel system continuously protects NPCs from apoptosis and preserves extracellular matrix synthesis. In the rat model, the HA/CS hydrogel inhibits inflammatory cascade and remarkably alleviates IVDD . Fig. 3 Chondroitin sulfate-based biomaterials for intervertebral disc regeneration and repair. (A) Injectable self-antioxidant hydrogels grafted with inflammation inhibitory chondroitin sulfate formed via dynamic boronate ester bonding between furan/phenylboronic acid and furan/dopamine-modified hyaluronic acid and mechanically enhanced by Diels-Alder reaction-induced secondary crosslinking, and (i) CLSM images of double-crosslinked HA/CS0 and HA/CS2.0 hydrogels (Adapted from Ref. ). (B) Injectable Disc-Derived ECM Hydrogel functionalized with chondroitin sulfate via amine groups of collagen and (ii) Assessment of the structural stability of cell free ECM-derived hydrogels and SEM images (Adapted from Ref. ). Fig. 3 Section title: Applications of chondroitin sulfate-based materials in NP regeneration Educational score: 4.1290411949157715 Domain: biomedical Document type: Study Language: en A prospective treatment attempt involves resecting the compromised NP tissue and restoring it with NPCs encapsulated in situ forming hydrogel. Riahinezhad et al. designed such a hydrogel using thiolated chondroitin sulfate crosslinked 4-arm [poly (ethylene glycol)-b-poly (trimethylene carbonate)-acrylate] via Michael type reaction. This hydrogel delivers NPCs with high viability and proliferating ability. These cells exhibit collagen type Ⅱ deposition instead of collagen type Ⅰ. The instantaneous moduli of this hydrogel are adjusted according to the native NP and resistant to cyclic compressive loading . Section title: Applications of chondroitin sulfate-based materials in NP regeneration Educational score: 4.184758186340332 Domain: biomedical Document type: Study Language: en An innovative graft copolymer, poly (N-isopropyl acrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS) combines the thermoresponsive behavior of the latter with the biocompatibility and bioactivity of CS to form a moisture-absorbing and fluid substance below the lower critical solution temperature and precipitated hydrogel above it . However, this combination has inadequate solution viscosity and adhesiveness. Christiani et al. embedded alginate microparticles into PNIPAAm-g-CS to enhance surface roughness and toughness. The structure provides an optimized platform for sustaining, multiplying, and differentiating adipose-derived stem cells (ADSCs) into native NP-mimicking phenotype due to incorporated GDF-6, a potent chondrogenic factor. The ex vivo implantation of this formation into degenerated porvine IVDs demonstrated promising results in restoring the compressive and neutral zone stiffness to levels seen in undamaged discs . Section title: Applications of chondroitin sulfate-based materials in NP regeneration Educational score: 4.206576347351074 Domain: biomedical Document type: Study Language: en Chondroitin sulfate and type Ⅱ collagen constitute the major ECM of NP. Borrelli et al. designed a self-assembled hydrogel of decellularized ECM functionalized with chondroitin sulfate. Of note, the inclusion facilitates a rounded morphology and higher sGAG synthesis of nasal chondrocytes which have been recognized as a promising cell source for NP regeneration with their robust matrix synthesis capacity . Basically, the effect of chondroitin sulfate on the chondrogenic phenotype of mesenchymal stem cells has been reported to be associated with Smad1/5/8 signaling inhibition and p38 signaling upregulation . Section title: Hyaluronic acid Educational score: 4.491150856018066 Domain: biomedical Document type: Review Language: en Hyaluronic acid (HA) is a nonsulfated, linear polyanion mucopolysaccharide and the only glycosaminoglycan that is ubiquitous in almost all mammalian animals, with additional production capabilities in certain bacteria . This material is widely present in the extracellular matrix. It plays a vital part in diverse biological processes, including cell development, migration, as well as influencing the physiological activities of the human body . Specifically, CD44, the best-characterized HA receptor, mediates cell migration of MSCs through interaction with extracellular HA, and its expression on activated lymphocytes aids in the primary rolling to the inflammatory sites in a HA-dependent manner . Hyaluronic acid is a versatile and extensively used material in tissue engineering because of its biocompatibility, biodegradability, nonimmunogenicity, and viscoelastic properties . Its applications span from scaffold fabrication and drug delivery platforms to wound care, ophthalmology, and orthopedics. The extensive study and successful utilization of HA and its derivatives underscore their considerable potential in advancing regenerative therapies and improving patient outcomes in various medical fields. Section title: Applications of hyaluronic acid-based materials in NP regeneration Educational score: 4.459833145141602 Domain: biomedical Document type: Study Language: en One ideal biomaterial as a novel treatment for IVD regeneration is able to support the expansion, differentiation, and function of encapsulated cells while providing sufficient mechanical stability. It has been shown that HA grafted with poly (N-isopropyl acrylamide) supports high cell viability and maintains differentiation potential . HA crosslinked with 1,4-butanediol diglycidyl ether (BDDE) has superior comprehensive mechanical testing performance . Therefore, Guo et al. combine HA-BDDE and HA-pNIPAM to form an interpenetrating polymer network as a cell carrier. The IPN hydrogel demonstrates rheological properties similar to native NP tissue. This network promotes GAG production and new matrix deposition of loaded NPCs after implanted ex vivo in organ cultured IVD . Using hyaluronic acid and type Ⅰ collagen to mimic the native nucleus pulposus tissue, encapsulated human BMSCs exhibit high gene expression of NP marker under a differentiation medium containing GDF5 and TGFβ1. Of note, cells tend to have a rounded morphology to NPCs . Similarly, Gansau et al. incorporated collagen and hyaluronic acid into fibrin-based hydrogel. Cell viability and proliferation are enhanced due to the addition of ECM components. Furthermore, incorporating HA elevates sGAG accumulation and inhibits collagen expression . Granular hydrogels have become favorable biomaterials with injectability and microscale porosity in tissue engineering. Muir et al. designed a hyaluronic acid granular hydrogel while encapsulating radiopaque zirconium oxide nano-powder. This radiopaque hydrogel can be monitored over time to track its location, degradation, and interaction with surrounding tissues, providing visualization of the placement of the hydrogel during injection. The large animal goat model of IVDD confirms that the granular hydrogel impedes disc height loss, increases proteoglycan production, and decreases collagen deposition . Section title: Applications of hyaluronic acid-based materials in NP regeneration Educational score: 4.248628616333008 Domain: biomedical Document type: Study Language: en Simple biomaterials or scaffolds may lack the ability to enhance cellular functions and direct stem cells to specific lineages. Therefore, biological factor-loaded biomaterials harness the power of active molecules to improve the efficacy of medical treatments. Pentosan polysulfate (PPS), a semi-synthetic GAG-like polysaccharide, has been proven to boost viability and induce MSCs differentiating to chondrocytes, and induce NP-like ECM production . Frith et al. incorporated soluble PPS into polyethylene glycol and hyaluronic acid hydrogel system and encapsulated MPCs. The composites form NP-like tissue, particularly type Ⅱ collagen deposition. This hydrogel is immunologically tolerant using a rat subcutaneous implantation model . Afterward, the research group determined that a covalently bound PPS possesses a strong potential to mediate IVD regeneration . Pereira et al. attempted to add stromal cell-derived fator-1 (SDF-1) to an HA-poly (N-isopropylacrylamide) hydrogel to attract Hu-MSCs. The composite enhances the migration of MSCs into compromised discs in comparison to HAP or SDF-1 alone . Tsaryk et al. combined TGF-β3 delivery by gelatin microspheres with collagen-low molecular weight hyaluronic acid hydrogel. Of note, the material promotes nasal chondrocytes and MSCs to expand and differentiate without triggering inflammation or inducing cytotoxicity . Hyaluronic acid scaffold seeded with human Wharton's Jelly-derived MSC (WJ-MSC) expressing high levels of TGF-β receptor Ⅰ and TGF-β receptor Ⅱ significantly restore the disc hydration in a rabbit model compared to scaffold along or WJ-MSC-lowTR-seeded HA scaffold. The morphological and histological analyses are consistent with T2-weighted MRI analysis . Section title: Applications of hyaluronic acid-based materials in NP regeneration Educational score: 4.410383701324463 Domain: biomedical Document type: Study Language: en Targeting RNA molecules is a promising therapeutic strategy for IVDD. This approach focuses on modulating the expression and function of various RNAs including long noncoding RNAs (lncRNAs), circular RNAs (cirRNAs), and micro RNAs (miRNAs) to influence cellular processes including apoptosis, proliferation, and extracellular matrix remodeling in NPCs . Recent studies demonstrate that stimulators of interferon genes (STING) are critical in diverse inflammatory and degenerative diseases. Chen et al. revealed that the STING signaling pathway is an intervention target for IVDD. A siSTING delivery hydrogel of aldehyde HA and poly (amidoamine)/siRNA complex is fabricated and the hydrogel system alleviates intervertebral inflammation and degeneration in the IVDD rat model . Likewise, circSTC2 is elevated in degenerative NP tissue, and the silence of circSTC2 can restore the extracellular matrix anabolism and catabolism balance. Chang et al. grated circSTC2 silencing genes-loaded 1,2-dioleoyl-3-trimethylammonium-propane/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine cationic liposomes on methacrylated hyaluronic acid microspheres. With the excellent merits including degradability, dispersibility, swellability, and injectability of HAMA microspheres, lipoplexes are persistently released over 27 days. This psh-circSTC2-lipo@MS enhances ECM anabolism and impedes catabolism in NPCs in vitro as well as in an in vivo rat IVDD model . Fig. 4 Hyaluronic acid-based biomaterials for intervertebral disc regeneration and repair. (A) Injectable self-healing hydrogel with continuous siRNA delivery property and (i) Representative H&E and Safranin O and Fast Green staining images demonstrate siSTING@HP gel slowing the IVD degeneration progress (Adapted from Ref. ). (B) A two-part biomaterial adhesive strategy involving dual-modified glycosaminoglycan and interpenetrating network hydrogel for annulus fibrosus repair and (ii) Cross-sectional view of HAMA Aldehyde treated AF and phase-contrast images of HAMA Aldehyde treated AF cells. (iii) Picrosirius red/alcian blue staining (Adapted from Ref. ). Fig. 4 Section title: Applications of hyaluronic acid-based materials in NP regeneration Educational score: 4.119406700134277 Domain: biomedical Document type: Study Language: en HA can be combined with different biomaterials to ameliorate pain and inflammation due to its anti-inflammatory properties. Fragmentation of ECM accompanies IVDD. However, research indicates that hyaluronic acid fragments induce inflammatory and catabolic mediators such as interleukin-1β, IL-6, cyclooxygenase-2, MMP-1, and MMP-13. Through siRNA intervention or antibody neutralization, it is suggested that hyaluronic acid fragments result in IVDD via the Toll-like receptor 2 signaling pathway . Section title: Applications of hyaluronic acid-based materials in AF regeneration Educational score: 4.247552871704102 Domain: biomedical Document type: Study Language: en Currently, there are some challenges and limitations associated with using hydrogel systems for sealing annulus fibrosus defects. For example, many hydrogels exhibit poor adhesion to wet tissue and insufficient mechanical strength that may lead to potential dislodgement and failure to seal the defect effectively. Tyler et al. designed a combined regeneration system by integrating oxidized and methacrylated glycosaminoglycan and fibronectin-linked fibrin and poly (ethylene glycol) diacrylate. The former absorbs the latter interpenetrating network and binds this hydrogel to ECM in the IVD covalently. The results show that the dual-modified hydrogel presents excellent adhesive strength without any observable cytotoxic effects and demonstrates mechanical compliance that aligns with the herniation risk of the current clinical standard . Interestingly, HA itself demonstrates an anti-inflammatory action in AF-defected and IFNα 2 β stimulated discs by downregulating IFIT3, ADATS4, and pro-apoptotic IGFBP3. Moreover, HA intervention increases collagen Ⅰ and aggrecan. Therefore, HA exhibits an anti-inflammatory property and matrix modulatory effect in annulus fibrosus . Section title: Applications of hyaluronic acid-based materials in combined therapy of NP and AF regeneration Educational score: 4.336142063140869 Domain: biomedical Document type: Study Language: en Hyaluronic acid (HA)-poly (ethylene glycol) (PEG) composite hydrogels are a prominent area of research due to their unique properties. Jeong et al. used artificial neural network analysis to screen HA-PEG formulation to match NP and AF cells. It turns out that lower-molecular weight HA leads to the highest NPC and AFC numbers and higher sGAG production . Based on the concept of IVD as integrity, Sloan et al. attempted to repair AF defect with riboflavin cross-linked high-density collagen gel and repair NP with modified HA hydrogel. Qualitative T2 MRI shows that combined therapies restore disc water content and preserve intact IVD morphology. Particularly, HA injection into NP recovers 35 % and 40 % of the effective instantaneous and equilibrium moduli . Similarly, Mohd et al. confirmed that HA hydrogel attenuated IL-6 and IL-1β in degenerated IVD validated by upstream regulations of c-Fos, phosphorylated p38 MAPK, and phosphorylated NF-κB in both AF and NP and regulated ECM deposition, such as higher CS, collagen Ⅱ, and fibronectin, via Smad3-TGF-β1 signaling . It has been implied that CS increases ECM production and correlates with peripheral sensory innervation and nociceptor. However, whether there exists direct action of chondroitin needs to be fully elucidated. Furthermore, HA hydrogel implantation inhibited injury-induced peripheral sensory hyperinnervation and pro-nociceptive TRPV1 and Trk-A expression in AF and NP tissues . These results suggest that HA hydrogel has a potential therapeutic application and could be translated clinically in patients with discogenic back pain due to IVDD. Section title: Cellulose Educational score: 4.15704870223999 Domain: biomedical Document type: Study Language: en Cellulose is a linear polysaccharide in which D-glucose units are connected by β-(1,4) bonds . Due to its superior mechanical properties and biocompatibility, nanoscale cellulose has garnered significant attention in recent years . Cellulose nanomaterials can be classified into two types: cellulose nanostructure nanomaterials including cellulose microcrystals and microfibrils, and cellulose nano-objects including cellulose nanocrystals and cellulose nanofibrils . In comparison to the former nanocelluloses, cellulose nanocrystals and cellulose nanofibrils exhibit elevated specific surface areas and a more uniform distribution of particle sizes, therefore promoting the formation of mechanically robust and spontaneously organized structures such as hydrogel . Section title: Applications of cellulose-based materials in NP regeneration Educational score: 4.177396297454834 Domain: biomedical Document type: Study Language: en Cellulose-based scaffolds have remarkable merits such as non-immunogenicity, nontoxicity, strong biomechanical properties, as well as injectability and in situ gelling that, therefore, hold a potential for NP replacement . Anna et al. first encapsulated NPCs in photocrosslinked carboxymethylcellulose (CMC) in which CMC was functionalized with methacrylate groups and the latter endow water solubility. Under normal pH conditions, the deprotonation of the carboxylic acid within the carboxymethyl groups leads to the negative charge of hydrogel that resembles the negatively charged glycosaminoglycans of ECM of NP. In addition, this polymer demonstrates support for NP cell viability with the occurrence of spherical cells and the composition of chondroitin sulfate proteoglycan . Section title: Applications of cellulose-based materials in NP regeneration Educational score: 4.132716178894043 Domain: biomedical Document type: Study Language: en Varma et al. developed a dual-polymer network hydrogel composed of methacrylated carboxymethylcellulose and methylcellulose (MC). The thermogelling MC facilitates the gelation of CMC in situ consistently within intervertebral discs. After injection into IVDs following discectomy, the CMC-MC composite is able to recover IVD height and biomechanical properties . In a follow-up study to examine the sustaining stability of the structure, the CMC-MC hydrogel showed restoration of height and manifested similar herniation risk and fatigue resistance to those with standard nucleotomy. Additionally, the hydrogel injection only results in a limited foreign body reaction, evidenced by analyzing fibrous capsule formation and macrophage infiltration during a 12-week period . Section title: Applications of cellulose-based materials in NP regeneration Educational score: 4.065186977386475 Domain: biomedical Document type: Study Language: en Similarly, Gupta et al. introduced human MSCs encapsulated carboxymethylcellulose hydrogel stimulated with transforming growth factor-beta 3 (TGF-β3). This hydrogel system confirmed significant glycosaminoglycan and type Ⅱ collagen deposition that fit in with the ECM of NP . Furthermore, the research group dug into the impact of the duration of TGF-β3 exposure on matrix metabolism and the best macromer concentration of CMC hydrogel. The results showed that a lower macromer concentration (1.5 % weight/volume) demonstrates stronger NP-like ECM elaboration and improved mechanical functionality and short-term TGF-β3 exposure augments sufficient ECM accumulation . Section title: Applications of cellulose-based materials in NP regeneration Educational score: 4.235660552978516 Domain: biomedical Document type: Study Language: en Prior investigations have mainly focused on small animal models and short-term outcomes spanning just for a few weeks. Extensive long-term studies in relevant large animal models are imperative for translating into clinical application. An injectable chitosan CMC hydrogel system supplemented with adipose-derived stem cells (ASCs) was evaluated in an ovine model for 12 months. Of note, this scaffold proved a long-term stabilization that alleviated IVDD and sustained intervertebral height in comparison to injured and untreated IVDs . Incorporation of cellulose nano-objects with synthetic polymer (e.g. poly (ethylene glycol) (PEG)) matrix can produce hydrogels that have improved mechanical stability. Schmocker et al. developed a photopolymerized PEG dimethacrylate (MA) cellulose nanofibrils (CNFs) composite hydrogel. The material was finely tailored to fit the elastic modulus and water content of native NP. By utilizing a newly designed device to realize in situ injection and photopolymerization, the composite hydrogel is capable of rebuilding disc height within an ex vivo bovine organ model even after 0.5 million loading cycles and histological analyses indicate a superb tissue integration of this hydrogel implant . Section title: Applications of cellulose-based materials in AF regeneration, combined therapy of AF and NP Educational score: 4.108968734741211 Domain: biomedical Document type: Study Language: en Celluloses are usually used as reinforcement by incorporating cellulose nanofibrils and nanocrystals into synthetic or natural polymers. Pereira et al. fabricated an injectable nanocellulose reinforced methacrylated gellan-gum (GGMA) composite hydrogel for AF substituting. The hydrogel system manifests compressive modulus consistent with native AF tissue and improved matrix entanglement with augmented scaffold stiffness. In vitro experiments indicated that nanocomposite hydrogel enhanced cell survival of encapsulated bovine AFCs and maintained AFCs physiological morphology . Section title: Applications of cellulose-based materials in AF regeneration, combined therapy of AF and NP Educational score: 4.351714134216309 Domain: biomedical Document type: Study Language: en Individual NP and AF regeneration strategies have been broadly developed to alleviate IVDD. However, the integrity of IVD implies that individual therapy is insufficient and emphasizes the importance of combined AF and NP therapies. Hydrogels based on a cellulose-alginate dual network for the AF and a cellulose hydrogel for the NP are designed to replace IVD. This scaffold increased endogenous MSC homing and adhesion due to functionalized SKP peptide and RGD (Arg-Gly-Asp) peptide and induced MSCs differentiation to chondrocyte and NP-like phenotype with the delivery of growth differentiation factor 5 (GDF-5). Moreover, many other advantageous merits include enhanced compressibility, shape retention capability and comparable biomechanical strength. Of particular, the rat caudal IVDD model demonstrated that the implant impeded the degeneration progress and significantly reestablished both AF and NP . Similarly, Yang et al. created a more complex IVD implant that consists of type Ⅱ collagen-based NP and bioprinted bacterial cellulose-based AF. The former involves rat NPCs while the multilamellar bacterial cellulose layers with micropattern in ± 30° alternative direction contain AFCs. Animal studies confirmed that this tissue-engineered IVD can integrate with the adjacent vertebral and withstand tensile stresses for as long as 3 months . Fig. 5 Cellulose-based biomaterials for intervertebral disc regeneration and repair. (A) Peptide-functionalized (RGD and SKP peptides) cellulose/alginate double network hydrogel and (i) T2‐weighted MRI imaging of the caudal spine (marked with red boxes) (Adapted from Ref. ). (B) Reverse reconstruction and bioprinting of bacterial cellulose-based functional IVD consist of micropatterned bacterial cellulose aligned in ± 30° direction as AF and gelated type Ⅱ collagen as NP and (ii) a-d) Phase contrast microscopy imaging of microgrooves. e-h) SEM image for the BC membrane. n-q) Alexa Fluor 488 phalloidin/Hoechst staining. iii) a,b) Designed pattern of the AF template in opposing orientations ( ± 30°) within adjacent lamellae. c,d) SEM transection images for AF. e) Live/dead staining of APCs (Adapted from Ref. ). Fig. 5 Section title: Alginate Educational score: 4.227465629577637 Domain: biomedical Document type: Study Language: en Alginate, a naturally occurring polysaccharide extracted from brown seaweed and certain bacteria, features linear chains of repeating units of β-D-mannuronic acid and α-L-guluronic acid, which are connected together by 1,4-glycosidic bonds . Previous studies have shown that Alginate can reduce ROS levels in cells by chelating metal ions. Moreover, phosphorylation of IκB-p65 complex is downregulated by inhibiting nuclear translocation of P65. It may also reduce phosphorylation of P-Erk1/2 and P-JNK in a concentration-dependent manner . Its unique characteristics, determined by the ratio of mannuronic acid to guluronic acid, allow it to form hydrogels that can be tailored for specific uses, including cell encapsulation, delivering drugs, wound repair, and scaffold fabrication . The ability to customize its mechanical and biological properties makes alginate an essential material for advancing regenerative medicine and other therapeutic applications. Section title: Applications of alginate-based materials in NP regeneration Educational score: 4.151822090148926 Domain: biomedical Document type: Study Language: en Due to its excellent biocompatible property, alginate hydrogel is one of the most extensively employed biomaterials and has been shown to maintain a juvenile rounded morphology . Different gelling crosslinker concentrations may lead to distinct mechanical strength and degradability. Kalaf et al. determined that a ratio of 60 mM calcium carbonate to 120 mM glucono-δ-lactone is identified as having the most optimal properties compared to 2 % alginate crosslinked with calcium chloride. Injecting in situ gelling 2 % alginate into an enzyme-induced comprised IVD reestablishes functionality by reducing height reduction during prolonged cyclic loading and remains contained within the disc with no leakage at the injection area . Section title: Applications of alginate-based materials in NP regeneration Educational score: 4.294565677642822 Domain: biomedical Document type: Study Language: en Simple alginate hydrogel may present insufficient to rebuild compromised IVD. BMSCs or bone marrow aspirate concentrate (BMAC) loaded ultra-purified alginate (UPAL) gels have been delineated as effective in the repair of IVD defects . Similarly, Tilotta et al. trapped human NPCs in alginate spheres and then stimulated them with Wharton's Jelly MSC-derived extracellular vesicles. The intervention leads to cell content increase and cell death decrease. Nitrites, increasing when NPCs face oxidative stress, drop drastically after extracellular vesicle treatment. Moreover, proteoglycan content, indicated by Alcian blue staining, is upregulated. Therefore, extracellular vesicles ameliorate IVD degeneration and inflammation while enhancing ECM production . Traditional simple alginate hydrogels lack mechanical strength when implanted into animal models. Zeng et al. seeded MSCs in alginate and reinforced with macroporous poly (ethylene glycol) diacrylate (PEGDA) microcryogels (PM). The system creates 3D microscale niches and shows improved elasticity. Reinforced alginate hydrogel impedes cell leakage and enhances cell survival. In a canine animal model, PM-alginate hydrogel rescues IVDD after 6 months compared to other treatments . Section title: Applications of alginate-based materials in NP regeneration Educational score: 4.525650978088379 Domain: biomedical Document type: Review Language: en Preserving the juvenile phenotype of NPCs is crucial for IVD regeneration. Syndecan binding peptide (AG73) has been shown to promote NPCs adhesion and the RGD peptide motif has been shown to modulate the mechanotransduction in NPCs . Tan et al. designed alginate hydrogels combined with AG73 and cyclic RGD. The results show that peptides hydrogel demonstrate high cell viability, ECM synthetic property, and induce NPC phenotype, like N-Cadherin . With the aim of regulating local inflammatory microenvironment, Jiang et al. developed a hydrogel composite composed of sodium alginate, silicate ceramics, and poly (N-isopropylacrylamide). Poly (N-isopropylacrylamide) crosslinks in response to body temperature and forms an interpenetrating network with sodium alginate. Silicate ceramics (SC) release calcium ion that promotes alginate sodium crosslink. Magnesium ion from SC contributes to M2 macrophage polarization and regulates the immune microenvironment in a controlled manner, hence enhancing cell matrix production in which collagen Ⅱ and aggrecan are elevated . Similarly, Wu et al. add melatonin, recently shown to possess antioxidant and anti-inflammatory action, to mesoporous bioactive glasses (MBG) and sodium alginate (SA) composite hydrogel. MBG provides sufficient mechanical strength and acts as a melatonin reservoir to facilitate continuous release. The melatonin-MBG-SA hydrogel ameliorates IVD inflammation including TNF, ADAMTS5, MMP-3, and MMP-13 triggered by IL-1β by interacting with cell membrane melatonin receptors (MT1 and MT2). Additionally, melatonin enhances cell viability and facilitates continuous cell differentiation of MSCs by scavenging ROS and activating Nrf2 signaling. In vivo rat model shows that this composite hydrogel effectively inhibits inflammation and restores mechanical stability . Koo et al. used an alginate construct loaded with collagen and hyaluronic acid to form a shape-memory structure (SMS). The findings indicate that the SMS is effective in reducing pain, maintaining hydration, and preserving the structural integrity and function of the IVD matrix, together with supporting cell viability and the production of essential matrix components . Fig. 6 Alginate-based biomaterials for intervertebral disc regeneration and repair. (A) Injectable mesoporous bioactive glass/sodium alginate hydrogel loaded with inflammation suppressor melatonin and (i) Immunofluorescence analysis of COL-II expression in NPCs (Adapted from Ref. ). (B) Shape-memory collagen/alginate scaffold and in vivo procedures including nucleotomy, implantation of SMSs, and injection of HA, and (ii) Immunofluorescent analyses of collagen type II (Adapted from Ref. ). (C) Engineered high-strength biohydrogel consists of oxidized sodium alginate, gelatin, antagomir-204–3p, and Zn 2+ as a multifunctional platform to deliver nucleic acid. iii) SF staining images of IVD (Adapted from Ref. ). Fig. 6 Section title: Applications of alginate-based materials in NP regeneration Educational score: 4.276427268981934 Domain: biomedical Document type: Study Language: en Natural sodium alginate is usually gelated by calcium chloride in which endotoxins are relatively high with regard to in vivo implant. Ultra-purified alginate (UPAL) has been suggested to have a favorable lower concentration of endotoxins. Of note, UPAL hydrogel shows superior biomechanical characteristics and does not exhibit material extrusion following discectomy. Furthermore, UPAL gel enhances anabolism in NP, and maintains the moisture of IVDs in rabbits and sheep models. Surprisingly, the gel recruits endogenous NP progenitor cells in NP tissues . Subsequently, the same research group encapsulated BMSCs into UPAL gel. Co-culture of NPCs and BMSCs demonstrates elevated ECM synthesis, phenotypic markers, and bioactive factors. Combined BMSCs and UPAL hydrogel display augmented potential to rebuild degenerated IVDs . With the aim to administrate into the IVD by quick injection in situations involving persistent low back pain, a mixture of rapidly expanding clones of human MSCs and non-gelling UPAL solution is shown to impede inflammatory cytokine production and improve nociceptive behavior of rats . Of note, these UPAL gels are presently carried out in an initial human clinical trial in young patients, aged 20 to 40, after discectomy . Section title: Applications of alginate-based materials in NP regeneration Educational score: 4.110878944396973 Domain: biomedical Document type: Study Language: en Utilizing bioactive materials to deliver nucleic acids proposes a promising treatment for IVD regeneration. Antagomir-204–3p (AM) is capable of restraining NPCs apoptosis with lower immunogenicity. Chen et al. used zinc-oxidized sodium alginate-gelatin (ZOG) to distribute AM in which the former is able to crosslink with NP. In vivo results demonstrate that ZOG-AM rejuvenates the intervertebral disc height, sustains disc hydration, and upholds tissue integrity . Section title: Applications of alginate-based materials in AF regeneration Educational score: 4.130437850952148 Domain: biomedical Document type: Study Language: en Cell therapy holds potential for IVD repair, but the biomaterials used for cell delivery, which can seal annulus fibrosus defects and restore biomechanical function, often exhibit subpar biological performance. With the aim to keep the mechanical and biological performance balance, Panebianco et al. fabricated a composite hydrogel system utilizing AFCs seeded-oxidized alginate microbeads (MBs) loaded into fibrin (FibGen) hydrogels through genipin-crosslinking. AFCs encapsulated in microbeads demonstrate high cell survival, which is vital for long-term healing while high-modulus fibrin hydrogels provide immediate stabilization to degenerated IVDs. Subsequently, the FibGen-MB hydrogels are functionalized with RGD peptides and demonstrate significantly reduced AFC apoptosis and maintained phenotypic gene expression. The cell-encapsulated FibGen-MB-RGD hydrogels are assessed during a prolonged bovine caudal IVD organ model and exhibit a low risk of herniation . Section title: Applications of alginate-based materials in combined therapy of AF and NP Educational score: 4.1823930740356445 Domain: biomedical Document type: Study Language: en Inflammatory cytokines including interleukin-1 and tumor necrosis factor α are elevated in IVDD and participate in inflammation cascade and ECM degradation. Kakutani et al. determined that concurrent treatment inhibits IL-1β and TNF-α production of NPCs, AFCs, and explants encapsulated in alginate by using IL-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor receptor-1 (sTNFR1) and promotes proteoglycan and collagen production in the NPCs and AFCs. Additionally, NPCs and AFCs produce these two cytokines in autocrine or paracrine manners via feedback regulation . Du et al. designed a biomimetic annulus fibrosus and nucleus pulposus composite consisting of circumferential poly (ε-caprolactone) microfibers laden with AFCs and an NPCs encapsulated alginate hydrogel core. The artificial IVD exhibits increased ECM deposition and organization, as well as strengthened mechanical strength . Section title: Chitosan Educational score: 4.593944549560547 Domain: biomedical Document type: Study Language: en Chitosan is a biopolymer obtained from chitin, an abundant polysaccharide present in the exoskeletons of crustaceans and in fungal cell walls. Chitosan is formed by deacetylating chitin, a process that removes acetyl groups from the polymer chain . Structurally, chitosan is composed primarily of β-(1–4)-linked D-glucosamine units (deacetylated units) and N-acetyl-D-glucosamine units (acetylated units). The extent of deacetylation (DD) determines the ratio of glucosamine to N-acetylglucosamine units in the polymer chain . An acid hydrolysis product of chitosan, chitosan oligosaccharide (COS), has been shown to have significant anti-inflammatory properties. It can inhibit LPS-induced inflammatory cytokines such as TNF-α and IL-6 and nitric oxide secretion in a dose-dependent manner, thereby suppressing phosphorylation of JNK and translocation of p65, a subunit of NF-κB, into the nucleus . The unique combination of biocompatibility, biodegradability, antimicrobial properties, and ability to form films and gels of chitosan makes it a versatile biomaterial in diverse fields, for example, biomedicine, food technology, agriculture, and industrial applications . Section title: Applications of chitosan-based materials in NP regeneration Educational score: 4.365435600280762 Domain: biomedical Document type: Study Language: en Based on the specific hypothesis that the cationic chitosan, due to its positive charge, creates an ideal environment for the entrapment of large quantities of newly synthesized anionic proteoglycans. Roughley et al. found that various formulations of cell-encapsulated chitosan hydrogels preserved a considerable portion of the proteoglycan generated by NPCs inside the hydrogel matrix, therefore suggesting the capability of chitosan as a platform for cell-loaded supplementation aimed at restoring disc function and structure . Furthermore, thermosensitive chitosan-glycerophosphate hydrogel has been proved to promote MSCs secreting proteoglycans and collagens in a proportion that is similar to that of nucleus pulposus cells . Among these different formulations, a physical crosslinked hydrogel that contains 2 % (w/v) chitosan, 0.075M sodium hydrogen carbonate, and 0.1M β-glycerophosphate proves to simulate the human nucleus pulposus tissue in mechanical properties and stimulates the highest production and retention of glycosaminoglycans by the loaded cells within the gel . Through N-hexanoylation of glycol chitosan, Li et al. developed a new hydrogel system with tunable thermos-sensitivity and enhanced stability. Biochemical serum profiles and histological examination of hexanoyl glycol chitosan-treated rats demonstrate excellent biocompatibility compared to saline-treated rats. The hydrogel maintains its stability for over a month in an ex vivo porcine system, supporting its potential use in IVDD . Section title: Applications of chitosan-based materials in NP regeneration Educational score: 4.466951370239258 Domain: biomedical Document type: Study Language: en Inflammation is critical in the pathogenesis of IVDD, with macrophages and inflammatory pathways significantly contributing to disc degeneration . Chronic low-grade inflammation and cellular senescence further exacerbate the condition. Targeting inflammatory processes offers a promising strategy for the treatment and management of IVDD . Diclofenac, one of non-steroidal anti-inflammatory drugs (NSAIDs), has been reported to impede the pro-inflammatory polarization of human macrophages. Teixeira et al. evaluated the anti-inflammatory action of nanoparticles (NP) of chitosan and poly-(γ-glutamic acid) with diclofenac (Df). After internalization of IVD cells, nanoparticles inhibit IL-6, IL-8, PGE2, and MMPs expression and upregulate aggrecan and type Ⅱ collagen production . Subsequently, the same research group improved the nanocomplexes at pH 7.4 instead of 5.0 in that they found NPs under such an environment promote ECM anabolism including type Ⅱ collagen and sulfated glycosaminoglycan production . Furthermore, via systematic analysis of immune cell responses, the results show that pro-inflammatory intradiscal administration leads to aggravated IVD lesions, as indicated by increased CD4 + T cells (CD4 + CD8-TcR + CD161-) in the lymph nodes and blood and more pro-inflammatory cytokines, such as IL-1β, IL-6, and COX-2. In contrast, intradiscal anti-inflammatory administration slows the breakdown of the injured IVD and is associated with elevated systemic myeloid cell level (CD11b + MHCⅡ+) . Fig. 7 Chitosan-based biomaterials for intervertebral disc regeneration and repair. (A) Anti-oxidant and anti-inflammatory effect of mesenchymal stem cell-derived exosomes derived from C57BL/6 mice in an IVDD rabbit model and possible signal pathway. (i) Immunocytochemistry of NLRP3 and TXNIP. (ii) TEM to evaluate the microscopic structure of mitochondria (Adapted from Ref. ). (B) In situ forming an injectable Chitosan/PEG hydrogel shaped by Schiff base reaction with light exposure under 405 nm LED and (iii) Immunohistochemistry staining of Aggrecan and collagen II (Adapted from Ref. ). (C) Core-shell oxygen-releasing fibers via coaxial electrospinning, actions of PFTBA on the viability and functions of AFSCs, and implantation of the PFTBA core-shell scaffold in AF repair after discectomy and (iv) Fluorescence microscopy of the core-shell structure. (v) X-ray, MRI and Micro-CT (Adapted from Ref. ). Fig. 7 Section title: Applications of chitosan-based materials in NP regeneration Educational score: 4.341644287109375 Domain: biomedical Document type: Study Language: en Traditionally, simple chitosan hydrogel possesses low mechanical strength, therefore limiting its use to biomedical applications. Huang et al. introduced a fast in situ crosslinking injectable chitosan/polyethylene glycol (PEG) hydrogel with improved machinal strength via incorporating a photo-crosslink of methacrylate and Schiff base reaction between these two components simultaneously. In vitro experiments have proved the biocompatibility and proliferation of NPCs. Micro-CT and MRI at 4 and 8 weeks claim promising effects on retarding the IVDD in rat tails . Similarly, Adoungotchodo et al. fabricated a chitosan-based hydrogel incorporating gelatine and Link N (LN), a peptide that naturally occurs in cartilage and the ECM of IVD. Rheological and compressive mechanical tests demonstrate that gelatine mildly affects hydrogel gelling but significantly strengthens mechanical properties in compression. Moreover, LN induces notably high glycosaminoglycan production in degenerative environments . Li et al. used poly (l-lactic acid) to enhance mechanical strength by distributing the former throughout chitosan scaffolds with human umbilical cord mesenchymal stem cells encapsulated. In vitro cell culture claimed that this hydrogel network is suitable for cell adhesion and proliferation. After being inserted for lumbar fusion, chitosan/PLLA enchance bone connectivity between adjacent vertebrae and, in the meantime, aids in the regeneration of annulus fibrosus tissue . Section title: Applications of chitosan-based materials in NP regeneration Educational score: 4.173334121704102 Domain: biomedical Document type: Study Language: en Based on the distinctive biomechanical characteristics of native cellulose nanofibers (CNFs), a 3D printed, nature-inspired CNF-filled CHI hydrogel was proposed in which the former brings in essential mechanical reinforcement, whereas the latter acts as a biocompatible matrix that promotes cell growth. Because chitosan and cellulose are purely natural and are fabricated without any modification of components and the addition of a chemical crosslinker, this integrated hydrogel matrix supports 3D cell seeding with superior cell viability. The composite hydrogel opens up new possibilities for designing demanding engineered tissue of intervertebral discs . Section title: Applications of chitosan-based materials in AF regeneration Educational score: 4.118124008178711 Domain: biomedical Document type: Study Language: en To date, there has been limited information available regarding strategies to address the challenge of hypoxia in annulus fibrosus (AF) defect sites. However, in a recent study, researchers have explored the use of perfluorotributylamine (PFTBA) core-shell fibers (10 % chitosan, chitosan: PCL, 1:6) fabricated via coaxial electrospinning. This approach aims to fabricate an oxygen-releasing platform designed to promote natural reconstruction processes in the annulus fibrosus following resecting disc. In vitro studies demonstrate that hydrogels facilitate proliferation, migration and ECM synthesis in annulus fibrosus stem cells under hypoxic conditions and in vivo studies show notable improvements in vertebral spacing and structural unity observed in animal models treated with these oxygen-releasing biomaterials . Section title: Applications of chitosan-based materials in combined therapy of AF and NP Educational score: 4.285333633422852 Domain: biomedical Document type: Study Language: en Tissue-engineered intervertebral discs (TE-IVDs) have been considered an effective therapeutic solution for treating IVDD. Yang et al. developed an artificial IVD employing chitosan hydrogel to mimic the central nucleus pulposus region, enclosed by a poly (butylene succinate-co-terephthalate) fiber layer to represent the inner annulus fibrosus and employing a poly (ether ether ketone) ring to simulate the outer annulus fibrosus with IVD cells seeded. The novel tissue-engineered IVD establishes a conducive setting for supporting the development of IVD cells. Overall morphology and biological functions of the scaffolds closely resemble those of natural porcine IVDs . Afterward, Yuan et al. constructed similar TEIVDs using poly (butylene succinate-co-terephthalate) copolyester as inner annulus fibrosus (IAF) while solid PBST as outer annulus fibrosus (OAF), and using chitosan as NP. AF and NP cells are loaded onto the corresponding scaffolds. Of note, the IVD cells express cell-specific extracellular matrix, therefore suggesting that TE-IVD is biologically functional. X-ray and MRI examinations indicated that the intervertebral disc space was maintained 4 weeks later after TE-IVD was engrafted into New Zealand white rabbits . Section title: Conclusions and perspectives Educational score: 4.561685562133789 Domain: biomedical Document type: Review Language: en Polysaccharide-based materials hold significant promise as therapeutic agents for IVDD, offering a versatile and biocompatible platform for tissue regeneration. Of these materials, hyaluronic acid and chondroitin sulfate, two key components of the NP's extracellular matrix, possess the strongest ability to stimulate the nucleus pulposus environment with the ability to retain water and resist compressive forces. Chitosan has superior adhesivity to alginate, which has moderate adhesivity, and hyaluronic acid. With regard to biomechanical strength, alginate stands out with its tunable mechanical strength and stability. Moreover, compared to other inorganic materials or synthetic polymers which may require harsher processing conditions and yield harmful byproducts, polysaccharides can be processed under mild conditions such as low temperature, neutral pH and degrade via enzymatic hydrolysis. In the meanwhile, polysaccharides possess excellent bioactivity and inherent hydrophilic properties to retain hydration similar to native NP tissue. Of note, while proteins like collagen and synthetic polymers like PLGA offer tunability, polysaccharides like HA or chitosan can reach a unique balance of bioactivity and mechanical properties. To conclude, these polysaccharide-based biomaterials, including hyaluronic acid, chitosan, and alginate have demonstrated the ability to mimic the extracellular matrix of the NP, support cell survival, and enhance matrix synthesis, making them ideal candidates for disc repair and regeneration. Their capacity to deliver bioactive molecules directly to the degenerative site as well as their cost-effectiveness further amplifies their therapeutic potential. Section title: Conclusions and perspectives Educational score: 4.019969940185547 Domain: biomedical Document type: Review Language: en Nevertheless, several challenges still remain despite of all these advances. The intricate biomechanics of the IVD necessitate the use of materials that are capable of providing support for cellular functions while simultaneously withstanding the mechanical loads and stresses that are inherent to the spinal environment. The AF and NP are anatomically and functionally interdependent and the integrity of NP and AF in IVD are needed to be considered. For instance, a weakened AF may lead to NP herniation, while a degenerated NP can lead to AF collapse. Herein, the combined therapy targeting both the NP and AF is critical for achieving comprehensive and durable outcomes. Furthermore, the transition of these materials from laboratory to clinical settings requires comprehensive investigations into their long-term biocompatibility, degradation profiles, and potential immunogenic responses. The development of injectable and minimally invasive polysaccharide-based systems is also of great importance in order to ensure clinical applicability and patient compliance. Section title: Conclusions and perspectives Educational score: 4.037980556488037 Domain: biomedical Document type: Review Language: en In the future, the integration of advanced technologies, including three-dimensional bioprinting, nanotechnology, and gene editing, may provide new avenues for enhancing the efficacy of polysaccharide-based therapies. Polysaccharide-based materials can be customized to suit the specific requirements of both AF and NP, for instance, customized stiffness and elasticity through chemical modifications, functionalized polymer grafts, or in combination with other materials to fabricate composites, which may pave the way for meeting combined AF and NP therapy. 3D bioprinting can facilitate the precise fabrication of complex disc structures, while nanotechnology may be employed to design nanoparticles for targeted drug delivery within the disc environment. Gene editing technologies, such as CRISPR, may facilitate the modification of cells encapsulated within these hydrogels to produce specific proteins or growth factors that enhance tissue regeneration. Section title: Conclusions and perspectives Educational score: 3.791417121887207 Domain: biomedical Document type: Review Language: en In conclusion, polysaccharide-based materials represent a promising and adaptable approach for the therapy of IVDD. Continued research and technological integration are essential to overcome current limitations and realize their full potential in clinical settings. Advancing these materials and strategies may facilitate the development of effective, long-lasting solutions for patients suffering from intervertebral disc degeneration. Section title: CRediT authorship contribution statement Educational score: 0.9500595331192017 Domain: other Document type: Other Language: en Xin Wang: Writing – original draft, Software, Resources. Yixue Huang: Writing – original draft, Software, Resources. Yilin Yang: Resources. Xin Tian: Resources. Yesheng Jin: Resources. Weimin Jiang: Funding acquisition, Conceptualization. Hanliang He: Writing – review & editing, Funding acquisition, Conceptualization. Yong Xu: Writing – review & editing, Funding acquisition, Conceptualization. Yijie Liu: Writing – review & editing, Funding acquisition, Conceptualization. Section title: Declaration of competing interest Educational score: 0.9818206429481506 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Study | biomedical | en | 0.999997 |
PMC11699362 | Section title: Introduction Educational score: 3.467787027359009 Domain: biomedical Document type: Other Language: en The umbilical cord is a delicate, adaptable structure that associates the embryo to the mother through the placenta from the 6th week of pregnancy until birth. It contains one vein, which carries oxygenated, nutrient-rich blood to the fetus, and two arteries which carry deoxygenated, nutrient-depleted blood away from the fetus. The stump continuously dries, shrinks, and isolates from the body, typically somewhere in the range of 5 and 15 days after birth with shading change from a yellow-green to dark as it dries out. During this period, the umbilical cord ought to be kept perfect and dry to dodge contamination . Section title: Introduction Educational score: 2.864753007888794 Domain: biomedical Document type: Other Language: en Care of the umbilical cord is the series of care applied in handling of the umbilical cord after delivery of the newborn. This includes clamping, cutting, and serial daily care of the umbilical stump post-delivery. These actions, if not meticulously carried out will contribute significantly to the newborn risk of neonatal infections and mortality . Umbilical cord infections contribute significantly to neonatal deaths. In sub-Saharan Africa, neonatal infections are the most common cause of death among children under age five. Poor and unhygienic cord care practices are often to blame . Section title: Introduction Educational score: 2.176600456237793 Domain: biomedical Document type: Other Language: en In countries with limited resources, umbilical cord infection continues to be a major cause of neonatal morbidity and poses a significant risk of mortality . However, best practices for umbilical cord care (UC) remain controversial in developed countries with aseptic perinatal care and dry cord care . Section title: Introduction Educational score: 3.9121527671813965 Domain: biomedical Document type: Review Language: en Numerous further intercessions to forestall umbilical string contamination have been attempted. The two that have been studied most extensively are 70° alcohol and 4 % chlorhexidine, administered in different dosage forms (alcoholic or aqueous solution, gel or powder). Other antiseptics used for the purpose are triple dye (a combination of 3 disinfectant solutions frequently used in the United States), povidone, iodine, and salicylic acid, among others. Topical antibiotics such as silver sulfadiazine, tetracyclines, or neomycin have also been used. However, the application of any CHX to the umbilical cord of the newborn led to a 23 % reduction in all-cause neonatal mortality . Section title: Introduction Educational score: 3.8252065181732178 Domain: biomedical Document type: Study Language: en WHO recommends dry cord care for nations with good obstetric care and low neonatal mortality rates. Recent studies suggest applying chlorhexidine on the umbilical cord stump during the first week of life for neonates delivered at home in settings with high NMR of 30/1000 live births or higher . The Ethiopian government introduced the Community Based Newborn Care (CBNC) program in 2013, to improve maternal and newborn health outcomes. Chlorhexidine was included as one of the components in CBNC after consensus building, and it was manufactured locally. So Chlorhexidine is a feasible and acceptable intervention in the Ethiopian setting . In addition to this and antiseptic mothers' awareness about cord care practices and maternal higher educational level were important factors in maintaining the cord in a healthy state . Section title: Introduction Educational score: 2.406970500946045 Domain: biomedical Document type: Study Language: en Cord infections in newborns range from 3 to 5% in developing countries and 0.5 % in developed ones. The WHO estimates over 4 million neonatal deaths occur each year, with 3.4 million in the first week, and the highest number of deaths within the first 24 h of life . However, there are no recent studies regarding mothers' cord care practices including harmful traditional practices that might strongly contribute to newborn infection which leads to neonatal mortality and morbidity in Ethiopia as well as in the study area. Section title: Introduction Educational score: 2.0484025478363037 Domain: biomedical Document type: Other Language: en Neonatal mortality is high worldwide, especially in sub-Saharan Africa. Neonatal sepsis and tetanus are major causes of neonatal death, with neonatal sepsis responsible for over 15 % of neonatal deaths globally and is the third leading cause of death among infants in their first month of life . Section title: Introduction Educational score: 1.912734031677246 Domain: biomedical Document type: Other Language: en . Some traditional substances, such as cow dung, herbal preparations, ash, mud, and oils, are often applied to promote healing and hasten cord separation . However, the use of CHX in these situations may be considered to replace the application of a harmful traditional substance to the cord stump . Section title: Introduction Educational score: 2.06717848777771 Domain: biomedical Document type: Other Language: en Bacterial colonization of the cord can result in omphalitis, thrombophlebitis, cellulitis, or necrotizing fasciitis. Various topical substances continue to be used for cord care around the world to mitigate the risk of serious infection . Section title: Introduction Educational score: 3.8555452823638916 Domain: biomedical Document type: Study Language: en Even though Chlorhexidine application delays the time to cord separation, its application to the cord reduces the risk of neonatal mortality and omphalitis in infants born at home in high-NMR settings . A single intervention of 4 % CHX has been displayed by numerous studies to effectively reduce the incidence of omphalitis-related mortality by approximately 25 % . Section title: Introduction Educational score: 2.0633537769317627 Domain: biomedical Document type: Study Language: en The umbilical cord is a common entry point for bacteria causing neonatal sepsis. Harmful traditional practices related to umbilical cord care are influenced by cultural beliefs of mothers. However, there is limited information available on this topic in the study area and in Ethiopia. This study aims to fill this gap. Section title: Study design Educational score: 2.245750904083252 Domain: biomedical Document type: Study Language: en A mixed quantitative community-based cross-sectional study, supplemented with a qualitative study design was conducted. Section title: Study area and period Educational score: 2.1877992153167725 Domain: biomedical Document type: Study Language: en The study was conducted from April 15 to May 15, 2021, in Arsi Zone Hetosa district. The Hetosa District is one of the largest districts of Arsi Zone, Oromia Regional State of Central Ethiopia. It is located 160 Km from Addis Ababa, the capital city of Ethiopia. The district is typical of the zone in terms of population density, socio–cultural and economic state, and demographic conditions. It has a total of 25 (twenty-two rural and three urban) kebeles in number. The area lies between 08° 08′N-08°13′ E latitude and 39° 14′ N −39° 23′ E longitude with an elevation range from 1500-4170 m above sea level with an estimated total population of 178,229 and 36,597 households according to the 2007 national census which was conducted by the Central Statistical Agency of Ethiopia (CSA). The estimated total number of women of reproductive age group and women who gave birth in the last six months at the time of data collection in the district (both rural and urban) was 39,210 and 2248, respectively. Section title: Source population Educational score: 1.852466344833374 Domain: biomedical Document type: Study Language: en All mothers who gave birth in the last six months at the time of data collection in the Hetosa district were considered as a source population for the quantitative part of this study population. Section title: The study population for quantitative data Educational score: 1.8476794958114624 Domain: biomedical Document type: Study Language: en All mothers who gave birth in the last six months at the time of data collection in selected kebeles of the Hetosa district were considered as a study population. Section title: The study population for qualitative data Educational score: 1.946057915687561 Domain: biomedical Document type: Study Language: en All postnatal mothers and all traditional birth attendants (TBAs), all health extension workers (HEWs), and all grandmothers in selected kebeles were considered as a study population. Section title: Inclusion criteria for quantitative data Educational score: 1.7999117374420166 Domain: biomedical Document type: Study Language: en All mothers who gave birth in the last 6-month period preceding the study were included in the study. Section title: Exclusion criteria quantitative data Educational score: 1.4955767393112183 Domain: biomedical Document type: Other Language: en All mothers who stay in the study area for less than 6 months. Section title: Exclusion criteria quantitative data Educational score: 1.6403995752334595 Domain: biomedical Document type: Other Language: en All mothers who gave birth to died fetuses or stillbirths. Section title: Exclusion criteria quantitative data Educational score: 1.5887690782546997 Domain: biomedical Document type: Other Language: en All mothers whose newborns stay in NICU more than three days after delivery. Section title: Exclusion criteria for the qualitative part Educational score: 1.608776569366455 Domain: biomedical Document type: Study Language: en Participants interviewed in the quantitative survey were excluded from in-depth interviews. Section title: Sample size determination for quantitative study Educational score: 3.920994997024536 Domain: biomedical Document type: Study Language: en The sample size for this particular study was determined by using a single population proportion formula using a basic assumption of a 95 % confidence level and, a 5 % margin of error by considering the proportion of safe cord care was 68.3 % with a study conducted in Nekemte city Oromia region, Ethiopia . n i = z 2 P ( 1 − P ) d 2 , ( 1.96 ) 2 0.683 ( 1 − 0.683 ) ( 0.05 ) 2 = 333 Where, Section title: Sample size determination for quantitative study Educational score: 2.3721015453338623 Domain: biomedical Document type: Other Language: it Z = standard normal distribution value = 1.96 at Section title: Sample size determination for quantitative study Educational score: 2.178570508956909 Domain: biomedical Document type: Study Language: en α value of 0.05. Section title: Sample size determination for quantitative study Educational score: 2.1175296306610107 Domain: biomedical Document type: Study Language: en W = margin of error = 0.05. Section title: Sample size determination for quantitative study Educational score: 2.158841848373413 Domain: biomedical Document type: Study Language: en Using Design effect = 1.5. Section title: Sample size determination for quantitative study Educational score: 2.5439319610595703 Domain: biomedical Document type: Study Language: en Since it is a multi-stage sampling technique by using the design effect and multiplying by 1.5 becomes 500 and Contingency (for non-response = 10 %) = 50 the final sample size is n f = 550. Section title: Assumptions Educational score: 1.8502628803253174 Domain: biomedical Document type: Other Language: it Ni = initial sample size; 333; Section title: Assumptions Educational score: 1.6695539951324463 Domain: biomedical Document type: Other Language: ro NF=Final sample size = 550; Section title: Assumptions Educational score: 2.1290528774261475 Domain: biomedical Document type: Study Language: en Confidence level which will be 95 %; Section title: Assumptions Educational score: 1.9441970586776733 Domain: biomedical Document type: Other Language: en P = proportion = 68.3 %; Section title: Assumptions Educational score: 2.3472068309783936 Domain: biomedical Document type: Study Language: en d = the margin of error will be taken as 5 %. Section title: Sample size determination for qualitative Educational score: 2.022554874420166 Domain: biomedical Document type: Study Language: en Qualitative data were collected using a total of twelve In-depth interviews which involved postnatal mothers and from Key Informants. Key informant interviews were conducted among Traditional Birth Attendants (TBAs), grandmothers, and Health Extension Workers (HEWs) in the community until information saturation was obtained. Section title: Sampling procedure for quantitative data Educational score: 3.6395041942596436 Domain: biomedical Document type: Study Language: en Multi-stage sampling technique was used to select the study participants. The study was conducted in eight Kebele's of hetosa district which is 30 % of the total kebeles. From twenty-five Kebele's found in the district, eight (seven rural and one urban) Kebele's were selected using simple random sampling method. The sample size was allocated to each Kebele proportional to the number of women who gave birth in the last six months from selected Kebele. Skip interval was determined for each kebele by dividing the estimated population by the respective sample size (i.e. Kth = N/n). The expected total number of women who gave birth in the last six months in selected kebeles was N = 1241. Hence, each participant was selected by systematic random sampling with a skip interval of 2 at each kebele. Using the lottery method, 1 was selected and used as a starting number based on the mother's registration book. Subsequently, every other mother was included until the desired sample size was achieved . K = N n f K = 1241 550 = 2.25 ≈ 2 Where, N = total Number of mothers who gave birth in the last six months in selected kebeles. Fig. 1 schematic representation of sampling technique to assess cord care practices among mothers who gave birth in the last six months in Hetosa district, Arsi zone, Ethiopia, 2021. Fig. 1 Section title: Sampling procedure for quantitative data Educational score: 1.7924124002456665 Domain: biomedical Document type: Other Language: ro n f = final sample size; Section title: Sampling procedure for quantitative data Educational score: 1.6636143922805786 Domain: biomedical Document type: Other Language: en K=Sampling interval. Section title: Sampling procedure for qualitative data Educational score: 3.406512498855591 Domain: biomedical Document type: Study Language: en The purposive sampling technique was used for the qualitative study. The open-ended questionnaire was prepared to obtain an in-depth understanding of neonatal cord care practices and associated factors. A total of 12 in-depth interviews (IDIs) were conducted. The IDIs involved 4 mothers, 3 traditional birth attendants (TBAs), 3 Health Extension Workers (HEWs), and 2 grandmothers all of whom were selected purposively based on their close relation with mothers and neonates, involvement in the community and an assumption to be the richness of information on the topic of the study. The number of IDIs was determined based on the level of saturation of the required information. Section title: Dependent variables Educational score: 1.7259526252746582 Domain: biomedical Document type: Other Language: ro ❖ Umbilical cord care practice Section title: Independent variables Educational score: 2.471025228500366 Domain: biomedical Document type: Study Language: en ❖ Socio-demographic variables: Age of respondents, Marital status, Residence, Educational status, Religion, Ethnicity, Monthly income, and Occupation ❖ Obstetrical and maternal health service variables: Antenatal care, Place of antenatal care, Number of pregnancies, Number of live births, Number of live children, Place of birth, Postnatal checkup, Birth attendance, and Sex of child. ❖ Cultural and traditional variables: substances application on cord, Type of substance applied, Frequency of application, Time of application, Source of advice about substance applied ❖ Mothers' Knowledge of umbilical cord care practice and source of information about cord care practice. Section title: Operational definitions and terms of definition Educational score: 2.266920566558838 Domain: biomedical Document type: Other Language: en Umbilical cord care practice: All types of care of the umbilical cord are administered by a mother postpartum, including topical applications of various substances to the umbilical cord stump from birth to when it naturally falls off . Section title: Operational definitions and terms of definition Educational score: 2.228868007659912 Domain: biomedical Document type: Other Language: en Dry cord care : Refers to care of the cord whereby no chemical substance is applied to the cord, only cleaning with plain water and soap until it falls off . Section title: Operational definitions and terms of definition Educational score: 2.6797068119049072 Domain: biomedical Document type: Other Language: en Good cord care: Defined as keeping the cord, clean and dry without application of any substance on the cord stump except medically indicated medications like chlorhexidine after cutting the umbilical cord . Section title: Operational definitions and terms of definition Educational score: 2.7512567043304443 Domain: biomedical Document type: Other Language: en Poor cord care: was defined as when the substance was applied to the cord stump of a newborn other than Chlorhexidine after cutting the umbilical cord. Section title: Operational definitions and terms of definition Educational score: 1.767422080039978 Domain: biomedical Document type: Other Language: en Good knowledge of mothers: Knowledge was "good" for mothers who correctly responded to 3 or more knowledge related questions . Section title: Operational definitions and terms of definition Educational score: 1.754181981086731 Domain: biomedical Document type: Other Language: en Poor knowledge of mothers: Knowledge was "poor" for mothers who correctly responded to less than 3 knowledge related questions correctly. Section title: Data collection tool and procedure for quantitative data Educational score: 2.661670207977295 Domain: biomedical Document type: Study Language: en A pre-tested, structured questionnaire was used to collect quantitative data. The instrument was derived from standard data collection tools and related literature. It was pre-tested for consistency of responses by taking 5 % of the sample size, conducted in a similar population but out of the selected kebeles. Necessary modifications were made accordingly before the actual data collection. To maintain validity, the questionnaire was prepared in English then translated into Afan Oromo and Amharic versions and back-translated to English by the principal investigator and language experts to ensure consistency and accuracy. The data were collected by trained 8 BSC holders with health-related fields from selected Keble's. Two supervisors were assigned to facilitate data collection. During data collection, the participants were told the significance of the research, and the consent of the respondents was taken. Section title: Data collection tool and procedure for qualitative data Educational score: 1.92924964427948 Domain: biomedical Document type: Study Language: en To generate qualitative information, an in-depth interview (IDI) was performed/conducted by the principal investigator. Each interview was audio recorded and taken after obtaining oral voluntary consent from the interview participants. Section title: Data quality control for quantitative data Educational score: 2.086195707321167 Domain: biomedical Document type: Study Language: en The two-day training was given to all data collectors on instructions on how to fill out the questionnaire and supervisors to facilitate data collection. The principal investigator& supervisors were closely following the day-to-day data collection process and ensure completeness and consistency. All the completed questionnaire was returned daily. Data were checked for completeness, accuracy, clarity, and consistency before being entered into the software. Proper coding and categorization of data were maintained for the quality of the data to be analyzed. Section title: Data quality assurance for qualitative data Educational score: 2.2129528522491455 Domain: biomedical Document type: Study Language: en Open-ended IDI questions taken from the literature were used for qualitative data collection The data were collected by the principal investigator by audio recording and note-taking. Trustworthiness or quality of the data analysis was ensured by data source triangulation by interviewing different bodies in the community. Furthermore, allocating sufficient time; to collect data and maintaining an objective and impartial view further added to the reliability of the data. Section title: Data Processing and analysis for quantitative data Educational score: 4.087383270263672 Domain: biomedical Document type: Study Language: en The pre-coded data were entered into Epi data version 3.1 software and then it was exported to SPSS version 25 for statistical analysis. A score of one was given for good cord care practice and zero for poor cord care practice. A binary logistic regression was used to identify the association of the independent variables with the dependent variable. Each variable that has p-value less than 0.25 was added to the final model to control the confounders. Variables that have a p-value <0.05 with a 95 % confidence interval in the final model were declared statistically significant. Knowledge of mothers on newborn cord care was assessed using a series of six close-ended questions and a composite variable was generated from these questions to categorize mothers as having “Good/poor” knowledge. A scoring system was used to analyze responses to closed-ended questions on Knowledge: 1 = coded as correct response and 0 = coded as incorrect response. Accordingly, mothers who correctly responded to at least 3 questions were categorized as having a good knowledge of newborn cord care. The descriptive data were presented using frequency, tables, figures, mean, and standard deviation. Hosmer and Lameshow goodness of fit test was conducted to test the model fitness and the model was adequate (p = 0.266). Multicollinearity was checked by using VIF and it was <10. Section title: Data Processing and analysis for qualitative data Educational score: 3.1342248916625977 Domain: biomedical Document type: Study Language: en Primarily, audio-recorded data were heard repeatedly until the principal investigator became intimately familiar with the contents. The audio-recorded qualitative data were transcribed into the English language. Then, codes or terms were identified and tallied to come up with some categories, which were later used to establish themes based on the objective of the study. Unique concepts were identified and trials were made to elaborate more and report on the final result. Finally, thematic analysis was done manually and the findings were triangulated with the quantitative one. Section title: Data Processing and analysis for qualitative data Educational score: 1.7215825319290161 Domain: biomedical Document type: Other Language: en Ethical approval was obtained from the Institutional Ethical Review Committee (IERC) of Debre Markos university (DMU), college of health science. Following the approval by IERC, an official letter of cooperation was written to the Hetosa Woreda health administration office, and in turn, the district health administration office wrote letters to each selected kebeles administration office to get permission and cooperation. The written informed consent from the respondent was obtained. The confidentiality and privacy of the respondents was assured. Participants were given the chance to ask any doubt about the study and made free to refuse or stop the interview at any moment they wanted. Section title: Socio-demographic characteristics Educational score: 3.969616174697876 Domain: biomedical Document type: Study Language: en A total of 541 mothers who gave birth in the last six months participated in this study giving a response rate of 98.36 %. The mean (mean ± SD) age of the respondents was 28.41 ± 5.72 years. Of all participants, 488 (90.2 %) mothers were married and 471 (87.1 %) were Oromo in ethnicity. Among the total participants, 263 (48.6 %) mothers attended primary school and about 286(52.9 %) women were orthodox in religion. About occupational status housewife 397 (73.4 %) take a larger proportion. The majority 405(74.9 %) mothers were rural residents and 224 (53.5 %) respondents used radio as a mass media source of information ( Table 1 ). Table 1 Socio-demographic characteristics of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 1 Variable Category Freq Variable Category Freq Age ≤24 years 135(25.0 %) Maternal occupation Housewife Farmer Government employer Other(merchant, daily labourer, student) 397(73.4 %) 21(3.9 %) 69(10 %) 25–32 years 281(51.9 %) ≥33years 125(23.1 %) Marital status Single 26(4.8 %) Do you have a source of Information/mass media Yes No 419(22.6 %) 122(77.4 %) Married 469(86.7 %) Other (windowed, divorced, separated) 46(8.5 %) Ethnicity Oromo 471(87.0 %) Type of information source you have Radio Television 224(53.5 %) 195(46.5 %) Amhara 42(7.8 %) Other(Tigree, Gurage, Woliyta) 28(5.2 %) Religion Orthodox 285(52.7 %) Monthly income ≤1999 2000–3999 ≥4000 282(52.1 %) 154(28.5 %) 105(19.4 %) Muslim 189(34.9 %) Protestant 56(10.4 %) Other (catholic) 11(2.0 %) Education level No formal education 81(15.0 %) Residence Rural Urban 405(74.9 %) 136(25.1 %) primary 263(48.6 %) secondary and above 197(36.4 %) Section title: Obstetric and maternal service factors Educational score: 1.9144909381866455 Domain: biomedical Document type: Study Language: en Attendance of ANC was high among the respondents with 505 (93.3 %) reporting to have attended at least one ANC visit. Most 255(50.5 %) respondents had made four or more ANC visits in their most recent delivery. Section title: Obstetric and maternal service factors Educational score: 3.9296491146087646 Domain: biomedical Document type: Study Language: en From the total mothers who participated in this study 320(59.1 %) were multigravidas. The majority of the respondents delivered their babies at a health facility 418 (77.3 %) while 123 (22.7 %) had home deliveries. Almost all of the respondents delivered their babies at health facilities attended by health professionals. On the other hand majority of those who had their birth in the home were attended by families 46(37.7). Among the total participants, 216 (39.9 %) mothers had postnatal care after their deliveries of recent baby. Slightly more than half of the respondents 295(54.5) gave male babies ( Table 2 ) Table 2 Obstetric characteristics of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 2 Variable Category Frequency Percent % Number of ANC visit One 23 4.6 Two 80 15.8 Three 147 29.1 ≥four 255 50.5 Place of ANC Private clinic 72 14.3 Hospital 58 11.5 Health center 284 56.2 Other (health post) 91 18.0 Gravidity Primigravida 142 26.2 Multigravida 320 59.2 Grand multipara 79 14.6 Place of birth Home 123 22.7 Health facility 418 77.3 Birth attendant Health professional 418 77.3 TBA 45 8.3 Family 46 8.5 Friend 8 1.5 Other 24 4.4 Baby's sex Male 295 54.5 Female 246 45.5 ANC = antenatal care, TBA = traditional birth attendant. Section title: Source of information and knowledge of mothers on cord care Educational score: 3.7005269527435303 Domain: biomedical Document type: Study Language: en Slightly more than half 298(55.1 %) respondents received information regarding umbilical cord care and most of them 217(72.8 %) received the information from health professionals either before or after the delivery of their recent baby. Among the participants who had ANC visits only 82 (16.2 %) said they were informed about umbilical cord care during their ANC visit ( Table 3 ) Table 3 Source of information of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 3 Variable Category Frequency Percent % Received information regarding UCC before or after delivery Yes 298 55.1 No 243 44.9 A person who gave the information regarding UCC Health professional 217 72.8 Family 31 10.4 Neighbor 28 9.5 TBA 12 4.0 Mass media 7 2.3 Other (friend) 3 1.0 A place where the information received regarding UCC Health facility 194 65.1 Home 67 22.5 Neighbor 30 10.1 Other 7 2.3 Umbilical cord care education during ANC Yes 82 16.2 No 423 83.8 ANC = antenatal care, UCC = umbilical cord care. Section title: Source of information and knowledge of mothers on cord care Educational score: 3.877648115158081 Domain: biomedical Document type: Study Language: en With regard to knowledge, 91.9 % of the respondents acknowledged that they always wash their hands with soap and water before umbilical cord care. However, 283 (52.3 %) of them knew that no substance was applied to the cord stump except chlorohexidine. Of the participants, 450(83.2 %) and 396(73.2 %) knew that cord-cutting and cord-tying material should be sterile respectively ( Table 4 ). Table 4 Knowledge of mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 4 Variables Category Frequency Percent(%) Hand wash with soap and water before UCC Yes 497 91.9 No 44 8.1 No substance should be applied on the cord stump except chlorohexidine Yes 283 52.3 No 258 47.7 The diaper should not cover the cord Yes 185 34.2 No 356 65.8 Newborn bathing should be sponge bath till the cord is cut off Yes 152 28.1 No 389 71.9 Cord-cutting material should be sterile Yes 450 83.2 No 91 16.8 Cord tying material should be sterile Yes 396 73.2 No 145 26.8 Overall knowledge Good 380 70.2 Poor 161 29.8 UCC = umbilical cord care. Section title: Source of information and knowledge of mothers on cord care Educational score: 2.020496129989624 Domain: biomedical Document type: Study Language: en Regarding knowledge of handling umbilical cords around 70.2 % of study participants had good knowledge and 29.8 % had poor knowledge Fig. 2 Knowledge level about umbilical cord care among mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Fig. 2 Section title: Umbilical cord care practices among mothers Educational score: 2.3616249561309814 Domain: biomedical Document type: Study Language: en Overall good cord care practice was found to be 53.4 % [95%CI, 49–58] Section title: Umbilical cord care practices among mothers Educational score: 2.0075268745422363 Domain: biomedical Document type: Study Language: en Of the respondents practiced good cord care, 217(40.1 %) did provide dry cord care and respondents, 324 (59.9 %) applied substances on the cord. Section title: Umbilical cord care practices among mothers Educational score: 2.8838887214660645 Domain: biomedical Document type: Study Language: en Most study participants around 217(40.1 % of them applied dry cord care, 206(46.7 %) of them applied butter,110(24.9 %) of participants applied Vaseline (petroleum jelly), 7(1.6 %) lotion, 3(0.7 %) food oil, 1(0.2 %) alcohol and 5(1.1 %) of them apply applied commercially available substance such as TTC. But 109(24.7 %) of the respondents applied Chlorhexidine as single or in combination with other substances. However, only 72(13.3 %) applied Chlorhexidine alone appropriately (once daily for seven days) . Fig. 3 Common substances applied to the cord stump among mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Fig. 3 Section title: Umbilical cord care practices among mothers Educational score: 3.104607343673706 Domain: biomedical Document type: Study Language: en Almost all participants applied those substances for different reasons. Among them, 123(22.7 %) applied substances to moisturize and prevent dryness of the cord stump whereas, 116(21.4 %) applied to facilitate cord separation. Others 84(15.5 %) applied ∗that substance to promote of the respondents who applied the substance, 129(23.8 %) mothers started to apply the substance on the second day, and 80(24.7 %) applied once daily. Regarding a person applying a substance on the cord, in 189(58.3 %) cases the substance was applied by the baby's mother and of those respondents who applied substance 210(64.8 %) always washed their hands before they applied the substance. Among those respondents who applied substance on the cord stump majority of them 222(68.5 %) had received information about the substance they applied and most of them 133(59.9 %) received the information from family members. Section title: Umbilical cord care practices among mothers Educational score: 2.8583338260650635 Domain: biomedical Document type: Study Language: en Various instruments were used to cut the cord. Common instruments used to cut the cord among mothers who deliver at health facilities were surgical blades and boiled/new scissors. However, among participants delivered at a health facility, 180(42.9 %) respondents didn't remember the material used to cut the cord. But 110(99.1 %) mothers who deliver at home use new razor blades. However, 13(2.4 %) used old scissors/old razor blades in case of home delivery. Regarding materials used to tie the cord, a cord clamp was used to tie the cord in half of the respondents 274(50.6 %), otherwise, 105(19.4 %) participants didn't remember the material used to tie the cord. About 18(14.6 %) mothers who deliver at home didn't use anything to tie the cord or leave the cord without tying. Section title: Umbilical cord care practices among mothers Educational score: 3.3024888038635254 Domain: biomedical Document type: Study Language: en Among study participants, the majority of mothers wash their child's body by immersion of whole body in water. Out of those interviewed 195 (36.0 %) correctly stated that the cord should be dry after washing their baby's body and majority of them dry their baby by loosely covered using a clean piece of cloth. Of the participants in this study, 480(88.7 %) didn't notice any complication on the cord. However, some of the mothers 61(11.3 %) notice complications such as reddening of the umbilicus 28(5.2 %), pus at the umbilicus 20(3.7 %), cord bleeding 10(1.8 %), swelling of the umbilicus 3(0.6 %). Most of the mothers 444(82.1 %) did allow the cord to separate on its own. But some of them 97(17.9 %) interfere with the cord separation process or try to separate the cord rather than allow the cord to separate on its own. Section title: Qualitative results Educational score: 2.455242156982422 Domain: biomedical Document type: Study Language: en For the qualitative component study, four postnatal mothers, three traditional birth attendants (TBA), three health extension workers (HEW), and two grandmothers’ participants were involved in a total of twelve in-depth interviews. The age of the participants ranged from 22 to 70 years with a mean age of 46 years. Participants' educational levels varied from no formal education to college. Regarding their occupation all mothers involved in this study were housewives, two of TBA and all grandmothers were farmers and one TBA was a merchant in their profession. Otherwise, all health extension workers were government employers. The result of this finding was used as a supplement for quantitative study by exploring common cultural cord care practices and factors associated with their practice. Section title: Umbilical cord tying and cutting Educational score: 2.6947877407073975 Domain: biomedical Document type: Study Language: en The majority of the interviewees in the qualitative findings stated that the cord was cut with a new blade and tied by thread or string if delivery occurred at home. Some of them reported that the boiled new razor blade was used instead if delivery was not emergent. Most of them believed that old blades or scissors should not be used to cut the cord, instead, it is better to buy the new one and keep it as the pregnancy reaches to term. However, there is variability in what is used to tie the cord such as sewing thread and strips of Cloth” netelacherk” in the local language and some of them believe that it is not necessary to tie the cord. Most of the time the cord was cut at a length of one or two joints of a finger but there is variation in cord tying practice. “ …. while I attend labor, I always used a new razor blade that I always captured in my pocket which I always boiled before use and I never used razor blade that was used before and I tied the cord with sewing thread or strip of cloth netelacherk in absence of it” (IDI with A 55 years old TBA). “I always measure two joints of fingers to the newborn side and cut to prevent bleeding. It never bleeds if I leave two joints of my finger to the newborn side and I never tie the cord. But the reason behind to the cord bleeding comes from to short cord i.e. if the cord cut less than two finger joints”. (IDI with 70 years old TBA) “I have given nine children at home and I never faced any complication until my recent delivery in which all attendants measured the cord with finger joints and cut with a new razor blade that I bought from the market or a knife-like blade given from health post and the cord to the newborn side was never tied” (IDI with 41 years old mother). Section title: Fresh butter Educational score: 2.1222283840179443 Domain: biomedical Document type: Study Language: en In line with the quantitative finding, most of the participants in the qualitative study believe that the application of fresh butter is mandatory on and around the stump, whether delivery occurs at home or in a health facility. Section title: Fresh butter Educational score: 2.334749698638916 Domain: biomedical Document type: Other Language: en As reported by the majority of mothers and grandmothers, applying fresh butter to the cord stump immediately after cutting or a few days later will protect the baby from different harm. “… during all my deliveries I always applied fresh butter to the cord after I wash my newborn, which it prevents the cord from different hurts such as sticking to the cloth and associated pain with dryness of the cord. It is also used to facilitate its separation in which mostly not exceed no more than three days to fall if fresh better is applied continuously, and then it is better to hit the separated cord to the neck which prevents the newborn from sleep disturbance” (A 38 years old IDI participant mother). Section title: Vaseline/petroleum jelly Educational score: 1.9440734386444092 Domain: biomedical Document type: Other Language: en According to their opinion, Vaseline can replace fresh butter if it is not available or used instead of using butter stayed more than three days. If butter is not fresh or stays more than three days it may cause soreness or infection to the umbilical cord site. “… In the absence of fresh butter commercially available Vaseline which has no any smell can be used instead since butter stays for more days is too bad to apply” (A 65 years old IDI TBA participant). Section title: Alcohol Educational score: 1.690964698791504 Domain: biomedical Document type: Other Language: en Others believe that alcohol helps to stop bleeding sometimes caused by inappropriate cord cutting i.e. the cord might bleed heavily if it is too short or less than two joints of a finger. But they never notice that the reason behind cord bleeding is associated with cord tying. “In the past, we called the traditional birth attendant (TBA) in our village if the cord didn’t stop bleeding. Thus, once TBA applies alcohol that she took from the health facility, the cord stops bleeding immediately and never bleeds again” (A 60-year-old IDI participant's grandmother). Section title: Alcohol Educational score: 2.101267099380493 Domain: biomedical Document type: Study Language: en Although most mothers weren't aware of as chlorhexidine helps with cord care, all health extension workers who participated in this study believed that nothing should be applied to the cord except medically indicated substances chlorhexidine. Although cord care education during antenatal and postnatal care has a great contribution to preventing harmful cord care practices, the education regarding cord care wasn't given strictly. Section title: Alcohol Educational score: 1.7805436849594116 Domain: biomedical Document type: Other Language: en In addition, the availability of chlorhexidine helps the health extension worker to educate and remind the mother how to give care to her newborn cord which also helps to replace harmful substances applied at home. But currently, these antiseptics have not reached every health post. “I educate mothers not to apply other harmful substances such as cow butter and Vaseline other than chlorhexidine while I distribute this antiseptic, but in the absence of it I forgot to educate them about the care given to the cord at the time of their last antenatal visit” (IDI with 28 years old HEW). Section title: Reason for substance application Educational score: 1.9548802375793457 Domain: biomedical Document type: Study Language: en In line with the quantitative study, most of the participants in the qualitative study believed that, if the cord didn't separate within a day, it may cause pain while it dries and sticks to the cloth. Section title: Facilitate cord separation Educational score: 2.207730531692505 Domain: biomedical Document type: Study Language: en Most of the participants in the qualitative study believed that, if fresh butter is not applied to the cord, the cord didn't separate within a day or it may take more than a week, which may lead th∗e cord to dry and stick to the cloth. “I recommend mothers to apply fresh butter or Vaseline if the cord doesn’t separate within three days, because if the cord doesn’t fall within three days it becomes dry and causes severe pain, otherwise it is not obligatory to apply that substance if the cord falls within three days” (IDI with 55 years old TBA). Section title: Facilitate cord separation Educational score: 1.544944167137146 Domain: other Document type: Other Language: en Others believe that the separation of the cord depends on the mother's lifestyle or on what the mother eats or drinks otherwise the cord falls within a few days if the mother is rich in what she eats or drinks. “Sometimes mothers apply Vaseline if the cord didn’t fall within three days but this will not happen if the mother is prosperous and drinks milk” (IDI with 65-year-old grandmother). “On the rest of my children, I didn’t apply anything to the cord stump and the cord fell within four to five days but on my recent child, I applied Vaseline and the cord fell within three days as I was told by TBA in our village” (IDI with 32 years old mother). Section title: Facilitate cord separation Educational score: 1.6847425699234009 Domain: biomedical Document type: Other Language: en Even if health extension workers told mothers not to apply anything to the cord, some mothers especially those who are aged or who have several pregnancies didn't stop to apply those harmful substances. Rather they would like to practice what they have already done before rather than accept education given by health professionals . “Some mothers said you recommend not to apply anything to the cord, but I applied fresh butter to the cord since I already applied it to the rest of my children and it didn’t hurt any of them or I never saw any complication rather it facilitated it’s separation to fall within few days” (IDI with 28 years old HEW). Section title: Facilitate cord separation Educational score: 1.7059274911880493 Domain: biomedical Document type: Other Language: en This indicates that the acceptability of mothers from what they receive from health professionals is less than from what they already experience or from what they have already seen or heard from expert persons around them. This indicates that the mothers trust fewer health professionals than those other persons around them. Section title: Facilitate cord separation Educational score: 1.4406412839889526 Domain: biomedical Document type: Other Language: en According to most of the mother's ideas, the substance they apply on the cord was informed by a person assisting by their recent birth especially TBA in case of home delivery. Section title: Prevent the cord not to drying and sticking to the cloth Educational score: 2.1081020832061768 Domain: biomedical Document type: Study Language: en The other reason for substance application to the cord stump was the fear of the cord drying and associated pain. Most of them believed that, once the cord dries it may cause pain while it dries and sticks to the cloth. “If we apply fresh butter or Vaseline the cord never dries and becomes soft otherwise once the cord dries, it will stick to the cloth, which leads to severe pain that the newborn cannot tolerate” (IDI with 22 years mother). “I always prepare fresh butter during all my delivery. If we never apply butter to the cord, the cord becomes as hard as a bone the cord sticks to the cloth, and the baby will cry due to the pain cord sickness of the cloth. Thus, the butter prevents it from dryness” (IDI with 41-year-old mother). Section title: Factors associated with good cord care practice Educational score: 1.8278074264526367 Domain: biomedical Document type: Study Language: en In the study, all the preliminary assumptions such as model fitness and collinearity were. Section title: Factors associated with good cord care practice Educational score: 3.6503078937530518 Domain: biomedical Document type: Study Language: en Checked and found it to be satisfactory. Consequently, in bivariable logistic regression, the age of the mother, educational status of the mother, marital status, mother's occupation, having mass media, gravidity, place of birth, and knowledge status of the mother were included in multivariable analysis. However, after controlling the effect of confounding in multivariable analysis the final result confirmed that mothers' age, education, place of birth, and level of knowledge on umbilical cord care were found to be statistically significant at p-value <0.05. Section title: Factors associated with good cord care practice Educational score: 4.147165298461914 Domain: biomedical Document type: Study Language: en Hence, those who had primary, secondary, and above educational status had 5.26 times (AOR = 5.26, 95 % CI: 2.50, 11.04) and 3.63 times (AOR = 3.63, 95 % CI: 1.61, 8.18) more likely to practice good cord care than mothers who had no formal education, respectively. The odds of having a good cord care practice among age groups of ≤ 24years and 25–32years was 4.56 times (AOR = 4.56, 95 % CI; 2.08, 9.96) and 3.70 times (AOR = 3.70, 95 % CI; 1.98, 6.94) higher than mothers whose age ≥ 33 respectively. Hence younger mothers practice good cords as compared to older ones. The odds of delivering in a health facility was 5.09 times (AOR = 5.09, 95 % CI; 2.95, 8.78) was found to significantly associate good practice of cord care than among those who delivered outside the health facility and mothers who had good knowledge of newborn cord care practice were eight times more likely to practice good cord care than those who had poor knowledge (AOR = 8.58, 95 % CI; 5.09, 14.46) ( Table 5 ) Table 5 Bivariable and multivariable logistic regression model predicting the likelihood of good cord care practice among mothers who gave birth in the last six months in Arsi zone, Hetosa district, Ethiopia, 2021, (n = 541). Table 5 Variables Practice COR (95%CI) AOR (95%CI) P-value Good(%) poor (%) Age group (years) ≤24 92(31.8 %) 43(17.1 %) 7.08(4.08, 12.28) 4.56(2.08, 9.96) 0.000 a 0.000 a 25–32 168(58.1 %) 113(44.8 %) 4.92(3.04, 7.94) 3.07(1.98, 6.94) ≥33 29(23.2 %) 96(38.2 %) 1 1 Educational status No formal education 15(5.2 %) 66(26.2 %) 1 1 0.000 a 0.002 a Primary 148(51.2 %) 115(45.6 %) 5.66(3.07, 10.43) 5.26(2.50, 11.04) Secondary and above 126(43.6 %) 71(28.2 %) 7.80(4.15, 14.68) 3.63(1.61, 8.18) Mothers' occupation Housewife 192(66.4 %) 205(81.3 %) 0.33(0.18, 0.58) 0.46(0.20, 1.06) Farmer 10(3.5 %) 11(4.4 %) 0.32(0.11, 0.88) 1.03(0.24, 4.33) Government employer 36(12.5 %) 18(7.1 %) 0.70(0.32, 1.54) 0.81(0.28, 2.30) Other 51(17.6 %) 18(7.1 %) 1 1 Marital status Single 20(6.9 %) 6(2.4 %) 1 1 Married 240(83.1 %) 229(90.9 %) 0.31(0.12, 0.79) 0.59(0.13, 2.23) Other 29(10.0 %) 17(6.7 %) 0.51(0.17, 1.52) 1.20(0.26, 5.49) Access to mass media Yes 237(82.0 %) 182(72.2 %) 1.75(1.16, 2.63) 1.58(0.91, 2.73) No 52(18.0 %) 70(27.8 %) 1 1 Gravidity Primigravida 101(34.9 %) 41(16.3 %) 6.39(3.46, 11.76) 1.41(0.58, 3.41) Multigravida 166(57.4 %) 154(61.1 %) 2.79(1.63, 4.78) 0.83(0.39, 1.73) Grand multipara 22(7.6 %) 57(22.6 %) 1 1 Birthplace Health facility 260(90.0 %) 159(63.1 %) 5.24(3.30, 8.31) 5.09(2.95, 8.78) 0.000 a Home 29(10.0 %) 93(36.9 %) 1 1 Knowledge level Good 260(90.0 %) 120(47.6 %) 9.86(6.24, 15.56) 8.58(5.09, 14.46) 0.000 a Poor 29(10.0 %) 132(52.4 %) 1 1 a = p-value <0.05, COR = crude odds ratio and AOR = adjusted odds ratio. Section title: Discussion Educational score: 2.6104180812835693 Domain: biomedical Document type: Study Language: en Although the majority of women 380(70.2 %) had good knowledge of cord care, only 53.4 % [95%CI, 49%–58 %] of women had good cord care practice. Among 289(53.4 %) of the respondents had good cord care, 217(40.1 %) did provide dry cord care and 72(13.3 %) applied Chlorhexidine appropriately. The finding of this study was almost nearly similar to the study done in Chencha district, southern Ethiopia 52.9 % . Section title: Discussion Educational score: 3.669318437576294 Domain: biomedical Document type: Study Language: en But this finding was higher than the study conducted in Hossana town, Hadiya zone, southern Ethiopia, 32.9 %, in Garissa county, Kenya which was 43 %, a population survey in eastern Uganda 38 % and in Cameroon 19.7 % [ 18 , , , ]. And this difference might be due to relatively a recent increase in antenatal care visits and institutional delivery services. The difference can also be a result of chlorhexidine provision among mothers. In addition, the current community-based interventions by health extension workers might have contributed to the reduction of harmful cord care practices among mothers in the study area. This is supplemented by qualitative findings in Pemba Tanzania which revealed that there was consensus among respondents that CHX liquid cord cleansing could be successfully implemented in the community with appropriate education and awareness . Section title: Discussion Educational score: 2.1609323024749756 Domain: biomedical Document type: Study Language: en Inversely this finding was lower than the study conducted in rural Sidama Zone, Southern Ethiopia 73 %, in Nekemte City, western Ethiopia 68.3 %, in Jimma Zone, Southwest Ethiopia 86.5 % . The possible explanation for this difference might be due to the presence of socio-cultural differences that exists across the country. The discrepancy can also be a result of differences in residency women that are urban residents will have information that could assist them in making decisions regarding healthy behaviors including maternal and child health education, and promotion including newborn cord care practice. Section title: Discussion Educational score: 2.7135419845581055 Domain: biomedical Document type: Study Language: en This study finding was also lower than the studies carried out in Ibadan, Nigeria 61.4 %, 77.8 %, 2013 edition of the Nigeria Demographic and Health Survey (NDHS) 72.2 % . The reason for this difference might be the socio-cultural difference, geographical area, economic status, and difference in life standard across the countries. The other possible reason also might be due to the difference in the variables included in measuring cord care practice. Some studies included only cord cleaning or the substance used for cord cleaning and others included the substance applied and the materials used to cut and tie the cord. However, in this study, the substance applied after cord cutting was used to measure mothers' cord care practice. Section title: Discussion Educational score: 2.3325231075286865 Domain: biomedical Document type: Study Language: en This finding was supplemented by qualitative findings. In this study 59.9 % of mothers responded that substances were applied on the stump; the overall percentage of respondents who applied substances on cord stump is higher than that found in four regions of Ethiopia (Amhara, Oromia, SNNPR, and Tigray) in 21 % and butter is the most commonly used substance in both studies . Section title: Discussion Educational score: 2.062669038772583 Domain: biomedical Document type: Study Language: en With regard to the reason for application this study was consistent with a systematic review held in low and middle-income countries in which the desire to promote healing and hasten cord separation are the underlying beliefs related to the application of substances to the Section title: Discussion Educational score: 2.2868993282318115 Domain: biomedical Document type: Study Language: en Umbilical cord . With regard to the material used to cut the ∗cord, this study was similar to the study conducted in Tanzania in which all TBA used new razor blades to cut the cord . Some of the mothers and TBA never tied the cord in case of home delivery. This finding was consistent with the study conducted in Southern Ethiopia, rural Sidama zone in which 11.6 % of cases never tied the cord . Section title: Discussion Educational score: 2.068239450454712 Domain: biomedical Document type: Study Language: en In the current study, educated mothers were more likely to practice good cord care than those who had no formal education. A formally educated mother is more exposed and informed than an uneducated and as such can make better decisions regarding her child's health and will likely possess a much better health-seeking behavior. This study is supported by study findings conducted in Benin City, Edo State, Nigeria, and Calabar Metropolis Cross River State, Nigeria . Section title: Discussion Educational score: 2.0432322025299072 Domain: biomedical Document type: Study Language: en This finding is also in keeping with studies that show that the higher the level of maternal education, the better the health-seeking behavior and thus exposure to a better knowledge of childcare practices. This study is supported by study findings conducted in the Volta region of Ghana, in Abakaliki, Ebonyi State, South-East Nigeria, and in Yenagoa Local Government Area, Bayelsa State, Nigeria . Section title: Discussion Educational score: 3.2728426456451416 Domain: biomedical Document type: Study Language: en In this study, younger women were more likely to practice good cord care than those who are older women. The possible reason behind this can be those younger mothers were more likely educated than older ones. In addition, older mothers may not be equipped with the latest cord care guidelines which have been modified in recent times. Another explanation may be as the age of women increases the probability of having other previous pregnancies or children increases. This may lead women to apply more cultural practices received from their experience. This finding is opposed to studies conducted in Ibadan, Nigeria, in Benin City, Edo State, Nigeria, and the 2013 edition of the Nigeria Demographic and Health Survey (NDHS) which show that older women practice better cord care than younger mothers . This difference may be a result of the fact that older mothers tend to have delivered more times thann younger mothers and as such are more experienced in child care as they may have learned hard lessons as well following harmful cord care over the years. Section title: Discussion Educational score: 2.276271104812622 Domain: biomedical Document type: Study Language: en This study finding also supported the qualitative study that older mothers are more frequently like to apply some non-recommended substances which is harmful to the newborn. This finding is consistent with a study conducted in the voltage region of Ghana . This might be associated with mothers' less trust in health workers or due to their past influence from what they see, hear, or exercise. According to the interview with health extension workers, aged mothers refused to accept the recommended cord care practices. Section title: Discussion Educational score: 2.440131425857544 Domain: biomedical Document type: Study Language: en According to this study finding, mothers who deliver in the health facility were more likely to practice good cord care than those who deliver outside the health facility. The possible reason for this is that as the possibility of the mother delivering in a health facility increases, the opportunity for this mother to aware more of her newborn care increases. This is due to Facility childbirth had a strong association with appropriate newborn care probably because of the ease with which medical advice on appropriate newborn practices including cord care can be obtained and given . Section title: Discussion Educational score: 2.119105577468872 Domain: biomedical Document type: Study Language: en This finding is supported by study findings conducted in two communities of Plateau State, North Central Nigeria, in Yenagoa Local Government Area, Bayelsa State, Nigeria, and rural contemporary Nigeria setting which show that mothers who deliver in recognized health facilities are more likely to practice better cord care. Section title: Discussion Educational score: 2.116086959838867 Domain: biomedical Document type: Study Language: en According to this study finding, mothers who had overall good knowledge were more likely to practice good cord care than mothers who had poor overall knowledge. The possible explanation for this is that once the mother knows the recommended practices on the newborn umbilical cord, the possibility of practicing what she already knows is higher than what she didn't know. This study finding was supported by the study employed in Chencha District, Southern Ethiopia, and Hossana town, Hadiya zone, southern Ethiopia . Section title: Discussion Educational score: 2.2061986923217773 Domain: biomedical Document type: Study Language: en However this finding is inconsistent with studies conducted in Ibadan, Nigerians, in which knowledge about cord care practice has no significant change in their practice . The inconsistency can be due to, Mothers who are aware of the effects of harmful cord care practices are less readily going to practice it. It can also be associated with a low level of mothers' trust in what they receive from health professionals than what they know or hear. This might be due to the influence of culture around them. The most common influencers in the community can be families such as mothers and grandmothers or traditional birth attendants most of the time they have a great acceptability to the community. Section title: Conclusion Educational score: 2.081897497177124 Domain: biomedical Document type: Study Language: en This study indicated that the magnitude of newborn cord care practice was low among mothers who gave birth in the last six months in the district. Section title: Conclusion Educational score: 2.2703378200531006 Domain: biomedical Document type: Study Language: en Age, educational status, place of delivery, and knowledge level regarding umbilical cord care were statistically significant predictors of good cord care practice. Section title: Conclusion Educational score: 1.9495229721069336 Domain: biomedical Document type: Other Language: en Therefore, age, education, health facility delivery, and having good knowledge of umbilical cord care expose the mother to a good cord care practice. Section title: Health care professionals Educational score: 1.7527086734771729 Domain: biomedical Document type: Other Language: en Provide health education regarding newborn cord care during at all levels of contact with the mother with increased reminders during the antenatal period. Section title: Health care professionals Educational score: 1.7356568574905396 Domain: biomedical Document type: Other Language: en Good health-seeking behavior and delivery at health facilities to effectively promote good cord care practice should be encouraged. Section title: Health care professionals Educational score: 1.60995614528656 Domain: biomedical Document type: Other Language: en Update health care providers' skills and knowledge or in-service re-education on appropriate umbilical cord care. Section title: Health care professionals Educational score: 1.9638375043869019 Domain: biomedical Document type: Other Language: en Should encourage mothers to apply chlorhexidine appropriately if available otherwise encourage dry cord care. Section title: Health facility Educational score: 1.7813581228256226 Domain: biomedical Document type: Other Language: en Facilities should have a standard protocol for umbilical cord care which should be communicated to all women and careers by their healthcare providers. Section title: District health bureau Educational score: 1.4742720127105713 Domain: biomedical Document type: Other Language: en Strengthen health extension workers to counsel and advise the mothers about cord care practice. Section title: District health bureau Educational score: 1.325277328491211 Domain: other Document type: Other Language: en Arrange different community-based interventional strategies such as community-based education to address the problem. Section title: District health bureau Educational score: 1.688536286354065 Domain: biomedical Document type: Other Language: en Increase the availability of chlorhexidine which replaces the application of other harmful substances. Section title: Healthcare managers and policymakers Educational score: 1.1006301641464233 Domain: other Document type: Other Language: en Strengthen policies and strategies focused on women's education. Section title: Healthcare managers and policymakers Educational score: 1.4715639352798462 Domain: biomedical Document type: Other Language: en Use this study as an input to carry out further investigation and make the problem addressed through different strategies. Section title: Educators Educational score: 1.2702745199203491 Domain: other Document type: Other Language: en Use the findings in teaching-learning process. Section title: Researchers Educational score: 2.1907856464385986 Domain: biomedical Document type: Other Language: en More studies, analytical and interventional should be done to obtain the determinants of harmful cord practices and to make further recommendations to decrease the prevalence of harmful cord care. Section title: Researchers Educational score: 1.9396836757659912 Domain: biomedical Document type: Other Language: en A study on assessing cord care practices at health facilities is needed to identify the care provided by health care providers. Section title: Researchers Educational score: 1.6412428617477417 Domain: biomedical Document type: Other Language: en A similar or comparative study needs to be done in the country for ease of generalization. Section title: Strength Educational score: 2.146831512451172 Domain: biomedical Document type: Study Language: en Since the study conducted use of community-based study and inclusion of rural kebeles which increase the generalizability of the study is the strength of this study. It also tries to address the cultural cord care practice of mothers with qualitative findings in the community. Section title: Limitation Educational score: 2.2804179191589355 Domain: biomedical Document type: Study Language: en The cross-sectional nature of the study makes it impossible to establish a temporal relationship between newborn cord care practices and identified risk factors. Section title: CRediT authorship contribution statement Educational score: 0.9905561804771423 Domain: other Document type: Other Language: en Genat Balcha Abdi: Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Bekalu Kassie Alemu: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Tensae KassaYizengaw: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Beker Ahmed Hussein: Writing – review & editing, Writing – original draft, Validation, Data curation. Section title: Data availability statement Educational score: 0.9666326642036438 Domain: biomedical Document type: Other Language: en D ata supporting the findings of this study are available in Figshare at URL . Additional data and materials related to this study are available from corresponding author upon reasonable request . Section title: Ethical approval Educational score: 1.6319109201431274 Domain: biomedical Document type: Other Language: en This study was approved by Institutional Research Ethical Review Committee (IRERC) of Debre Markos University, college of health science with reference number HSC/R/C/ser/co/318/11/13, dated April 01, 2021 with relevant guidelines and regulations Following the approval by IRERC, an official letter of cooperation was written to the Hetosa Woreda health administration office, and in turn, the district health administration office wrote letters to each selected kebeles administration office to get permission and cooperation. Written informed consent was obtained from all participants prior to data collection. The confidentiality and privacy of the respondents was assured. Participants were given the chance to ask any doubt about the study and made free to refuse or stop the interview at any moment they wanted and no study participants below the age of 18 years old. Section title: Funding Educational score: 1.0171712636947632 Domain: other Document type: Other Language: en A total fund of 2500 Ethiopian birr with a project code of HSC/R/C/ser/co/318/11/13, dated April 01, 2021 was received from the 10.13039/501100021567 Debre Markos University Office of Research and Publication directorate director. The funding agency was not involved in the study's conception and design, data collection, participant enrollment, statistical analysis, data interpretation, or manuscript development. Section title: Declaration of competing interest Educational score: 0.9818205833435059 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Other | biomedical | en | 0.999997 |
PMC11699366 | Section title: Background and aims Educational score: 4.072709083557129 Domain: biomedical Document type: Study Language: en In the absence of disease-modifying therapies for Parkinson's disease (PD), much research focuses on improving quality of life, health, and wellbeing. A new care model, Proactive and Integrated Management and Empowerment in Parkinson's (PRIME) , is a multicomponent intervention being evaluated within a randomized controlled trial (RCT) which is enrolling patients with parkinsonism, and therefore includes those with frailty, multimorbidity and/or cognitive impairment as well as informal caregivers . This intervention aims to improve the experience of living with PD and align care with ‘what matters most’ to individuals . We sought to assess benefit of this innovative care model using an outcome which is aligned to the project aims, captures the hypothesised impact and is meaningful to patients. Section title: Background and aims Educational score: 3.6890060901641846 Domain: biomedical Document type: Other Language: en Patient-reported outcome measures (PROMs), which collect outcomes directly from the people who experience them are increasingly being used alongside or instead of biomarkers and measures of morbidity, mortality and healthcare use . Many of these are disease-specific which has limitations, particularly in older people, as well as those with multiple long-term conditions, disability or short life expectancy in whom there are often trade-offs when considering multiple interventions/treatments . Section title: Background and aims Educational score: 3.97422456741333 Domain: biomedical Document type: Study Language: en Given the complexity and heterogeneity of PD, PROMs focusing on a single domain, such as motor symptoms, are often not appropriate and risk overlooking the spectrum of experience. Conventional outcome measures may not be validated for proxy completion on behalf of someone lacking capacity and, whilst some outcomes have been endorsed for use in PD dementia , floor and ceiling effects may render them unsuitable for use in individuals with intact cognition, precluding their use in a trial recruiting across the disease spectrum. When considering a complex intervention like the PRIME-UK care model, there is a need for PROMs which capture the downstream effects of the multiple components, which are aiming to produce impact across physical, psychological and social domains. Section title: Background and aims Educational score: 4.076376914978027 Domain: biomedical Document type: Study Language: en Given the limitations of traditional PROMs, novel outcomes that can more holistically capture personal experiences and wellbeing have been proposed. Cools et al . suggested happiness as an outcome in PD studies but this also has limitations since wellbeing is a multi-dimensional construct encompassing psychological wellbeing, not simply the pursuit of happiness . Traditional outcomes can be made more patient-centred by reframing them into goal-orientated outcomes: instead of using a pain inventory for arthritis, we may assess pain control sufficient to carry out an activity important to the patient . Instead of disease-free survival, the goal-orientated outcome would be survival until a personal milestone, or there may be no survival outcome if this were not a patient priority . Goal attainment scaling (GAS) requires individuals to set meaningful goals with progress subsequently rated on a standardised scale and a formula applied to calculate an aggregated score . In addition to clinical use as an intervention to motivate behavioural change, goal-orientated approaches have been used to evaluate interventions in research settings, including amongst people with multiple sclerosis , older adults living with frailty and PD patients [ , , , , ]. We aimed to select a suitable primary outcome measure and adapt it for use within the PRIME-UK RCT. Section title: Selection of a primary outcome measure Educational score: 3.7272379398345947 Domain: biomedical Document type: Study Language: en The PRIME RCT, approved by the London-Harrow Research Ethics Committee , aimed to enrol a ‘real-world’ population to make the findings generalisable to clinical practice. The outcome measure therefore needed to be suitable for use in participants across the spectrum of disease stage and phenotype, including those with cognitive impairment and multimorbidity, as well as meaningful to patients and able to capture the effect of a complex intervention acting across multiple domains. Section title: Selection of a primary outcome measure Educational score: 4.115504264831543 Domain: biomedical Document type: Study Language: en Goal-orientated outcomes offered a way to elicit an individual's goals, and generate a numerical score to allow assessment of attainment over time and comparison between patients with multiple personalised goals. From a literature review, we identified four goal-based measures which have been used to evaluate interventions, rather than goal-setting as an intervention itself, each of which we deemed to have pros and cons based on the practicality of use within a trial, prior use in PD and psychometric properties ( Table 1 ). We selected the Bangor Goal-setting Interview (BGSI) as the PRIME RCT primary outcome measure because it was developed for research purposes, rather than for clinical settings with subsequent adaptation, and has advantages for this context ( Table 1 ), including a freely-available manual and the flexibility to adapt it to specific settings . It has also been shown to be feasible for use with people with PD dementia and Dementia with Lewy Bodies . Table 1 Interventional studies, including trials, which included a goal-orientated outcome measure. Table 1 Goal-orientated outcome measure Brief description of measure Examples of uses including study population Pros Cons Goal attainment scaling (GAS) Main problem area/domain is identified during an interview with the patient and goal(s) agreed jointly; the expected outcome defined as achievement level 0 is agreed and descriptors are pre-agreed for level −1/-2 and +1/+2, representing a worse or better outcome than expected respectively. Each goal is rated on the 5-point scale at follow up. - A single blind RCT of cognitive training, transfer training and psychomotor training in people with PD and MCI - A pilot, double-blind RCT of memantine versus placebo for PD dementia - A service evaluation of dance classes for PD - An RCT of comprehensive geriatric assessment as an adjunct to usual care in frail older patients - Goals can be weighted to account for the importance to the patient and/or anticipated difficulty - There is no requirement to record baseline scores (although these are usually rated as −1) - Performance is judged against predefined levels. - Potentially time-consuming to define pre-determined levels for each of the 5 outcome score levels (−2 to +2) - Zero and minus scores can be discouraging for patients - If baseline score is recorded as −1 (which allows for deterioration to −2), it is not possible to record if a goal is partially achieved (but does not warrant the expected outcome level of 0) . Patient-specific functional scale (PSFS) A self-reported measure in which patients list up to five activities they struggle to do because of their condition/problem. For each, they rate from 0 to 10 their ability to perform the activity at baseline and then subsequently at follow-up. - A prospective, observational pre-post study of goal-orientated rehabilitation in individuals with a neurological condition (including PD and MS) - RCTs in populations with COPD , low back pain and cervical radiculopathy . - May be less time-consuming and require fewer resources than a semi-structured interview as the process is patient-directed - Reported to have good reliability, validity and responsiveness in a range of musculoskeletal conditions including mechanical back pain . - Does not involve a semi-structured interview so the patient is not guided/supported through the process which may be challenging for some patient groups including those with cognitive impairment - Lack of evidence for use in neurological disease . Bangor Goal-setting interview (BGSI) The patient sets goals in a collaborative interview with the option for an ‘informant’ to provide input. Goal attainment descriptors are pre-defined to indicate what would constitute e.g. 0, 50, 100 % attainment. Current attainment is rated on a 1 to 10 scale, with option to rate importance and motivation to change. Attainment is rated from 1 to 10 at follow-up. - Primary outcome in a single blind pilot RCT of cognitive rehabilitation versus relaxation therapy for PD dementia/Lewy Body Dementia - Primary outcome in a single-blind RCT of goal-orientated cognitive rehabilitation in early-stage dementia, the’GREAT’ trial - Developed for research purposes and the manual includes specific guidance on use of BGSI in an RCT - The manual and recording sheet are freely available for researchers to use and include resources to guide the interview, written in an accessible format, suitable for individuals with cognitive impairment - Incorporates the option, not only for an informant to support the process, but also for a caregiver to independently rate attainment - The structure allows some flexibility to adapt the measure for use in different settings - Descriptors set at baseline can help with subsequent rating of attainment - Potentially time-consuming to define the descriptors at baseline - Limited information on the psychometric properties Canadian Occupational Performance Measure (COPM) Semi-structured interview in which the patient is guided to consider 3 areas: productivity, self-care and leisure. Patients choose up to 5 problems and rate current performance and satisfaction on a scale of 1–10, where a higher score represents greater performance or satisfaction. Performance and satisfaction are rated again at follow-up. - A case series (pre and post testing) of an 8-week treatment program to train for the use of compensatory external aids to achieve personalised goals - Evidence supports the reliability, construct validity and responsiveness . - Specification of the 3 areas (productivity, self-care and leisure) may not be relevant to all individuals or interventions - The measure and related resources must be purchased GAS: goal attainment scaling; RCT: randomized controlled trial; PD: Parkinson's Disease; PSFS: patient-specific functional scale; MS: Multiple Sclerosis; BGSI: Bangor Goal-setting Interview; COPM: Canadian Occupational Performance Measure. Section title: Selection of a primary outcome measure Educational score: 2.757145404815674 Domain: biomedical Document type: Study Language: en We adapted the BGSI manual processes for use in our trial. Our sample size calculation was based upon a minimal clinically important difference (MCID) of 2 points on the BGSI, as used in the GREAT trial , based on recommendations for the Canadian Occupational Performance Measure (COPM), a similar rating scale . Section title: Operationalizing goals as an outcome measure Educational score: 3.449220657348633 Domain: biomedical Document type: Study Language: en 3.1 Use of the Bangor Goal-setting Interview within the PRIME-RCT Agreement of goals before randomization avoids prior knowledge of intervention status biasing goal selection . During a collaborative interview the participant sets three to five goals with a trained researcher, with input from a family member/caregiver (an ‘informant’), particularly for participants with cognitive impairment . Unlike the original BGSI, we do not pre-specify domains within which participants must identify goals since these are expected to be highly personalised. Section title: Operationalizing goals as an outcome measure Educational score: 4.03525972366333 Domain: biomedical Document type: Study Language: en After goal selection, percentage descriptors are specified to indicate what would constitute, for example, 50 % goal attainment. The participant is then asked to rate their current attainment, perceived importance and readiness to change numerically for each goal, with an option for an enrolled caregiver to independently rate attainment. The four steps are summarised in Fig. 1 , described in more detail below and illustrated in Table 2 with an example. Fig. 1 The procedure for using the Bangor Goal-Setting Interview within the PRIME randomized controlled trial prior to randomization. Fig. 1 Table 2 Applying the adapted Bangor Goal-setting interview to set and follow up a goal within the PRIME randomised controlled trial, where text in italics indicates how the researcher would guide the participant through the process. Table 2 STEP 1 Specify the area (domains not pre-specified a ) “Parkinson's can impact on daily life in various ways. There are different things that people may want to change to make their lives more enjoyable. We are asking everyone taking part in this trial to come up with several goals that they would like to achieve. A goal might be something that you currently find difficult to do and wish you could do more easily, or without getting frustrated. Or a goal might be something that you are currently not doing and would like to do more of. Or it might be something that you would like to learn how to do.” Going to the gym STEP 2 SMART goal statement (100 % attainment) I will go to the gym to exercise 3 times per week for 45–60 min Description of current attainment (0 % attainment) I have membership but do not go at all Goal attainment descriptor b (50 %) I will go 1–2 times per week or 3 times for a shorter session (e.g. 20 min s) STEP 3 Rating of attainment Image 1 1 Rating of importance b Image 2 7 Rating of readiness to change b Image 3 6 STEP 4 (e ach 3 monthly follow up conducted by blinded assessor b ) Free text record of current activity in relation to goal “Last time we spoke about going to the gym. Your goal was to go to the gym 3 times per week to exercise for 45–60 min. How are things going with that?” “Due to a recent small fracture of my right ankle I had to wear a boot and rest for 6 weeks, but I'm gradually starting to exercise again. In the last month, I have gone to the gym 4 times for 30–40 min” Participant raw attainment score (without reminder of prior rating in step 3 b ) “What score would you give yourself on a scale of 1-10 for how well you are doing now? 10 would be that you are doing it very successfully, 1 would be that you are not doing it at all or not successfully at all." 3 Calibrated attainments scores a “At your baseline visit, you said you had membership but were not going to the gym and you gave yourself an attainment score of 1. We decided at that time that achieving your goal by 50 % or making it half way to your goal would be going to the gym 1–2 times per week or going 3 times for shorter sessions ( e.g. 20 min). Based on what you have just told me, it sounds like you are more than 50 % of the way to achieving your goal. I wonder whether the new score should be somewhere around 6- would you agree with this?" Interviewer-decided attainment score calibrated from baseline attainment score using % descriptors6 Calibrated score as decided by participant following discussion5 SMART: Specific, Measurable, Achievable, Relevant, Time-bound. a Indicates an aspect of the process which is specific to the operationalisation of BGSI within PRIME-RCT. b Indicates an aspect of the process which was specified as an option within the BGSI manual and which was incorporated into the PRIME-RCT. Section title: Follow-up of attainment Educational score: 2.103133201599121 Domain: biomedical Document type: Study Language: en At follow-up, the participant describes their current activity in relation to each goal. They then rate their attainment numerically, without reminder of their prior rating. The researcher uses the percentage descriptors defined at baseline to decide on a researcher-determined calibrated attainment score which reflects the description of current attainment. In dialogue with the participant, the researcher assists them to decide on the participant-determined calibrated score. Section title: Follow-up of attainment Educational score: 1.8205522298812866 Domain: biomedical Document type: Other Language: en Sometimes goals cannot be attained due to factors beyond the participant's control e.g. a dance class not attended because of cancellation; giving a low attainment score would not reflect participant wellbeing, so the goal rating is omitted, and an explanation recorded. However, to avoid overestimation of goal attainment, low attainment is recorded if the reason relates to patient wellbeing; for example, they did not attend the dance class due to an injury. This distinction is an adaptation to the BGSI. Section title: Frequency of data collection Educational score: 2.6678409576416016 Domain: clinical Document type: Other Language: en A key consideration was the frequency of goal follow-up, acknowledging that participants may select goals which are achievable in different timeframes and may subsequently wish to add another goal to replace an event-related goal. In the PRIME RCT, participants specify at baseline the timeframe in which they intend to achieve each goal. Regardless, all goals are followed up 3-monthly by a blinded assessor. Participants are reminded not to reveal their treatment allocation during the call and any unblinding is recorded. Section title: Derivation of the score Educational score: 3.6665897369384766 Domain: biomedical Document type: Study Language: en The primary outcome is change in participant-rated attainment, calculated as shown in Fig. 2 . Whereas for classical measurement instruments a cut-off score may indicate a particular diagnosis and individual scores can be compared to normative data, intervention evaluation using a goal-based outcome relies on comparison of two group means . Follow-up at all 3-monthly timepoints will facilitate a secondary longitudinal analysis to determine whether any improved goal attainment is maintained. Secondary analyses will include change in caregiver-rated attainment and for the three goals rated as most important. Fig. 2 A worked example showing the process for calculating the change in attainment for one participant who had set three goals. Fig. 2 Section title: Conclusion Educational score: 3.7384212017059326 Domain: biomedical Document type: Study Language: en We have described how a goal-orientated outcome measure was adapted for use in a trial involving individuals with complexity, frailty and multimorbidity. As the trial progresses, we will gain crucial real-world experience of applying this in people with parkinsonism, including the time and resources required. We will also gather quantitative data on the numbers of goals set, typical domains chosen, agreement between patient and caregiver scores and the proportion unable to rate attainment (e.g. due to cognitive impairment). Section title: Conclusion Educational score: 3.9048962593078613 Domain: biomedical Document type: Study Language: en The lack of a defined MCID for the BGSI is a limitation; determining this in a parkinsonism population would add to its utility and help to inform whether BGSI is better able to detect a meaningful difference than some other outcomes, as suggested [ , , ]. Whilst the evidence for responsiveness is encouraging , evidence for the validity and reliability of goal-orientated outcome measures is limited and relates mainly to the COPM . Future work could establish whether an improvement in goal attainment is associated with downstream benefits such as reduced unplanned hospitalisation, which would demonstrate predictive validity. Section title: Conclusion Educational score: 3.827770948410034 Domain: biomedical Document type: Study Language: en There are many potential benefits to wider use of goal-orientated outcome measures in clinical trials. They provide a holistic outcome not limited to specific conditions and with universal relevance, important for trials with broad inclusion criteria, enrolling heterogenous populations including adults with frailty and multimorbidity. Above all they aim to provide a meaningful measure of “what matters”. Knowledge gained from use of the adapted BGSI in the PRIME 10.13039/100014144 RCT will help to support its implementation in conditions besides neurodegenerative disease, the focus of its use so far. Section title: CRediT authorship contribution statement Educational score: 0.9588629603385925 Domain: other Document type: Other Language: en Emma Tenison: Writing – original draft, Methodology, Conceptualization. Katherine Lloyd: Writing – review & editing, Project administration, Methodology. Yoav Ben-Shlomo: Writing – review & editing, Supervision, Methodology, Conceptualization. Emily J. Henderson: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization. Section title: Trial registration number Educational score: 1.0281779766082764 Domain: biomedical Document type: Other Language: vi NCT05127057. Section title: Grant support Educational score: 1.025671124458313 Domain: other Document type: Other Language: en 10.13039/501100000324 Gatsby Charitable Foundation , GAT3676 . Section title: Funding sources Educational score: 1.0777229070663452 Domain: other Document type: Other Language: en The PRIME-UK programme is funded by the 10.13039/501100000324 Gatsby Charitable Foundation . Section title: Declaration of competing interest Educational score: 1.028397798538208 Domain: other Document type: Other Language: en The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ET is funded by a 10.13039/501100000272 National Institute for Health and Care Research Academic Clinical Lectureship and has received a speaker honorarium from the Neurology Academy. Section title: Declaration of competing interest Educational score: 0.9470882415771484 Domain: other Document type: Other Language: en KL is in receipt of PhD fellowship funding from The Gatsby Foundation. Section title: Declaration of competing interest Educational score: 0.9684327244758606 Domain: other Document type: Other Language: en EH is HEFCE funded by University of Bristol for her academic work and has received honoraria from the Neurology Academy and travel support from Bial. Section title: Declaration of competing interest Educational score: 1.0354756116867065 Domain: other Document type: Other Language: en YBS is partly funded by National Institute for Health and Care Research Applied Research Collaboration West (NIHR ARC West) and University of Bristol and has received funding from Parkinson's UK, Royal Osteoporosis Society, MRC, HQIP, Templeton Foundation, Versus Arthritis, Wellcome Trust, National Institute of Health Research, Gatsby Foundation. | Study | biomedical | en | 0.999999 |
PMC11699391 | Section title: Introduction Educational score: 3.0414602756500244 Domain: biomedical Document type: Review Language: en As with many other diabetic complications, the poor bone quality and increased fracture risk in diabetes has already aroused great concerns. 1 , 2 It was well recognized that hyperglycemia exposure inhibits proliferation and differentiation of osteoblasts, 3 but its specific mechanism is still not clear, and no definitive pharmaceutical remedies for diabetic osteoporosis are available except for those traditional medications to treat primary osteoporosis. Section title: Introduction Educational score: 4.19386100769043 Domain: biomedical Document type: Study Language: en Energy metabolism is an important physiological basis for maintaining cell function. The function of mitochondria, as the hub of energy metabolism, is the key to maintaining normal life activities. Mitochondrial dysfunction-related diseases have expanded from rare monogenic disorders to common polygenic diseases, including metabolic, cardiovascular, neurodegenerative, and neuromuscular diseases in nowadays. 4 , 5 Lots of effort was made to explore signaling pathways to reverse mitochondrial dysfunction. 6 Recent studies have suggested a mutual cross-talk between bone remodeling and glucose homeostasis. 7 , 8 So far, the best characterized metabolic pattern for osteoblast maturation is the glucose metabolism reprogramming from oxidative phosphorylation (OXPHOS) toward aerobic glycolysis, coupling with diminished mitochondrial respiration. 9 , 10 Suppression of glucose metabolism restricts osteoblast differentiation. 11 Thus, it is interesting to investigate whether and how the disturbed metabolic reprogramming causes impaired osteoblastogenic differentiation and poor bone quality in diabetes, and how to reverse it. Section title: Introduction Educational score: 4.789687156677246 Domain: biomedical Document type: Review Language: en Mitophagy is a mechanism that eliminates damaged mitochondria and controls mitochondrial quality and quantity. Simultaneously, mitophagy mediates metabolic reprogramming, either into an OXPHOS phenotype or into a glycolytic phenotype, to satisfy the metabolic requirements of cells. 12 , 13 Mitophagy dictates stem cell fate by facilitating glycolytic phenotype swift. 14 The deficiency of mitophagy induces reactive oxygen species (ROS) stress, and leads to the development of neurodegenerative disorder, 15 cardiomyopathy, 16 and acute kidney injury. 17 In type 2 diabetes patients, mitophagy pathway gene expression was downregulated. 18 In β-cells, decreased glucose-responsive insulin secretion was associated with impaired mitophagy. 19 Mitophagy is mediated by PTEN-induced kinase 1 (PINK1)/Parkin pathway. PINK1 is a mitochondrial serine/threonine-protein kinase. It can sense mitochondrial stress, accumulate on defective mitochondria, recruit Parkin, and finally trigger mitophagy. 20 Loss of PINK1/Parkin may contribute to several diseases, such as Parkinson’s disease. 21 As to bone metabolism, PINK1-mediated mitophagy is involved in cartilage degeneration seen in osteoarthritis. 22 , 23 , 24 Section title: Introduction Educational score: 3.5092618465423584 Domain: biomedical Document type: Study Language: en Therefore, our research interest is what role mitophagy plays in hyperglycemia-induced osteoblastogenic defects and whether the intervention of related mechanisms can restore diabetic osteoporosis. Section title: High glucose level inhibited osteogenic differentiation and respiration rate of MC3T3-E1 cells Educational score: 4.154201507568359 Domain: biomedical Document type: Study Language: en To investigate the effect of high glucose level on the osteoblastogenesis of MC3T3-E1 cells in vitro , alkaline phosphatase (ALP) and Alizarin red staining were performed. Alizarin red staining in H-Glu group showed significantly fewer mineralization nodules compared with L-Glu group in the late stage (14 days, 21 days) of osteogenic differentiation. However, there was no significant difference in ALP staining between the two groups in the early stage (1 day, 7 days) of osteogenic differentiation . Furthermore, the mRNA expression levels of Runx2, Osx, Col1a1, and ALP were reduced in H-Glu group at day 14, while there was no significant difference between H-Glu and L-Glu group at day 1, and only Osx expression decreased significantly in the H-Glu group at day 7 . Consistently, the western blot (WB) results showed that the protein levels of Runx2 and Osx were decreased in H-Glu group at day 14 . These results suggest that high glucose inhibits the late stage of osteogenic differentiation in MC3T3-E1 cells. Figure 1 High glucose inhibited osteoblastogenic differentiation and respiration rate of MC3T3-E1 cells The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu group was 25 mmol/L. (A) ALP staining was performed in L-Glu and H-Glu cultured MC3T3-E1 cells after 1 and 7 days of osteogenic induction. Alizarin red S staining was performed in L-Glu and H-Glu cultured MC3T3-E1 cells after 14 and 21 days of osteogenic induction. Scale bars, 500 μm. (B–D) MC3T3-E1 cells were cultured in an osteogenic medium for 1, 7, and 14 days. Expressions of osteogenic markers at different time points were determined by RT-qPCR. (E) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of Runx2 and Osx were determined by WB. (F–Q) MC3T3-E1 cells were cultured in osteogenic medium for 1, 7, and 14 days. Mitochondrial oxidative capacity and glycolytic capacity were measured in real time. Basal respiration, ATP-linked respiration, maximal respiration, spare respiratory capacity, basal, proton leak, glycolysis, glycolytic capacity, and glycolytic reverse were calculated by WAVE software. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: High glucose level inhibited osteogenic differentiation and respiration rate of MC3T3-E1 cells Educational score: 4.385751724243164 Domain: biomedical Document type: Study Language: en Since the disruption of energy metabolism in osteoblasts may lead to impaired osteoblast function, 25 , 26 we then detected two key energy metabolic pathways, aerobic respiration and glycolysis in MC3T3-E1 cells using the Seahorse extracellular flux analyzer. On day 1 of osteogenic differentiation, no noteworthy distinctions of basal respiration, maximal respiration, spare respiration capacity, and ATP-linked respiration, which are pivotal parameters that underscore mitochondrial functionality, were observed between H-Glu group and L-Glu group . As to cellular glycolytic function, H-Glu group exhibited elevated glycolysis and glycolytic capacity compared to the L-Glu group, with no statistically significant variance in glycolytic reversal between the two groups . Subsequent to 7 days of osteogenic induction, exclusive reductions in maximal respiration and glycolysis were discerned in H-Glu group relative to L-Glu group . Following a 14-day osteogenic differentiation period, the mitochondrial stress test revealed a substantial attenuation in basal, maximal, and ATP-linked respiration in H-Glu cells as contrasted with those in L-Glu group . Simultaneously, outcomes from the glycolytic rate examination exhibited notable diminutions across glycolysis, glycolytic capacity, and glycolytic reversal in H-Glu cells in comparison to L-Glu counterparts . Collectively, these findings provide evidence that the late stage of osteogenic differentiation is notably affected by a discernible inhibition of osteoblastic mitochondrial function induced by the high glucose level. Section title: High glucose level did not affect mitochondrial biogenesis in MC3T3-E1 cells Educational score: 4.148841857910156 Domain: biomedical Document type: Study Language: en Mitochondrial proliferation may indicate the engagement of feedback loops to compensate for compromised mitochondrial function. Alterations in gene expression profile involved in mitochondrial biogenesis, and changes of concentrations and activities of candidate respiratory complexes in cells may lead to energetic dysfunction. 27 Hence, the peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1α), nuclear respiratory factors (NRF-1 and NRF-2), and mitochondrial transcription factor A (TFAM) that are the key regulators of mitochondrial biogenesis were determined in osteoblasts. The results showed that there was no difference in the expression of the aforementioned genes between H-Glu and L-Glu groups . Figure 2 High glucose did not affect mitochondrial biogenesis but elevated ROS production and decreased mitochondrial membrane potential (MMP) in MC3T3-E1 cells (A) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. The total DNA of the cells was extracted. Using HK2 as reference gene, expression ND1 and 16s were determined by RT-qPCR. (B) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of mitochondrial biogenesis markers were determined by RT-qPCR. (C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of respiratory chain complexes were determined by WB. (D–F) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. ROS generation was observed by using flow cytometry following staining with DCFH-DA. (G and H) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 50 μm. Data presented as mean ± SD. n = 3 biological replicates. A t test was used for comparison between two groups. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: High glucose level did not affect mitochondrial biogenesis in MC3T3-E1 cells Educational score: 4.40100622177124 Domain: biomedical Document type: Study Language: en Mitochondrial biogenesis involves the integration of multiple transcriptional pathways controlling both nuclear and mitochondrial gene expression. For example, mitochondrial DNA (mtDNA) transcription is activated by the family of PGC-1 proteins (PGC-1α and PGC-1β), from which PGC-1α is considered the master regulator of mitochondrial biogenesis. The pathway is initiated by PGC-1α activation, followed by stimulation of a series of nuclear transcription factors, that is the NRF-1 and NRF-2 and by the increase in expression of TFAM, the final effector of mtDNA transcription and replication. 28 Section title: High glucose level did not affect mitochondrial biogenesis in MC3T3-E1 cells Educational score: 4.1120405197143555 Domain: biomedical Document type: Study Language: en To further measure mitochondrial biogenesis, we quantitated relative mtDNA copy number by qPCR in MC3T3-E1 cells. As shown in Figure 2 B, mtDNA copy number in osteoblasts of H-Glu group remained similar to L-Glu group. Additionally, we examined the relative levels of the 5 OXPHOS complexes in mitochondria by WB. The result was in accordance with the mitochondrial biogenesis gene expression. Mitochondrial respiratory chain complex protein levels were not significantly different between the two groups . These results demonstrate that high glucose level does not affect the mitochondrial biogenesis of MC3T3-E1 cells and suggest that high glucose level may affect mitochondrial function through other mechanisms. Section title: High glucose level elevated ROS production and decreased MMP in MC3T3-E1 cells Educational score: 4.0977864265441895 Domain: biomedical Document type: Study Language: en Mitochondria is one of the main sources of ROS, as it utilizes oxygen for energy production. Mitochondrial dysfunction may lead to increased radical production and consequent “oxidative stress.” Therefore, cell ROS level is one of the recognized indicators to evaluate mitochondrial function. 29 The flow cytometry results in this study showed that the number of positively stained cells in H-Glu group was significantly higher than that in L-Glu group . Fluorescence intensity quantitative analysis revealed that the ROS level in H-Glu group was significantly higher than that in L-Glu group . Section title: High glucose level elevated ROS production and decreased MMP in MC3T3-E1 cells Educational score: 4.1490325927734375 Domain: biomedical Document type: Study Language: en Mitochondrial function can also be assessed through monitoring changes in MMP. A decrease in the MMP indicates mitochondrial dysfunction. 30 We examined the MMP using a cell-permeant dye tetramethylrhodamine (TMRM), which can accumulate in active mitochondria with intact membrane potentials. The TMRM signals in living cells were observed by confocal laser microscopy, quantitative analysis of TMRM fluorescence intensity in images showed that the average fluorescence intensity of cells in H-Glu group was 36% lower than that in L-Glu group. The MMP of H-Glu group cells were reduced . These results suggest that high glucose level impairs mitochondrial function in MC3T3-E1 cells. Section title: RNA-seq data analysis of differentially expressed genes in MC3T3-E1 cells cultured in L-Glu and H-Glu Educational score: 4.209380149841309 Domain: biomedical Document type: Study Language: en The aforementioned results demonstrated that high glucose level inhibited the late stage of osteogenic differentiation and impaired mitochondrial function of these cells. Then, what is its molecular mechanism? To address this question, MC3T3-E1 cells cultured in low glucose or high glucose osteoblastogenic induction media for 14 days were examined by RNA sequencing (RNA-seq) analysis . Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the major pathways involved in the differential genes between the two groups were mitophagy, autophagy, and cell cycle . Gene ontology (GO) functional enrichment showed that the major pathways involved in the differentially expressed genes were ubiquitin proteasome system and mitophagy . As shown in Figure 3 E, further analysis of mitophagy genes revealed that transcription levels of PINK1 and dynamic-associated protein 1 (Drp1) were significantly reduced in H-Glu group compared to L-Glu group. We further verified the expression of Drp1 and PINK1 by qPCR . It is worth mentioning that we also detected mitophagy marker genes of non-ubiquitination-dependent pathways by qPCR and there were no significantly difference between the two groups . These findings suggest that marker gene PINK1 of mitophagy signaling pathway and Drp1 may be important factors involved in the mechanism by which high glucose inhibits osteogenic differentiation. Figure 3 RNA-seq data analysis of differentially expressed genes in MC3T3-E1 cells cultured in L-Glu and H-Glu MC3T3-E1 cells were cultured in osteogenic medium for 14 days, then eukaryotic transcriptome sequencing was performed. (A) The volcano map showed significant differences in gene expression between the two groups. (B) The enrichment analysis of KEGG pathway based on the significantly different genes in the volcano map showed that the main pathways involved in the damage of osteoblasts by high sugar were mitochondrial autophagy, cell autophagy and cell cycle. (C) GO ontology functional enrichment showed that the main pathways involved in the damage of osteoblasts by high glucose were ubiquitin-proteasome system, ubiquitin-proteasome system, and mitochondrial autophagy. (D) The KEGG pathway loop map showed the main mechanisms of hyperglycemic damage: mitochondrial autophagy, autophagy, and cell cycle. (E) Genes of interest with significant differences. n = 4 biological replicates. (F) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of PINK1 and Drp1 were determined by RT-qPCR. (G–I) MC3T3-E1 cells were cultured in osteogenic medium for 1, 3, 7, and 14 days. Expressions of marker genes involved in ubiquitin-dependent mitophagy pathway at different time points were determined by RT-qPCR. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. A t test was used for comparison between two groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: High glucose inhibited PINK1/Drp1 signaling, leading to impaired osteogenic differentiation Educational score: 4.222012519836426 Domain: biomedical Document type: Study Language: en To further validate the functionality of PINK1 and Drp1 in osteogenic differentiation, we inhibited the expression of PINK1 and Drp1 in MC3T3-E1 cells by siRNA. Figures 4 A and 4B demonstrated that the expression of PINK1 and Drp1 genes was significantly knocked down. We substantiated the involvement of PINK1/Drp1 signaling in the process of osteogenic differentiation through gene knockdown under normal glucose culture. Evidently, the targeted suppression of PINK1 or Drp1 significantly inhibited the expression levels of Runx2 and Osx . We also observed that inhibiting the expression of PINK1 resulted in the downregulation of Drp1 protein. Conversely, inhibiting Drp1 did not affect the level of PINK1 protein, implying that PINK1 acts as the upstream signaling molecule of Drp1 . Consistently, the quantification of mineralized nodules within osteoblasts, following the downregulation of PINK1 and Drp1, exhibited a substantial reduction compared to the control group . Figure 4 High glucose inhibited PINK1/Drp1 signaling, leading to impaired osteoblastogenic differentiation (A and B) Expressions of PINK1 and Drp1 genes were determined by RT-qPCR after siRNA transfection. (C) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (D) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (E) Expression of PINK1 gene was determined by RT-qPCR after PINK1 plasmid transfection. (F) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (G) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 21 days after plasmid transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (H) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm. (I) MC3T3-E1 cells were cultured in osteogenic medium (L-Glu) for 14 days after siRNA transfection. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 50 μm. (J) MC3T3E1 cells were transfected with the mito-Keima plasmid and cultured in osteogenic medium (L-Glu) for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm. (K) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. Expression of PINK1, Drp1, Runx2, and Osx were determined by WB. (L) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (M) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 50 μm. (N) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 50 μm. (O) MC3T3-E1 cells were cultured in osteogenic medium (H-Glu) for 14 days after plasmid transfection and siRNA transfection. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 50 μm. Data presented as mean ± SD. A t test was used for comparison between two groups. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: High glucose inhibited PINK1/Drp1 signaling, leading to impaired osteogenic differentiation Educational score: 4.093708515167236 Domain: biomedical Document type: Study Language: en We further constructed an overexpressed PINK1 plasmid and transfected osteoblasts. Then, we induced osteogenic differentiation of MC3T3-E1 cells in high glucose medium. Figures 4 E and 4F showed that the expression of PINK1 gene and protein were significantly increased after transfection. Notably, this augmentation in PINK1 expression coincided with noteworthy enhancements in the expressions of Runx2 and Osx . Alizarin red staining showed that the number of mineralized nodules of osteoblasts overexpressing PINK1 increased significantly under high glucose medium culture . Section title: High glucose inhibited PINK1/Drp1 signaling, leading to impaired osteogenic differentiation Educational score: 4.091944217681885 Domain: biomedical Document type: Study Language: en We also examined the mitochondrial function of osteoblasts that had knocked down PINK1 and Drp1, respectively. In Figure 4 H, TMRM staining was performed on living cells in each group to detect MMP. The results showed that inhibiting the expression of either Drp1 or PINK1 could decrease the MMP of cells. In Figure 4 I, MitoSox was used to detect mitochondrial reactive oxygen species, and the results showed that knockdown of Drp1 or PINK1 could increase the superoxide level of cellular mitochondria. In Figure 4 J, MCT3E1 cells were transfected with pMito-Keima to detect mitophagy, and the results showed that mitophagy was weakened in cells with the Drp1 or PINK1 knocked down. Section title: High glucose inhibited PINK1/Drp1 signaling, leading to impaired osteogenic differentiation Educational score: 4.22469425201416 Domain: biomedical Document type: Study Language: en To verify Drp1 is a downstream target of PINK1, we further knocked down Drp1 in the presence of overexpression of PINK1 in MCT3E1 cells and examined the osteogenic phenotype and mitochondrial function of osteoblasts in high glucose medium. Figures 4 K and 4L showed that the protein levels of Runx2 and Osterix of cells in high glucose medium were significantly increased when PINK1 overexpressed, and the formation of mineralized nodules was increased; while Drp1 depletion significantly weakened this phenotype. Correspondingly, the MMP decreased and the level of mitochondrial superoxide of PINK1 overexpressed cells in high glucose medium increased significantly after Drp1 knockdown . Furthermore, the level of mitophagy of PINK1-overexpressed cells in high glucose medium was also significantly decreased after Drp1 was knocked down . Collectively, these findings indicate that the PINK1/Drp1 signaling exerts positive influence on mitochondrial function of osteoblasts and osteogenic differentiation. Overexpression of PINK1 can rescue the impaired osteogenic differentiation under high glucose medium. Section title: BMP9 reversed both osteogenic differentiation impairment and mitochondrial dysfunction induced by high glucose in MC3T3-E1 cells Educational score: 4.1718244552612305 Domain: biomedical Document type: Study Language: en The aforementioned results show that high glucose level inhibits osteogenic differentiation and impairs mitochondrial function of MC3T3-E1 cells, so we are interested in whether there are molecules that can protect the mitochondrial function and promote osteogenic differentiation of MC3T3-E1 cells exposed to high glucose level. Our previous mice studies have demonstrated that bone morphogenetic protein 9 (BMP9) is effective in improving both ovariectomized (OVX) and senile osteoporosis. 31 , 32 In this study, an obvious less mineralized nodule formation was observed in H-Glu group compared to L-Glu group. However, the impaired osteogenic differentiation induced by high glucose treatment was partially reversed by BMP9 treatment: mineralized nodule formation was increased after BMP9 treatment relative to L-Glu group . Moreover, expression levels of ALP, Runx2, Osx, Ocn, and Col1a1 were significantly increased after BMP9 treatment . Consistently, the WB results showed that the protein levels of Osx and Runx2 were increased after BMP9 treatment . Figure 5 BMP9 reversed both the osteoblastogenic differentiation impairment and mitochondrial dysfunction induced by high glucose in MC3T3-E1 cells The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu and H-Glu+BMP9 group was 25 mmol/L. The final concentration of BMP9 was 100 ng/ml. (A) Alizarin red S staining was performed in MC3T3-E1 cells after 21 days of osteogenic induction. Scale bars, 500 μm. (B) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of osteogenic markers were determined by RT-qPCR. (C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of Runx2 and Osx were determined by WB. (D and E) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Mitochondrial oxidative capacity was measured in real time. Basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity were calculated by WAVE software. (F and G) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 50 μm. (H and I) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 50 μm. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: BMP9 reversed both osteogenic differentiation impairment and mitochondrial dysfunction induced by high glucose in MC3T3-E1 cells Educational score: 4.1214752197265625 Domain: biomedical Document type: Study Language: en We used extracellular flux analysis to real-time monitor mitochondrial respiratory capacity in vitro . BMP9 treatment dramatically increased basal, ATP production-coupled respiration, maximal respiration, and the spare respiratory capacity . Furthermore, the result of TMRM signal showed that the MMP was significantly increased after BMP9 treatment . We also examined superoxide in the mitochondria of live MC3T3-E1 cells using mitochondrial superoxide indicator MitoSOX Red. In the MC3T3-E1 cells cultured in high glucose, superoxide production was higher than that in control cells, and BMP9 significantly reduced superoxide production . Collectively, these data indicate that BMP9 could improve mitochondrial function of MC3T3-E1 cells under high glucose medium culture. Section title: BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteogenic differentiation impairment in MC3T3-E1 cells Educational score: 4.171627044677734 Domain: biomedical Document type: Study Language: en To explore the regulatory mechanism of BMP9 on mitophagy, we inhibited the expression of PINK1 and Drp1 in cells by siRNA. After Drp1 or PINK1 depletion, the protein levels of Runx2 and Osterix of cells in H-Glu+BMP9 group were significantly decreased , and the formation of mineralized nodules was inhibited, as compared with only treated with BMP9 . Correspondingly, after PINK1 or Drp1 knockdown, the MMP decreased, and the level of mitochondrial superoxide of cells in H-Glu+BMP9 group increased significantly . Furthermore, the level of mitochondrial autophagy of cells in H-Glu+BMP9 group was also significantly decreased after Drp1 or PINK1 was knocked down . Figure 6 BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteoblast differentiation impairment in MC3T3-E1 cells The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu, H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 25 mmol/L. The final concentration of BMP9 in H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 100 ng/ml. (A) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (B) MC3T3-E1 cells were cultured in osteogenic medium for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 500 μm. (C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm. (D) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 20 μm. (E) MC3T3E1 cells were transfected with mito-Keima plasmid and siRNA and cultured in osteogenic medium for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm. (F) Representative electron micrographs show mitochondria and mitophagosomes (arrowhead) in MC3T3-E1 cells of different groups. Scale bars, 500 nm. Data presented as mean ± SD. A t test was used for comparison between two groups. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteogenic differentiation impairment in MC3T3-E1 cells Educational score: 4.157710552215576 Domain: biomedical Document type: Study Language: en We further observe the ultrastructure of mitochondria of MC3T3-E1 cells by transmission electron microscope. Figure 6 F showed mitochondria of L-Glu cells showed a bilayer-membrane rod-like structure with clear mitochondrial ridge. Mitochondria of cells in H-Glu group were swollen, and mitochondrial ridge structure was destroyed and disappeared. The ridge structure of some mitochondria in H-Glu+BMP9 group was blurred, and the damaged mitochondria could be seen to phagocytosed and degraded by double-layer membrane structure . While mitochondrial membrane structure was damaged but mitochondrial autophagy was not observed in PINK1 or Drp1 knockdown groups. Section title: BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteogenic differentiation impairment in MC3T3-E1 cells Educational score: 4.0504841804504395 Domain: biomedical Document type: Study Language: en Our results indicate that BMP9 effectively ameliorates osteogenic differentiation of cells under high glucose condition by activating PINK1/Drp1 signaling axis. Section title: BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteogenic differentiation impairment in BMSCs Educational score: 4.176314353942871 Domain: biomedical Document type: Study Language: en We conducted experiments to explore whether high glucose inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the mitophagy-associated PINK1/Drp1 signaling pathway and whether BMP9 has a reversal effect. After Drp1 or PINK1 depletion, the protein levels of Runx2 and Osterix of cells in H-Glu+BMP9 group were significantly decreased , and the formation of mineralized nodules was inhibited, as compared with only treated with BMP9 . Correspondingly, the MMP of cells in H-Glu+BMP9 group significantly decreased after PINK1 or Drp1 knockdown . Furthermore, the level of mitophagy of cells in H-Glu+BMP9 group was also significantly decreased after Drp1 or PINK1 was knocked down . The results indicate that BMP9 can also effectively ameliorate osteogenic differentiation of BMSCs under high glucose by augmenting the activity of the PINK1/Drp1 signaling axis. Figure 7 BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteoblast differentiation impairment in BMSCs The concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu, H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 25 mmol/L. The final concentration of BMP9 in H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 100 ng/ml. (A) BMSCs cells were cultured in osteogenic medium for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB. (B) BMSCs cells were cultured in osteogenic medium for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 1 mm. (C) BMSCs cells were cultured in osteogenic medium for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm. (D) BMSCs cells were transfected with mito-Keima plasmid and siRNA and cultured in osteogenic medium for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm. Section title: BMP9 treatment in diabetic mice enhances bone mineral density and improves bone microarchitecture Educational score: 4.070879936218262 Domain: biomedical Document type: Study Language: en To further verify the effects of BMP9 on bone mineral density (BMD) and bone quality in vivo , we constructed an streptozotocin (STZ) induced diabetes mice model. The design of the animal experiment is shown in Figure 8 A. All diabetic mice remained hyperglycemic until the end of the study. Although serum BMP9 concentration was higher in STZ+BMP9 group, blood glucose was similar between STZ and STZ+BMP9 group. . Figure 8 Diabetic mice models induced by STZ were treated with BMP9 (A) Flow charts for the establishment of STZ mice model. (B) Fasting blood glucose concentrations were measured and compared every two weeks from week 2 to week 18. (C) Fasting blood glucose concentrations at week 18 were compared between groups. (D) Serum BMP9 levels were detected at week 18. Data presented as mean ± SD. n = 8 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: BMP9 treatment in diabetic mice enhances bone mineral density and improves bone microarchitecture Educational score: 4.23948860168457 Domain: biomedical Document type: Study Language: en As shown in Figure 9 A, a 3D reconstruction of the cortical bone and trabecular bone at the distal femurs clearly showed thinner cortical bone and the breakage of cancellous bone in STZ diabetic mice, and the administration of BMP9 improved the cortical bone thickness and microarchitecture of the distal femur trabecular bone. Moreover, micro-CT analysis of the distal femur showed decreased cortical BMD in the STZ mice and improved BMD in the STZ+BMP9 group ( p < 0.05) . Other cortical bone parameters, including BV/TV and Ct.Th, were significantly decreased in STZ mice as compared with controls ( p < 0.05). The Ct.Th value of STZ mice treated with BMP9 increased compared to that in the STZ group ( p < 0.05) , while the difference in BV/TV value between the two groups was not statistically significant . Micro-CT analysis also showed that compared with mice in the control group, STZ mice exhibited obvious decrease in BV/TV, Tb.N, and BMD and increased Tb.Sp of the distal femur trabecular bone, while the BV/TV, Tb.N, and BMD of mice treated with BMP9 increased by 43% ( p < 0.05), 30% ( p < 0.05), and 29% ( p < 0.01), respectively . Consistent with the micro-CT analysis results, H&E staining of the distal femur sections confirmed a reduction in trabecular number and trabecular thickness in STZ mice compared with that of the control, and these reductions were attenuated by BMP9 treatment . Figure 9 BMP9 promoted mitophagy markers to the control levels in diabetic mice in vivo (A) Representative images derived from micro-CT analysis, including 2D image construction of distal femur, 3D images reconstruction of trabecular bone of distal femur, and 3D image reconstruction of the femoral midshaft corticoid bone. (B–D) Quantitative analysis of the vBMD, BV/TV and Ct.Th of corticoid bone by micro-CT. (E–I) Quantitative analysis of the BV/TV, Tb.N, Tb.Sp, Tb.Th, and vBMD of trabecular by micro-CT. (J) Representative images of HE-stained decalcified femur sections. Scale bars, 200 μm. (K–M) The right femur was isolated and subjected to biomechanical properties analysis. The maximum load, elastic modulus and SMI were evaluated for each group. (N) Representative images derived from micro-CT analysis, including 2D image construction and 3D image reconstruction of L3 lumbar vertebra. (O–S) Quantitative analysis of the vBMD, BV/TV, Tb.N, Tb.Th, and Tb.Sp of L3 by micro-CT. (T and U) The levels of serum bone turnover parameters PINP and CTX-I were detected by ELISA. (V) WB analysis of protein levels of mitophagy marker in skull. (W) Expressions of ALP, Osx, Drp1, and PINK1 in skull were determined by RT-qPCR. (X) Immunofluorescence analysis of Drp1 and PINK1 expression in femur sections. Scale bars, 10 μm. (Y) Immunofluorescence analysis of PINK1 expression in femur sections. Scale bars, 20 μm. (Z) Immunofluorescence results of double labeling of osteoblasts with PINK1 and Drp1 in femur sections. Scale bars, 20 μm. Data presented as mean ± SD. n = 8 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Ns, no significance. Section title: BMP9 treatment in diabetic mice enhances bone mineral density and improves bone microarchitecture Educational score: 4.082710266113281 Domain: biomedical Document type: Study Language: en Furthermore, compared with the control group, the maximum load and Young’s modulus of femur in STZ group were significantly decreased ( p < 0.05), the maximum load and elastic modulus of femur in STZ+BMP9 group were increased by 27.1% ( p < 0.01), and the value of elastic modulus was increased by 21.6% compared with STZ mice, but there was no statistical difference. The structural simulation index was not significantly different from that of STZ group . Section title: BMP9 treatment in diabetic mice enhances bone mineral density and improves bone microarchitecture Educational score: 4.1136322021484375 Domain: biomedical Document type: Study Language: en Additionally, we examined the parameters of lumbar vertebrae in mice. The bone density and 3D reconstruction images of the third lumbar vertebra in mice were shown in Figure 9 M. Compared with the normal control group, the lumbar-spine BMD in the STZ group decreased by 16.9% ( p < 0.01) and increased by 14.1% ( p < 0.01) in STZ+BMP9 group. The BV/TV, Tb.N, and Tb.Th values of lumbar cancellous bone in the STZ group were significantly decreased compared with the control group, and the Tb.Sp value was significantly increased in the STZ mice. Compared with the STZ group, the BV/TV and Tb.N values of lumbar cancellous bone in the STZ+BMP9 group were increased by 10.7% and 7.5%, respectively, and there was no significant difference compared with the control group. The Tb.Sp and Tb.Th values of lumbar vertebrae in STZ+BMP9 group were not significantly different from those in STZ group . Section title: BMP9 treatment in diabetic mice enhances bone mineral density and improves bone microarchitecture Educational score: 4.076971054077148 Domain: biomedical Document type: Study Language: en We then examined bone turnover markers in mice. Compared with the normal control group, the serum procollagen I N-terminal propeptide (PINP) level of mice in STZ group was significantly decreased ( p < 0.01), but was significantly increased in STZ+BMP9 group compared with the STZ mice group ( p < 0.01) . There was no significant difference in serum C-terminal telopeptide of type 1 collagen (CTX-I) concentration between three groups . These results suggest that BMP9 has the ability to ameliorate diabetic osteoporosis. Section title: BMP9 promoted mitophagy markers to the control levels in diabetic mice in vivo Educational score: 4.172711372375488 Domain: biomedical Document type: Study Language: en The favorable bone effect of BMP9 in diabetic STZ mice was investigated mechanistically. The protein from parietal bone in mice was quantified by WB. The results demonstrated that the expressions of mitophagy related proteins PINK1 and Drp1 were significantly increased in mice treated with BMP9, and the ratio of autophagy marker proteins LC3A/BⅡ and LC3A/BⅠ were significantly increased in STZ+BMP9 group . Moreover, qPCR results showed expression levels of ALP, Osx, Drp1, and PINK1 were significantly increased in BMP9 treatment group . We further stained the bone sections of the femur in mice by immunofluorescence. As shown in Figure 9 X, the expression of Drp1 protein in osteoblasts located on the bone trabecular surface of mice in STZ group was significantly decreased compared with that of control mice, and was significantly increased after BMP9 administration, which was consistent with the variation trend of PINK1 in Figure 9 Y. In order to further confirm the aforementioned results, double-label immunofluorescence staining was performed on the femur section. Osx was used to label osteoblasts and the expression of PINK1 in osteoblasts was detected. Compared with the control group, the expression of PINK1 in osteoblasts in the STZ group was significantly decreased. The expression of PINK1 in osteoblasts in STZ+BMP9 group was significantly higher than that in STZ group . These results suggested that BMP9 can promote the expression of mitophagy protein in bone tissue of diabetic mice, promoting mitophagy may be an effective means to improve the bone mass and quality in diabetic mice. Section title: Discussion Educational score: 4.095444679260254 Domain: biomedical Document type: Study Language: en In this study, we revealed that the suppressed PINK1/Drp1-mediated mitophagy is a molecular pathway responsible for impaired osteogenic differentiation in diabetes. Activation of mitophagy by BMP9 can mitigate osteoporosis induced by hyperglycemia. Section title: Discussion Educational score: 4.182399749755859 Domain: biomedical Document type: Study Language: en Osteoblast-mediated bone formation requires enough ATP, which is generated through OXPHOS and glycolysis. 33 Our research found that high glucose condition lasting to the late stage of differentiation could induce the decrease in basal and maximal respiration, ATP synthesis, and glycolytic rate of osteoblast. We also found mitochondrial dysfunctions, including the increased ROS, and decreased MMP in osteoblast cultured under high glucose level. Our RNA sequencing analysis suggested that impaired mitophagy might be the molecular mechanism for high glucose-induced inhibition of osteogenic differentiation. Section title: Discussion Educational score: 4.199438095092773 Domain: biomedical Document type: Study Language: en Upon mitochondrial stress, mitophagy is activated and can selectively eliminate the damaged mitochondria, and thus stabilizing cellular homeostasis. 34 Autophagy and mitophagy play a vital role in regulating osteoblasts’ proliferation and differentiation. Thus, we further explored the role of mitophagy in high glucose-induced inhibition of osteogenic differentiation and its related signaling pathways. PINK1 is an important promotor of mitochondrial autophagy. When MMP declines, the Ser228 and Ser402 groups of PINK1 will be phosphorylated, 35 upon activation of PINK1 phosphorylation, ubiquitin on the mitochondrial outer membrane can be phosphorylated, then initialing mitophagy. We found that siRNA knockdown of PINK1 inhibited the ameliorative effect of BMP9 to impaired mitophagy and osteogenic differentiation in high glucose conditions, while overexpression of PINK1 can reverse the inhibitory effect of high glucose level on osteogenic differentiation. Section title: Discussion Educational score: 4.390120506286621 Domain: biomedical Document type: Study Language: en Mitophagy and mitochondrial dynamics are close related. Drp1, a GTPase of the dynein superfamily, is the most important molecule in promoting mitochondrial fission. After Drp1 is recruited into the outer membrane of mitochondria, it will form helical oligomers, thereby inducing contraction and severing of the mitochondrial membrane. 36 Our RNA sequencing analysis showed the decreased expression level of Drp1 in osteoblasts cultured in high glucose medium, suggesting the potential interplay between Drp1 and PINK1-induced mitophagy in impaired osteogenic differentiation caused by hyperglycemia. Then, we conducted a series of experiments to address the regulatory effect between Drp1 and PINK1. We found that the expression of Drp1 was downregulated after the knockdown of PINK1. In contrast, the expression of PINK1 did not change following the inhibition of Drp1, suggesting Drp1 as a downstream signaling molecule of PINK1. Our results also demonstrated that Drp1 knockdown can inhibit osteogenic differentiation and mitophagy and impair mitochondrial function. PINK1 was shown to directly phosphorylate S616 of Drp1 to regulate mitochondrial fission, 37 which was consistent with our results. Taken together, we hypothesized that PINK1/Drp1-mediated mitophagy plays a critical role in osteogenic differentiation, and its suppression contributes to hyperglycemia-induced impaired osteoblastogenesis. To further verify this hypothesis, we then tried to find a molecule to activate this signaling pathway to maintain mitophagy of osteoblast in hyperglycemia and treat diabetic osteoporosis in vivo . Section title: Discussion Educational score: 4.247705936431885 Domain: biomedical Document type: Study Language: en BMP9, a member of the transforming growth factor beta (TGF-β) superfamily, regulates various physiological processes such as angiogenesis, glucose metabolism, neurogenesis, and tumorigenesis. Our previous research found that BMP9 promotes osteoblastogenesis by upregulating LGR6 and activating the Wnt/β-catenin pathway and suppresses osteoclast differentiation by inhibiting the nuclear factor-κB (NF-κB) pathway. 31 Our team’s other research also found that BMP9 reduces expression of senescent genes and alleviates senescence-associated secretory phenotype in bone microenvironment, simultaneously increasing bone mass and bone biomechanical properties in aged mice. 32 As a continuation of work, we investigated the regulatory role of BMP9 on mitophagy in osteoblast and its protective effect on diabetic osteoporosis in this study. In vitro , we observed that BMP9 improved the osteogenic differentiation of MC3T3-E1 cells under high glucose conditions. This protective effect of BMP9 on osteoblasts could be mediated by the regulation of mitophagy through its capacity to upregulate PINK1/Drp1 expression. Whereas knockdowns of PINK1 and Drp1 can abolish the bone protective function of BMP9 under high glucose conditions. Section title: Discussion Educational score: 4.36656379699707 Domain: biomedical Document type: Study Language: en Mitophagy dysfunction participates in pathogenesis diabetes and diabetes-related complications that include β-cell damage, 38 glucose intolerance, 39 cardiomyopathy, 40 insulin resistance, 41 and diabetes-associated cognitive dysfunction. 42 Previous study has found that non-imprinted protein 2 in the Prader-Willi/Angelman syndrome region (NIPA2), a mitophagy-related molecule, was decreased in the bone tissue of diabetic mice. 43 Our research found that PINK1/Drp1-mediated mitophagy was inhibited in the bone of diabetic mice and its activation by BMP9 ameliorated diabetic osteoporosis, indicating that PINK1/Drp1-mediated mitophagy may be a therapeutic target for diabetic osteoporosis. BMP9 is known to be a powerful promoter of osteogenic differentiation. Our research found that BMP9 rescued diabetic osteoporosis by addressing various suboptimal bone phenotypes, including promoting bone formation, improving bone density, enhancing the microstructure of cancellous and cortical bones, and ameliorating femur biomechanics. Notably, all these favorable bone effects of BMP9 in diabetes are not related to changes in blood glucose. These results suggest that BMP9 may act directly and specifically on bone tissue in diabetic mice, and this advantage makes BMP9 a potential therapeutic drug for diabetic osteoporosis. Section title: Discussion Educational score: 4.126983642578125 Domain: biomedical Document type: Study Language: en In conclusion, our findings suggest the suppressed PINK1/Drp1-mediated mitophagy is responsible for the impairment of osteogenic differentiation in diabetes. BMP9 facilitates osteogenic differentiation and maturation under diabetic conditions through activating PINK1/Drp1-mediated mitophagy, ultimately exerting a bone-protective effect in diabetic mice. Further research on PINK1/Drp1-mitophagy pathway and its agonists, such as BMP9, is a promising way to explore the potential pathogenesis and therapeutic strategy for osteoporosis in diabetes. Section title: Limitations of the study Educational score: 4.020401477813721 Domain: biomedical Document type: Study Language: en This study had several limitations. Firstly, the precise mechanism through which BMP9 activates PINK1/Drp1 signaling pathway requires further elucidation. Secondly, the late onset of skeletal phenotype in diabetic mice led to a limited number of extracted skull primary cells or mesenchymal stem cells with diminished activity. Thirdly, we did not use osteoblast conditional gene knockout mouse models to verify the impaired PINK1/Drp1-mediated mitophagy as a pathogenesis of diabetic osteoporosis. Section title: Lead contact Educational score: 1.0401784181594849 Domain: other Document type: Other Language: en Further information and requests for the resources and reagents should be directed to and will be fulfilled by the lead contact, Hong-yan Zhao ( hyanzhao@163.com ). Section title: Materials availability Educational score: 2.291783094406128 Domain: biomedical Document type: Study Language: en This study did not generate any new unique reagents and components. The siRNA oligonucleotide sequences were provided in Table S1 , and primer sequences of target genes were provided in Table S2 . Section title: Data and code availability Educational score: 0.9649666547775269 Domain: biomedical Document type: Other Language: en • RNA sequencing data have been deposited at Zenodo and are publicly available as of the date of publication. Accession numbers are listed in the key resources table . • This paper does not report the original code. • Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request. Section title: Acknowledgments Educational score: 0.9163002967834473 Domain: other Document type: Other Language: en This research was supported by the 10.13039/501100001809 National Natural Science Foundation of China and Shanghai Sailing Program . We sincerely appreciate their generous funding, which made it possible for us to conduct this research project. We would also like to express our gratitude for the technical support provided by the laboratory staff at Shanghai National Clinical Research Center for Endocrine and Metabolic Diseases. Finally, we would like to thank the anonymous reviewers for their insightful and constructive feedback on our manuscript, which has significantly improved its overall quality. Section title: Author contributions Educational score: 1.0411733388900757 Domain: other Document type: Other Language: en X.-j.C.: conceptualization, investigation, formal analysis, validation, writing – original draft, writing – review and editing. Y.-y.Y.: investigation, formal analysis, validation. Z.-c.P.: investigation, formal analysis, validation. J.-z.X.: investigation, validation. T.J.: investigation, validation. L.-l.Z.: investigation, validation. K.-c.Z.: methodology, writing – review and editing. D.Z.: methodology, writing – review and editing. J.-x.S.: methodology, writing – review and editing. C.-x.S.: methodology, writing – review and editing. L.-h.S.: methodology, writing – review and editing. B.T.: conceptualization, supervision, writing – review and editing, formal analysis. J.-m.L.: conceptualization, supervision, formal analysis, writing – review and editing. H.-y.Z.: conceptualization, supervision, formal analysis, funding acquisition, project administration, writing – review and editing. Section title: Declaration of interests Educational score: 0.878638505935669 Domain: other Document type: Other Language: en The authors declare no competing interests. Section title: Key resources table Educational score: 3.041226625442505 Domain: biomedical Document type: Other Language: en REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Anti-Hsp90 antibody Santa Cruz Cat# sc-13119 Rabbit Anti-PINK1 antibody Novus Cat# BC100-494S Rabbit Anti-Drp1 antibody Abcam Cat# ab184247 Rabbit Anti-Runx2 antibody CST Cat# 8486 Rabbit Anti-Osx antibody Abcam Cat# ab209484 Anti-VDAC1/Porin antibody Abcam Cat# ab306581 LC3A/B antibody CST Cat# 4108 HRP-conjugated Alpha Tubulin antibody Proteintech Cat# HRP-66031 HRP-linked Anti-Mouse antibody CST Cat# 7076 HRP-linked Anti-Rabbit antibody CST Cat# 7074 CF 488 tyramide Runnerbio China Cat# Bry-880488 AF 647 tyramide Runnerbio China Cat# Bry-880647 Chemicals, peptides, and recombinant proteins α-modified minimal essential medium Gibco Cat# 32571101 Fetal bovine serum Gibco Cat# 10091148 Penicillin/streptomycin Beyotime Cat# C0222 β-glycerophosphate disodium salt hydrate Sigma-Aldrich Cat# 50020 L-ascorbic acid Sigma-Aldrich Cat# A4544 Lipofectamine 2000 Invitrogen Cat# 11668027 Streptozotocin Sigma-Aldrich Cat# S0130 MitoSOX™ Red mitochondrial superoxide indicator Invitrogen Cat# M36008 Image-iT tetramethylrhodamine (TMRM) reagent Invitrogen Cat# I34361 Hoechst 33342 Invitrogen Cat# H3570 Seahorse XF DMEM medium Agilent Cat# 103575–100 Hematoxylin Staining Solution Beyotime Cat# C0107 Super sensitive ECL luminescence reagent Meilunbio Cat# MA0186 Tri reagent Sigma-Aldrich Cat# T9424 Recombinant Mouse BMP-9 Biolegend Cat# 553204 Critical commercial assays BCIP/NBT Alkaline Phosphatase Color Development Kit Beyotime Cat# C3206 Alizarin Red S Solution Solarbio Cat# G1450 Mitochondrial isolation kit Thermo Scientific Cat# 89874 Bicinchoninic acid (BCA) protein assay kit Thermo Scientific Cat# 23227 ROS assay kit Beyotime Cat# S0033S ELISA Kit for Procollagen I N-Terminal Propeptide (PINP) USCN Life Science Cat# SEA957Hu ELISA Kit for Cross Linked C-Telopeptide Of Type I Collagen (CTXI) USCN Life Science Cat# CEA665Hu Mouse BMP-9 ELISA Kit RayBiotech Cat# Q9WV56 Seahorse XF Cell Mito Stress Test Kit Agilent Cat# 103015–100 PrimeScript reverse transcript master mix TaKaRa Cat# RR036Q ChamQ Universal SYBR qPCR Master Mix Vazyme Cat# Q711-02 Total OXPHOS Rodent WB antibody cocktail Abcam Cat# ab110413 Bicinchoninic acid (BCA) protein assay kit Thermo Scientific Cat# 23227 Deposited data RNA sequencing data This paper Zenodo: https://doi.org/10.5281/zenodo.14174646 Experimental models: Cell lines MC3T3-E1 cells ATCC Cat# CRL-2593 Experimental models: Organisms/strains C57BL/6 mice Charles River Cat# 213 Oligonucleotides PINK1 siRNA GenePharma N/A Drp1 siRNA GenePharma N/A siRNA oligonucleotide sequences, see Table S1 This paper N/A Primer sequence of target genes in RT-qPCR, see Table S2 This paper N/A Recombinant DNA Mitochondria-targeted mKeima-Red expression plasmid MBL Cat# AM-V0251 PINK1 expression plasmid GenePharma N/A Drp1 expression plasmid Shanghai Xitubio Biotechnology N/A AAV-BMP9 Hanbio N/A Software and algorithms Zen software Zeiss N/A R (version 4.3.2) R Core Team https://www.r-project.org GraphPad Prism 9 GraphPad https://www.graphpad.com/features Other Instron 5569 Instron https://www.instron.com/en/products/testing-systems/out-of-production-systems/electromechanical SkyScan 1176 Bruker https://www.accela.eu/bruker-biospin/skyscan-1176 Seahorse Extracellular Flux Analyzer XFe96 Agilent https://www.agilent.com/cs/library/usermanuals/public/usermanual-xfe96-xf96-cell-characterization-cell-analysis-5994-0368en-agilent.pdf QuantStudio Dx Real-Time PCR Instrument Applied Biosystems https://www.thermofisher.com/hk/en/home/clinical/diagnostic-testing/instruments-automation/genetic-analysis-instruments/quantstudio-dx-systems.html NanoDrop ND-2000 spectrophotometer Thermo Scientific Cat# ND-2000C eBlot Touch Imager eBlot https://www.e-blot.com/ JEM-1400Flash electron microscope JEOL https://www.jeol.com/products/scientific/tem/JEM-1400Flash.php TissueFAXS system TissueGnostics, Austria https://tissuegnostics.com/products/scanning-and-viewing-software/tissuefaxs-imaging-software Section title: Cell line Educational score: 4.038376331329346 Domain: biomedical Document type: Study Language: en The preosteoblast line MC3T3-E1 cells (American Type Culture Collection) were cultured in α-modified minimal essential medium (α-MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Beyotime) at 37°C with 5% CO2. The culture medium was changed every two days. Section title: Cell line Educational score: 4.150334358215332 Domain: biomedical Document type: Study Language: en BMSCs were isolated from the bone marrow of mice. First, mice were euthanized, followed by the dissection of its tibia, femur, and humerus. The ends of the bones were cut off with sharp scissors. The bone marrow was then flushed out from the bone cavities using α-MEM containing 10% FBS. The aspiration of bone marrow was filtered through a 70-micron mesh, then centrifuged, washed, and resuspended in α-MEM containing 10% FBS. The culture dishes were placed at 37°C in a 5% CO2 incubator. Non-adherent cells were removed after 24–72 h by changing the culture medium. Once the culture reached 70–90% confluence, the cells were subcultured at a 1:3 split ratio. Section title: Mouse strain Educational score: 2.5886690616607666 Domain: biomedical Document type: Study Language: en Male C57BL/6 mice (Charles River), 9 weeks old, were maintained at the Experimental Animal Facility with temperatures of 22°C–24°C, humidity levels of 55%–60%, and a 12-h light/dark cycle. They were provided with water and a standard rodent chow diet. All animal experiments were conducted according to the guidelines for the humane use and care of laboratory animals and were approved by the Shanghai Jiao Tong University School of Medicine Animal Study Committee. Section title: Osteoblastogenic induction and bone morphogenetic protein 9 intervention Educational score: 4.0979766845703125 Domain: biomedical Document type: Study Language: en Osteoblastogenic induction medium consisted of α-MEM (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Beyotime), 10mM β-glycerophosphate disodium salt hydrate (Sigma-Aldrich), and 50μM L-ascorbic acid (Sigma-Aldrich). The osteoblastogenic induction media were formulated with two concentrations of glucose: low glucose (5.5 mmol/L) and high glucose (25 mmol/L). BMP9 intervention was achieved through the supplementation of 100 ng/mL BMP9 (Biolegend) in the osteoblastogenic induction media. MC3T3-E1 cells or BMSCs were cultured in the osteoblastogenic induction media to induce osteogenic differentiation. Based on the glucose concentrations of the media and BMP9 intervention during osteogenic differentiation, the MC3T3-E1 cells or BMSCs were assigned to three groups: the low-glucose group (L-Glu), the high-glucose group (H-Glu), and the high-glucose group supplemented with BMP9 (H-Glu + BMP9). Section title: Small interfering RNA (siRNA) and plasmid transfection Educational score: 4.008640289306641 Domain: biomedical Document type: Study Language: en We purchased commercial and customized siRNAs and plasmids: a PINK1 siRNA (GenePharma), a Drp1 siRNA (GenePharma), a mitochondria-targeted mKeima-Red expression plasmid (pMT-mKeima-Red, MBL), and a PINK1 expression plasmid (GenePharma). MC3T3-E1 cells were transiently transfected with siRNA or plasmids using Lipofectamine 2000 (Invitrogen), following the transfection protocol provided by the Lipofectamine 2000 reagent. The siRNA oligonucleotide sequences were listed in Table S1 . Section title: Alp staining assay and Alizarin red staining assay Educational score: 4.173403739929199 Domain: biomedical Document type: Study Language: en Alp staining of differentiated MC3T3-E1 cells was performed using a 5-bromo-4-chloro-3-indolyl phosphate/tetranitro blue tetrazolium chloride (BCIP/NBT) alkaline phosphatase color development kit (Beyotime) according to the manufacturer’s instructions. In brief, MC3T3-E1 cells were washed and fixed with 4% paraformaldehyde following the 7-day differentiation induction, and the staining solution was applied for 15 min at room temperature. Level of differentiation was quantified by visual examination of cells stained purple. Alizarin red S staining assay (Solarbio) was performed after 14 and 21 days of osteoblastic induction according to the manufacturer’s instructions. In brief, MC3T3-E1 cells were fixed with paraformaldehyde for 10 min. The mineralized nodules were stained with 0.2% Alizarin red and washed three times with PBS. Section title: Western blot and qPCR analyses Educational score: 4.160195350646973 Domain: biomedical Document type: Study Language: en Total protein was extracted by radioimmunoprecipitation (RIPA) lysis buffer (Biocolors, Shanghai, China) supplemented with Protease Inhibitor Cocktail (APExBIO, Houston, USA). Mitochondrial protein fraction was extracted using a mitochondrial isolation kit (Thermo Scientific) for cultured cells according to the manufacturer’s instruction. The concentration of protein was measured using a Bicinchoninic acid protein assay kit (Thermo Scientific), then the degenerated protein lysates were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked in 5% fat-free milk (Sangon Biotech) for 1.5 h at room temperature and then incubated overnight at 4°C with the primary antibodies: mouse anti-Hsp90, rabbit anti-PINK1, rabbit anti-Drp1, rabbit anti-Runx2, rabbit anti-Osx, anti-VDAC1/Porin antibody, LC3A/B antibody and antibodies of OXPHOS complexes I–V from total OXPHOS Rodent WB antibody cocktail (Abcam). HRP-conjugated secondary antibody incubation was performed at room temperature for 1 h. Immunoblotting signals were detected by super sensitive ECL luminescence reagent (Meilunbio, China) and visualized by the eBlot Touch Imager (eBlot, China). Section title: Western blot and qPCR analyses Educational score: 4.100516319274902 Domain: biomedical Document type: Study Language: en For RNA extraction, total RNA was extracted from tissues or cultured cells using Tri reagent (Sigma-Aldrich). The absorbance ratio at 260/280 nm and the RNA concentration of all samples were detected using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific). Reverse transcription was performed using the PrimeScript RT Master Mix (TaKaRa), and RT-qPCR was conducted using the ChamQ Universal SYBR qPCR Master Mix (Vazym). The measurements were taken using the QuantStudio Dx Real-Time PCR Instrument (Applied Biosystems, USA). The ΔΔCT method was used to evaluate the relative mRNA expression that was normalized to 36b4. Primer sequences were listed in Table S2 . Section title: Cell bioenergetic profiling Educational score: 4.166317939758301 Domain: biomedical Document type: Study Language: en Mitochondrial function was determined by measuring the oxygen consumption rate (OCR) in real time using a Seahorse Extracellular Flux Analyzer XFe96 (Agilent). Cells were seeded in an XF96 microplate at 3 × 10³ cells/well. The culture medium was changed to Seahorse XF DMEM buffer (Agilent) containing 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate, and the plate was incubated at 37°C in a non-CO₂ incubator for 1 h. The compounds from the Seahorse XF Cell Mito Stress Test Kit (Agilent) were loaded into the injection port to reach the final concentrations of 1 μM oligomycin, 2 μM carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP), 0.5 μM antimycin A, and 0.5 μM rotenone to detect respiratory parameters, including basal respiration, ATP production-coupled respiration, maximal respiration, and spare respiratory capacity. The OCR in each microplate well was normalized to the total protein of each well. Section title: Cell bioenergetic profiling Educational score: 4.130896091461182 Domain: biomedical Document type: Study Language: en The glycolytic function was evaluated by measuring the extracellular acidification rate (ECAR) in real time using a Seahorse Extracellular Flux Analyzer XFe96 (Agilent). Briefly, cells were seeded in an XF96 microplate at 3×10 3 cells/well. The culture medium was changed to Seahorse XF DMEM buffer (Agilent) and the plate was incubated at 37°C in a non-CO2 incubator for 1 h. The compounds were loaded into the injection port in sequence to reach the final concentration of 10mM glucose, 2μM oligomycin, and 0.1M 2-Deoxy-D-glucose (2-DG). The final ECAR results were normalized to total protein. Section title: ROS measurement Educational score: 4.1003594398498535 Domain: biomedical Document type: Study Language: en The intracellular levels of ROS in cells were assessed by ROS assay kit (Beyotime) that contains DCFH-DA fluorescent probe. Briefly, cells were seeded in 12-well plates and induced into osteoblastogenic differentiation for 14 days. Subsequently, washed cells were incubated with 10 μM DCFH-DA for 20 min in dark and then harvested and washed with PBS. Finally, the intensity of DCFH-DA fluorescence was determined by flow cytometry (BD FACSLyric) at 480 nm (excitation) and 530 nm (emission). Section title: Detection of mitochondrial membrane potential Educational score: 4.1403703689575195 Domain: biomedical Document type: Study Language: en MMP was assessed using Image-iT tetramethylrhodamine (TMRM) reagent (Invitrogen) following the manufacturer’s instructions. In brief, cells were seeded in 35 mm glass-bottom microwell dishes (MatTek) and induced to undergo osteoblastogenic differentiation for 14 days. Subsequently, the washed cells were incubated with 100 nM TMRM reagent and 0.1 mg/mL of the nuclear stain Hoechst 33342 (Thermo Scientific) at 37°C in an incubator for 30 min, followed by washing with PBS. Finally, the microscope was configured to image TMRM (Ex 548/Em 574) and Hoechst 33342 (Ex 350/Em 460), and images were acquired from multiple sites per well at 20× magnification for fluorescence expression using a laser scanning confocal microscope (LSM880, ZEISS). Section title: Mitochondrial superoxide measurement Educational score: 4.146015167236328 Domain: biomedical Document type: Study Language: en Mitochondrial superoxide in cells was detected by MitoSOX Red mitochondrial superoxide indicator (Invitrogen). Cells were seeded in 35 mm glass bottom microwell dishes (MatTek) and induced to undergo osteoblastogenic differentiation for 14 days. Subsequently, the washed cells were incubated with 5 μM MitoSOX and 0.1 mg/mL of the nuclear stain Hoechst 33342 (Thermo Scientific) at 37°C in an incubator for 10 min, followed by washing with PBS. Finally, the microscope was configured to image TMRM (Ex 510/Em 580) and Hoechst 33342 (Ex 350/Em 460), and images were acquired from multiple sites per well at 20× magnification using a laser confocal microscope (LSM880, ZEISS). Section title: Detection of mitophagy in vitro Educational score: 4.055600643157959 Domain: biomedical Document type: Study Language: en To visualize mitophagy using a mitophagy-specific probe, MCT3E1 cells were transfected with pMT-mKeima-Red (MBL). Keima in neutral conditions was observed using excitation of 430 ± 20 nm and emission of 624 ± 20 nm. Keima in acidic conditions was observed using excitation of 562 ± 20 nm and emission of 624 ± 20 nm. Images were obtained by an LSM880 confocal microscope (Zeiss). Section title: Transmission electron microscopy Educational score: 4.199743747711182 Domain: biomedical Document type: Study Language: en For primary fixation, MC3T3-E1 cells were enriched by centrifugation, then immersed in 2.5% glutaraldehyde and stored at 4°C for 24 h. The samples were washed with 0.1 M phosphate buffer for 15 min, three times, and then immersed in 1% osmium tetroxide for 2 h for post-fixation. For dehydration, the samples were washed with 0.1 M phosphate buffer for 15 min, three times, and then step-by-step immersed in dehydrating agents (50% ethanol, 75% ethanol, 80% ethanol, 95% ethanol, 100% ethanol, and 100% acetone). The samples were immersed in infiltration agent A (a 2:1 solution of acetone and 812 embedding agent) for 2 h, then in infiltration agent B (a 1:2 solution of acetone and 812 embedding agent) overnight. For embedding, the samples were placed into a mold with embedding medium and then baked at 37°C and 60°C for 12 h, respectively. The samples were cut into sections (60–80 nm thick). For lead-uranium double staining, the sample sections on the copper grid were immersed in a 2% uranyl acetate staining solution for 30 min in the dark, and then in lead citrate staining solution for 15 min. Image Acquisition: The organelle morphology was captured using the JEM-1400Flash electron microscope (JEOL). Section title: Bone morphogenetic protein 9 intervention for STZ-induced diabetic osteoporosis in the mouse model Educational score: 4.0210065841674805 Domain: biomedical Document type: Study Language: en Mice assigned to the STZ group were administered low doses of STZ at 40 mg/kg for five consecutive days to induce diabetic osteoporosis. Mice in the control group were injected with a sodium citrate solution. Mice assigned to the STZ+BMP9 group were administered STZ for five consecutive days, then were administered AAV-BMP9 (Hanbio) at week 5. Specimens were collected at week 18, and elevated BMP9 levels were maintained until the mice were euthanized. Section title: Micro-CT analysis and three-point bending test Educational score: 4.1212053298950195 Domain: biomedical Document type: Study Language: en All procedures involving micro-CT were performed according to the recommendation of the American Society for Bone and Mineral Research. 44 Briefly, the right femurs of mice were isolated, fixed in 4% paraformaldehyde for 48 h, and then maintained in 75% ethanol. Quantitative analysis of distal femoral metaphysis and midshaft femoral diaphysis were performed using a high-resolution ex vivo micro-CT scanner, Skyscan 1176 (Bruker). Using 2D data from scanned slices, 3D analysis was performed to calculate morphometric parameters by a microCT Software (Bruker). The following trabecular morphometric indexes were analyzed: vBMD, percentage of bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and structure model index (SMI). The cortical thickness (Ct.Th) was assessed at the midshaft of femurs. Section title: Micro-CT analysis and three-point bending test Educational score: 4.08202600479126 Domain: biomedical Document type: Study Language: en For biomechanical testing, the left femurs were cleaned of adherent tissue, wrapped in saline-soaked gauze, and tested immediately. A three-point bending test was carried out at the midshaft of the femurs using a mechanical testing machine, the Instron 5569 (USA). The elastic modulus, bending stiffness, maximum bending load, and fracture energy were evaluated. Section title: Serum levels of bone turnover markers Educational score: 4.065899848937988 Domain: biomedical Document type: Study Language: en Mice were fasted for at least 12 h before being euthanized. Blood was collected and allowed to clot at room temperature for 1 h, then centrifuged at 4,000 rpm for 15 min at 4°C. Serum samples were stored at −80°C. Serum levels of bone turnover markers were determined using enzyme-linked immunosorbent assay (ELISA) kits for mouse PINP (USCN, Wuhan, China), CTX-I (USCN, Wuhan, China), and BMP9 (RayBiotech, USA). Section title: H&E staining Educational score: 4.116410732269287 Domain: biomedical Document type: Study Language: en The fixed lumbar spines of mice were decalcified in 23% ethylenediaminetetraacetic acid at 4°C for 5–7 days and embedded in paraffin. Bone sections (4 μm) were deparaffinized and washed with water for 3 min. The bone section was stained with hematoxylin staining solution (Beyotime) for 10 min, then washed with water and hydrochloric acid ethanol for 30 s, counterstained with eosin solution, and dehydrated. Section title: TSA-enhanced multiplex immunohistochemistry staining Educational score: 4.12040901184082 Domain: biomedical Document type: Study Language: en The expression of Drp1 and PINK1 in bone was investigated using multiplex immunohistochemistry staining enhanced by tyramide signal amplification. Bone tissue was prepared into paraffin sections. The bone sections were blocked with 5% BSA and then washed three times with PBS. The sections were incubated overnight in a dilution of anti-Drp1 antibody. After washing three times with PBS, the sections were incubated in a dilution of HRP-linked anti-mouse secondary antibody. Following another round of washing with PBS, the sections were incubated in a dilution of AF 647 tyramide for 10 min. Consistent with the above process, the bone sections were stepwise incubated in a dilution of anti-PINK1 antibody, HRP-linked anti-mouse secondary antibody, and CF 488 tyramide. Finally, the bone sections were incubated in antifade mounting medium with DAPI. The bone sections were scanned using the TissueFAXS system (TissueGnostics, Austria). Section title: Quantification and statistical analysis Educational score: 3.041602849960327 Domain: biomedical Document type: Study Language: en Flow cytometry data were analyzed with FlowJo 10, and fluorescence intensity was quantified by ImageJ software. Section title: Quantification and statistical analysis Educational score: 2.753842353820801 Domain: biomedical Document type: Study Language: en Two-tailed Student’s t tests and two-tailed Mann-Whitney non-parametric tests were used for statistical analysis and were performed using GraphPad Prism 9. Specific statistical analyses used are described in the figure legends, and p -values are listed in the graphs. All data collection and analyses were conducted in a blinded fashion. | Review | biomedical | en | 0.999995 |
PMC11699392 | Section title: Introduction Educational score: 4.048327445983887 Domain: biomedical Document type: Review Language: en Radical prostatectomy (RP) is the primary treatment for prostate cancer. However, a considerable percentage of patients (approximately 15–45 %) experience biochemical recurrence (BCR) after the procedure . Accurately predicting the timing of postoperative BCR is crucial in determining subsequent treatment approaches. In 1999, a predictive model was established incorporating various factors such as preoperative prostate-specific antigen (PSA) levels, pathological Gleason scores (GS), extracapsular extension (ECE), seminal vesicle invasion (SVI), lymph node invasion, and positive surgical margins (PSM). This model remains a valuable tool for determining the appropriate treatment strategies. Kattan developed a nomogram capable of predicting the BCR in patients who underwent RP for prostate cancer. This nomogram was subsequently validated at multiple centers in the United States [ , , , ]. Several prediction models inspired by the Kattan postoperative nomogram were subsequently introduced [ , , ]. Section title: Introduction Educational score: 3.064730167388916 Domain: biomedical Document type: Study Language: en The primary focus of these models is to predict the probability of long-term BCR. However, it is noteworthy that approximately two-thirds of BCR cases manifest within the first 3 years after undergoing RP . Consequently, a model that can accurately forecast the BCR probability within this critical timeframe holds greater significance than previous models. Section title: Introduction Educational score: 4.081273078918457 Domain: biomedical Document type: Study Language: en Contrastingly, previous models have predominantly relied on clinical or pathological data obtained during the perioperative period, neglecting the inclusion of short-term postoperative PSA levels acquired during follow-up. Nonetheless, PSA is a serum marker requiring regular monitoring after RP. Previous studies have demonstrated the potential importance of short-term postoperative PSA levels in predicting BCR. [ , , ] In theory, prostatectomy completely eliminates prostate tumors, leading to a decline in PSA levels to an undetectable range within 6 weeks. However, persistent elevated PSA levels after prostatectomy suggest the presence of residual microtumor foci that require further management. Hence, this study aimed to develop a novel nomogram prediction model based on postoperative elevated PSA level recurrence. Section title: Introduction Educational score: 3.724148988723755 Domain: biomedical Document type: Study Language: en This model sought to enhance the accuracy of predicting the probability of BCR after robot-assited prostatectomy, simplifying existing clinical follow-up strategies and aiding in the early prediction of patients at high risk of BCR. Section title: Study participants Educational score: 3.696645736694336 Domain: biomedical Document type: Study Language: en A total of 1700 patients from University-based hospital who underwent robot-assisted radical prostatectomies between August 2009 and December 2022 were included in this study. Data were collected prospectively and entered into an electronic database by trained personnel. Regular quality control checks were performed to ensure the accuracy of the data. With a median follow-up of 61 months (range, 12–162 months). Consider excluding cases with observation periods less than one year from the analysis. Section title: Variable definition Educational score: 4.044508934020996 Domain: biomedical Document type: Study Language: en The primary focus of this study was to investigate BCR as a primary outcome. In this study, BCR was defined as the occurrence of two consecutive elevations in PSA levels after undergoing RP, with both measurements surpassing 0.2 ng/mL. However, it is important to note that there is currently no universally accepted international standard for the PSA threshold that indicates BCR following RP. Since the publication of the consensus by European urological experts in 2004, two distinct criteria have been widely used, and more researchers have recognized consecutive PSA levels above 0.2 ng/mL . Section title: Variable definition Educational score: 2.689516067504883 Domain: biomedical Document type: Other Language: en The threshold of 0.2 ng/mL was selected based on the consensus from the European Urological Association (EAU) guidelines. While we recognize the absence of a universally accepted threshold, the value of 0.2 ng/mL is commonly adopted by researchers and clinicians in numerous studies . Section title: Variable definition Educational score: 3.977433443069458 Domain: biomedical Document type: Review Language: en Due to the improved lymph node yield, better staging, and theoretical improvement in the control of micro-metastatic disease, guidelines have supported the use of pelvic lymph node dissection (PLND) in patients deemed to be at intermediate or high risk of lymph node involvement and cases with lymph node involvement were excluded. Neoadjuvant conventional androgen deprivation therapy agents (luteinizing hormone–releasing hormone (LHRH) analogues with or without first generation antiandrogens) in the setting of neoadjuvant therapy before radical prostatectomy for patients with clinically localized high risk-prostate cancer [goserelin monotherapy, cyproterone monotherapy and combination therapy with either leuprolide plus flutamide or goserelin plus flutamide]. Section title: Statistical analysis Educational score: 4.089951515197754 Domain: biomedical Document type: Study Language: en We used Cox regression analysis to identify the risk factors associated with BCR. The univariate regression analysis identified important variables, which were subsequently incorporated into the multivariate analysis. The multivariate analysis employed a backward stepwise regression approach, leading to the exclusion of non-significant variables. The remaining significant variables were utilized in constructing the nomogram model, employing the regression modeling strategy package in R software (Windows version 4.3.1; http://www.r-project.org/ ). The consistency index (C-index) of the nomogram model was computed, and a calibration curve was generated to compare predicted values from the nomogram with actual values. Internal validation was performed via 500 iterations of repeated sampling using the bootstrap method, while external validation utilized data from the validation group. The validation cohort was selected from a separate institution using the same inclusion criteria, ensuring that the group represents a comparable patient population. Section title: Results Educational score: 4.181612968444824 Domain: biomedical Document type: Study Language: en The clinical and pathological characteristics of patients are shown in Table 1 . Table 1 Clinical and pathological Features of patients. Table 1 Total Total 1700 Age, years Median (IQR) 67 (63,71) Age, number <65 561 (33.0) ≥65 1137 (66.9) Unknown 2 (0.1) PSA(ng/mL) Median (IQR) 7.5 (5.5–10.7) PSA (ng/mL) <10 1194 (70.2) 10–19.00 391 (23.0) ≥20 112 (6.6) Unknown 3 (0.2) BMI (Kg/m 2 ) <18.5 38 (2.2) 18.5–22.9 635 (37.4) 23–24.9 491 (28.9) 25–29.9 492 (28.9) ≥30 34 (2.0) Unknown 10 (0.6) Clinical T stage 1–2b 1287 (75.7) 2c 289 (17.0) 3–4 120 (7.1) Unknown 4 (0.2) Clinical Gleason score 6 393 (23.1) 7 872 (51.3) 8–10 431 (25.4) Unknown 4 (0.2) Preoperative potency No erection 660 (38.8) Erection 548 (32.2) Penetrate 462 (27.2) Unknown 30 (1.8) D'Amico risk classification High 698 (41.1) Intermediate 769 (45.2) Low 229 (13.5) Unknown 4 (0.2) Neoadjuvant treatment None 1090 (64.1) ADT 166 (9.7) Anti-A 178 (10.5) NACHT 246 (14.5) Unknown 20 (1.2) Nerve-sparing technique None 497 (29.2) Bilateral 207 (12.2) Unilateral 996 (58.6) Pathologic T stage 1–2b 393 (23.1) 2c 1026 (60.4) 3–4 281 (16.5) Pathologic Gleason score 6 167 (9.8) 7 1191 (70.1) 8–10 342 (20.1) Surgical margin Negative 1354 (79.6) Positive 346 (20.4) Extraprostatic extension Negative 1,469 (86.4) Positive 231 (13.6) Seminal vesicle involvement Negative 1606 (94.5) Positive 94 (5.5) Section title: Results Educational score: 4.199941158294678 Domain: biomedical Document type: Study Language: en Independent risk factors associated with BCR were identified By applying Cox regression to the clinical and pathological information of the modeling group ( Table 2 ). Univariate analysis revealed that PSA, clinical T stage, biopsy Gleason score, D'Amico risk classification, pathological T stage, pathologic Gleason score, extraprostatic extension, SVI, and positive surgical margins were independent risk factors for BCR. Multivariate analysis showed a significant association with BCR. Multivariate analysis showed that high PSA ≥20 ng/mL (HR: 1.93; p = 0.034), pathological T stage 3–4 (HR: 1.89; p < 0.001), pathological Gleason score 8–10 (HR: 5.43; p < 0.001), extraprostatic extension (HR: 1.41; p < 0.001), SVI (HR: 1.92; p = 0.018), and positive surgical margin (HR: 2.73; p < 0.001) were independent predictors of BCR considered the independent risk factors and established in the final model. Table 2 Independent predictors of BCR with Cox proportional hazards model. Table 2 Variables Univariate analysis Multivariate analysis Hazard ratio (95 % CI) P value Hazard ratio (95 % CI) P-value Age: 0.83 (0.59–1.17) 0.289 ≥65 vs < 65 PSA (ng/mL) <10 Ref. 10–19.99 1.51 (1.03,2.19) 0.033 1.18 (0.79–1.76) 0.424 ≥20 2.75 (1.65,4.59) <0.001 1.93 (1.05–3.53) 0.034 Clinical Gleason score 6 Ref. Ref. 7 2.77 (1.58,4.86) <0.001 1.84 (0.87–3.9) 0.11 8–10 4.2 (2.35,7.53) <0.001 1.81 (0.74–4.45) 0.195 Clinical T stage 1–2b Ref. 2c 1.86 (1.25–2.76) 0.003 1.24 (0.66–2.3) 0.504 3–4 2.67 (1.61–4.43) <0.001 1.71 (1.02–2.89) 0.043 D'Amico risk classification Low Ref. Ref. Intermediate 2.79 (1.26–6.2) 0.66 1.75 (0.75–4.07) 0.084 High 4.82 (2.2–10.56) <0.001 2.16 (0.9–5.2) 0.196 Pathologic T stage 1–2b Ref. Ref. 2c 0.74 (0.48–1.13) 0.216 0.66 (0.42–1.03) 0.173 3–4 2.87 (1.82–4.51) <0.001 1.89 (0.82–4.38) <0.001 Pathologic Gleason score 6 Ref. Ref. 7 3.61 (1.31–9.95) 0.011 2.74 (0.98–7.65) 0.054 8–10 8.67 (3.09–24.29) <0.001 5.43 (1.89–15.57) <0.001 Extraprostatic extension 2.72 (1.86–3.97) <0.001 1.41 (1.02–2.18) <0.001 Yes vs. No Seminal vesicle involvement 3.92 (2.4–6.39) <0.001 1.92 (1.12–3.29) 0.018 Yes vs. No Surgical margin 3.42 (2.44–4.79) <0.001 2.73 (1.87–3.98) <0.001 Yes vs. No Section title: Results Educational score: 4.055380344390869 Domain: biomedical Document type: Study Language: en The nomogram model serves as a graphical representation of the underlying regression equation. To establish a scoring system, we assigned scores based on the magnitude of the regression coefficients for each independent variable. These scores were then allocated to different levels of each independent variable. Consequently, for every patient, a comprehensive score was computed, enabling the determination of the probability associated with the outcome time for that specific patient. This calculation was facilitated by employing a conversion function that links the score to the corresponding probability of the outcome . Fig. 1 Nomogram predicting BCR after robot-assisted radical prostatectomy. A vertical line perpendicular to the horizontal axis was made according to the corresponding state of each predictor, and the intersection point between the vertical and upper score lines was the score of the predictor. The score of the five predictors was added to obtain the final score of the patient. Fig. 1 Section title: Results Educational score: 4.119897842407227 Domain: biomedical Document type: Study Language: en Internal validation of the prediction model showed that the C-index's accuracy was 0.743 (95 % confidence interval [CI]: 0.741–0.745). Calibration of the new model in the validation group also demonstrated good consistency between the predicted and actual values , indicating that the prediction model performed reasonably well in distinguishing individuals with varying outcome probabilities. Moreover, the area under the receiver operating characteristic (ROC) curve (AUC) was 0.744. This indicates that the model has a significant accuracy in predicting BCR in this context . Fig. 2 Calibration curve of the new model. Fig. 2 Fig. 3 The ROC curve of the prediction model. Fig. 3 Section title: Results Educational score: 3.8652474880218506 Domain: biomedical Document type: Study Language: en The BCR-free survival rate for the entire cohort at the median follow-up time was 25 %, and the 1-, 3-, and 5-year estimates were 90 %, 37.5 %, and 22.5 %, respectively. Fig. 4 shows the BCR-free survival curve after RARP. Fig. 4 Kaplan-Meier survival curve of biochemical recurrence-free survival. Fig. 4 Section title: Discussion Educational score: 4.46065616607666 Domain: biomedical Document type: Review Language: en BCR is a crucial factor in determining whether a change in treatment strategy is necessary in patients who have undergone RP. Approximately 34 % of patients may progress to distant metastasis without additional radiotherapy or endocrine therapy after experiencing BCR . Currently, a sole clinical approach is available for identifying the incidence of BCR. Nevertheless, research findings indicate that the rate of BCR within a decade after undergoing RP is 34.3 % . Throughout the years, researchers have dedicated their efforts to developing a statistical model that can precisely forecast the likelihood of BCR by utilizing clinical and pathological data from patients. The postoperative nomogram introduced by Kattan et al., in 1999 is widely recognized as the most prominent model in this field . Utilizing preoperative PSA levels and postoperative factors such as the Gleason score, ECE, SVI, and PSM, this nomogram demonstrated a high degree of effectiveness in estimating patients' probability of BCR. Subsequently, several scholars proposed a range of prediction models based on the foundation of the Kattan postoperative nomogram. These models incorporate specific variables that emphasize the robotic approach to enhance the overall prediction accuracy of the model [ , , , 17 ]. With efforts to enhance the model's prediction accuracy, the C-index was 0.743 (95 % CI: 0.741–0.745) indicating that the predictive model for BCR after robotic-assited prostatectomy performed well in discriminating between patients at different risk levels. These findings suggest that the model exhibits reasonably accurate ability to predict the occurrence of BCR within this population. Prediction models that amalgamate multiple indicators to anticipate disease occurrence or progression have been extensively used in various medical research domains. The “Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis” statement published in 2015 aimed to standardize the process of establishing prediction models . Furthermore, Alba et al. specified standard statistical methods for constructing a prediction model in 2017 . A reliable prediction model typically comprises two essential components: discrimination and calibration. Discrimination pertains to using an indicator to differentiate the risk of a specific event within a group, often assessed using the AUC or C-index. Calibration, another crucial evaluation index, reflects the agreement between predicted and actual values. Integrated Discrimination Improvement, proposed by Pencina et al., in 2008, is a statistical method used to compare two models . However, as reported by Wessler et al., only 63 % of prediction model studies reported discrimination, and 36 % reported calibration . The ROC curve and corresponding AUC offer valuable insights into the overall performance of the BCR prediction model, enabling clinicians and researchers to evaluate its predictive capabilities. These results indicate that the new model enhances the prediction performance, particularly in RARP. Section title: Discussion Educational score: 4.08486270904541 Domain: biomedical Document type: Study Language: en Moreover, about the sentinel node technique and its relevance to pelvic lymph node dissection (PLND) in prostate cancer [ , , ]. Unfortunately, data on variant histologies and aberrant growth patterns were not available for our cohort.According to the European Association of Urology guidelines, PSA levels are typically assessed every 6 months for the initial 3 years post-prostatectomy and yearly thereafter . A limitation of this study is that the long time span of the study may introduce variation due to changing surgical techniques and case volumes. External validation using independent data is crucial and we have alsosentien performed external validation. The predictive nomogram model provides a user-friendly and precise means to estimate the likelihood of achieving a BCR-free status within a three-year timeframe for each individual patient. This empowers healthcare providers to tailor follow-up strategies specifically to each patient, effectively minimizing the unnecessary allocation of medical resources. This model holds significant clinical utility, offering a valuable tool for optimizing patient care. Its potential benefits warrant further dissemination and widespread adoption in clinical practice. However, as the data were obtained from a single center, data from other centers is needed for further validation. Section title: CRediT authorship contribution statement Educational score: 1.0240274667739868 Domain: other Document type: Other Language: en Tanan Bejrananda: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Kiyoshi Takahara: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Dutsadee Sowanthip: Conceptualization. Tomonari Motonaga: Conceptualization. Kota Yagi: Conceptualization. Wataru Nakamura: Conceptualization. Masanobu Saruta: Conceptualization. Takuhisa Nukaya: Conceptualization. Masashi Takenaka: Conceptualization. Kenji Zennami: Conceptualization. Manabu Ichino: Conceptualization. Hitomi Sasaki: Conceptualization. Makoto Sumitomo: Conceptualization. Ryoichi Shiroki: Writing – review & editing, Writing – original draft, Supervision, Conceptualization. Section title: Informed consent Educational score: 1.2511179447174072 Domain: biomedical Document type: Other Language: hu N/A. Section title: Approval of the research protocol by an Institutional Reviewer board Educational score: 1.0348050594329834 Domain: other Document type: Other Language: en Approval number of research protocol by the Ethics Committee of the Fujita Health University Hospital (Approval No. HM19-265). Section title: Registry and the registration number of the study/trial Educational score: 1.2511179447174072 Domain: biomedical Document type: Other Language: hu N/A. Section title: Animal studies Educational score: 1.2511179447174072 Domain: biomedical Document type: Other Language: hu N/A. Section title: Declaration of competing interest Educational score: 1.06308114528656 Domain: other Document type: Other Language: en We, the undersigned authors of the manuscript titled "Biochemical Recurrence Prediction After Robot-Assisted Radical Prostatectomy (BCR-PRARP)," declare that: Section title: Declaration of competing interest Educational score: 0.9582133293151855 Domain: other Document type: Other Language: en 1. We have no conflicts of interest to disclose, either financial or non-financial, in relation to the research, authorship, or publication of this article. Section title: Declaration of competing interest Educational score: 1.0132629871368408 Domain: other Document type: Other Language: en 2. We have not received any financial support, funding, or sponsorship from any company, institution, or organization related to this study. Section title: Declaration of competing interest Educational score: 1.390007734298706 Domain: biomedical Document type: Other Language: en 3. The research was conducted independently, and all data used in this study were collected and analyzed without influence or interference from external parties. Section title: Declaration of competing interest Educational score: 0.9658511281013489 Domain: other Document type: Other Language: en 4. All co-authors have contributed significantly to the research, writing, and revision of this manuscript, and we have approved the final version of the manuscript for submission. Section title: Declaration of competing interest Educational score: 1.054585337638855 Domain: other Document type: Other Language: en 5. There are no personal relationships, affiliations, or associations with any organizations or individuals that could potentially bias the results or interpretation of this study. | Review | biomedical | en | 0.999997 |
PMC11699394 | Section title: Introduction Educational score: 3.869502067565918 Domain: biomedical Document type: Review Language: en Rhythm is ubiquitous in human behavior: music and dance are inherently rhythmic, and most daily activities depend on finely timed motor control to coordinate our movement with the environment. Cognition also partly relies on rhythm, as evidenced by temporal predictability in language, 1 and even memory and attention seem to be sustained by rhythmic brain processes. 2 , 3 , 4 Reciprocally, rhythm processing is cognitively demanding; for example, stabilizing synchronization with increasingly complex rhythmic patterns requires additional attentional load. 5 , 6 , 7 , 8 , 9 Section title: Introduction Educational score: 4.110029697418213 Domain: biomedical Document type: Review Language: en One prominent theory in psychology, dynamical systems theory, has sought to explain how the brain rhythmically deploys attention to the right moments in time. Dynamical systems theories assume that self-organized neural and behavioral systems coordinate perceptual and cognitive processes. 10 Oscillator models are central mathematical and conceptual tools in dynamical system theories, and are particularly suited to study the anticipatory processes sustaining attentional and rhythmic mechanisms in human behavior. 11 , 12 , 13 In these models, internal and behavioral oscillatory systems (e.g., electrical activity in populations of neurons, rhythmic movements like gait) synchronize to external events for more fluent processing. 14 , 15 , 16 , 17 , 18 , 19 Section title: Introduction Educational score: 3.948976516723633 Domain: biomedical Document type: Study Language: en These oscillations appear spontaneously (i.e., in the absence of any external cues) at different rates for each individual. 20 These spontaneous motor rhythms can be measured behaviorally by asking participants to tap at their own comfortable rate. Spontaneous rates have been shown to influence participants’ abilities to synchronize both to other people and music. 21 , 22 Specifically, participants more optimally synchronize to rates that are closer to their own spontaneous rates. These findings may arise because smaller phase adjustments require fewer attentional resources or because oscillatory attention processes respond more efficiently via resonance-like properties. However, neither of these possible attentional mechanisms has yet been directly explored. Section title: Introduction Educational score: 4.310218811035156 Domain: biomedical Document type: Study Language: en One simple yet reliable way to measure such attentional processes is via pupillary activity due to its tight coupling to noradrenergic activity from the locus coeruleus. 23 , 24 , 25 Greater task-evoked pupil dilations and pupil size have consistently been linked to increased attention allocation since the 1960s 26 , 27 , 28 , 29 and, more recently, oscillatory pupil activity (particularly in the delta range between ∼0.5 and 2 Hz) has been demonstrated to entrain to rhythms and appears to be related to movement. 30 , 31 , 32 Unlike electroencephalography, the event-related pupil dilation response is so sluggish (typically peaking between ∼500 and 1,200 ms post-event onset 33 , 34 ) that phase-aligned pupil dilations can be interpreted as entrained rather evoked. This is particularly relevant for testing theories like dynamical systems where the difference between evoked and entrained responses is crucial to the underlying neural mechanism. 35 , 36 , 37 , 38 , 39 Thus, pupillometry offers a robust and convenient way to investigate (1) whether synchronizing to rates beyond one’s spontaneous rate demands more attention and (2) whether attention is better entrained (and therefore more efficiently processed according to dynamical systems theory) to one’s own spontaneous rate relative to faster and slower tempi. Section title: Introduction Educational score: 4.11989164352417 Domain: biomedical Document type: Study Language: en To this end, we first recorded the spontaneous rates of 25 healthy adults in an unpaced tapping tapping (spontaneous motor tempo, SMT) and created three metronomes consisting of 60 woodblock sounds for each participant, one corresponding to their average SMT, one 20% faster, and one 20% slower than this rate. Participants were then tasked with either passively listening or synchronizing their finger taps to the metronomes across three trials per condition while their pupil size was recorded with an eye-tracker . We hypothesized that average pupil sizes would be larger while synchronizing relative to listening, particularly at the faster and slower tempi, reflecting increased attentional demands. Furthermore, we hypothesized that pupil activity would be better entrained (as assessed with phase coherence) at participants’ SMT where, according to dynamical systems theory, their internal oscillators are already primed to resonate for more efficient attentional processing. Figure 1 Experimental design The study design. Participants either tapped or listened to metronomes that matched their SMT or were 20% faster or slower. Half of the participants tapped to the metronomes first followed by listening to the metronomes (synchronization then perception) while the other half of the participants listened to the metronomes first followed by tapping to them (perception then synchronization). Section title: Results Educational score: 4.092920303344727 Domain: biomedical Document type: Study Language: en As in previous studies, 21 , 22 , 40 , 41 participants’ SMTs spanned a relatively broad range from 462 to 1,192 ms (ITI, M = 692.34, SD = 190.34) . Despite this considerable variability, all participants nevertheless managed to synchronize their taps to each of their three custom metronomes with equal success as indicated by equivalent vector lengths (tapping consistency, F (1,44) = 1.37, p = 0.26, η2G = 0.11) and directions (tapping precision, F (1,44) = 2.42, p = 0.10, η2G = 0.13) in all tempo conditions. Thus, the following pupillometry results reflect the attentional processes employed to achieve this performance rather than attentional differences related to errors or their corrections. Figure 2 Distribution of participants’ SMTs Mean individual spontaneous motor tempi, ordered from fastest to slowest. Error bars indicate standard errors of the mean. Section title: Synchronizing taps is more cognitively demanding than listening only beyond one’s SMT Educational score: 4.131186485290527 Domain: biomedical Document type: Study Language: en Pupil size by task and tempo condition are presented in Figure 3 . The repeated measures analysis of variance (ANOVA) on average task-evoked pupil sizes over the entire trial revealed a modest effect of task ( F (1,22) = 12.530, p = 0.002, η2G = 0.142), indicating that synchronizing one’s taps to the metronome was more cognitively demanding than passively listening to the same metronome. Crucially, there was also an interaction with tempo ( F (2,44) = 4.533, p = 0.016, η2G = 0.030) where post-hoc t tests demonstrated that the main effect of task was driven exclusively by the faster ( t (22) = 3.301, p = 0.004, d = 0.69) and slower ( t (22) = 4.434, p < 0.001, d = 0.92) metronomes evoking greater pupil sizes while synchronizing relative to listening whereas pupil sizes did not differ at their preferred rates ( t (22) = 1.165, p = 0.257). These results show that synchronizing to the metronome only required more attention than listening to it when its tempo was outside of one’s comfortable rate. Figure 3 Evoked pupil size by task and tempo Raincloud plots for average pupil size by task and tempo condition. Black diamonds denote the mean while error bars represent one standard error of the mean. Each point depicts a single subject’s average pupil size and gray lines connect these averages between Task conditions. ∗∗ p < 0.01, ∗∗∗ p < 0.001 (post-hoc t tests). Section title: Synchronizing taps results in greater attentional entrainment at all tempi Educational score: 4.125253677368164 Domain: biomedical Document type: Study Language: en An additional participant was missing too much data from the SMT condition, unbalancing the design, and thus had to be excluded. Nonetheless, the repeated measures ANOVA on entrained pupillary activity revealed a main effect of task ( F (1,21) = 5.000, p = 0.036, η2G = 0.052) with no effect of tempo ( F (2,42) = 1.131, p = 0.332) or interaction ( F (2,42) = 0.932, p = 0.402). This demonstrates that attention was more efficiently deployed to the frequency of the metronome while participants were synchronizing their taps relative to passively listening . Figure 4 Pupillary entrainment by task Raincloud plots depicting the main effect of task on entrained pupil activity. Black diamonds denote the mean while error bars represent one standard error of the mean. Each point depicts a single subject’s average phase coherence and gray lines connect these averages between task conditions. ∗ p < 0.05 (ANOVA). Section title: Evoked and entrained pupillary effects are not driven purely by movement Educational score: 4.091857433319092 Domain: biomedical Document type: Study Language: en One could argue that we might have observed the same pattern of results had we instructed participants to move without the presence of the metronome cues. That is to say that these effects could be mere motor effects rather than sensorimotor synchronization effects per se. As an additional control analysis, we analyzed participants’ average pupil dilations and entrained pupillary activity during their SMT recordings and compared this to their respective signals while listening or synchronizing to metronomes at this tempo. Two participants’ pupillary recordings during the SMT recordings were corrupted or missing and so were omitted from these control analyses. There was no effect of task on either the average evoked pupil size ( F (2,40) = 2.514, p = 0.094) or pupillary phase coherence ( F (2,40) = 1.823, p = 0.175). The results are plotted in Figure 5 in the following. Figure 5 Control analyses Raincloud plots for the control analyses only on the SMT condition while listening, synchronizing, and tapping without a metronome. Black diamonds denote the mean while error bars represent one standard error of the mean. Each point depicts a single subject’s average pupil size (A) or phase coherence (B) and gray lines connect these averages between Task conditions. There were no significant differences observed. Section title: Discussion Educational score: 4.1162495613098145 Domain: biomedical Document type: Study Language: en Here we tested whether synchronizing to metronomes at one’s preferred motor rate demands fewer cognitive resources (assessed by evoked pupil size) and whether it is associated with more efficient allocation of those resources (assessed by pupillary entrainment) compared to faster and slower rates. We found that synchronizing, relative to listening, evoked greater average pupil sizes at rates outside of participants’ preferred tempo, indicating that more attention was deployed to achieve the same level of synchronization to the faster and slower metronomes. Despite this overall increase in attention while synchronizing to faster and slower tempi, pupillary phase coherence was modestly greater while synchronizing compared to listening but in a similar manner across all tempi conditions. Section title: Discussion Educational score: 4.215829849243164 Domain: biomedical Document type: Study Language: en Our average evoked pupil size results demonstrate that more attentional resources are allocated to synchronize to tempi outside of one’s comfortable rate, confirming our first hypothesis. This is consistent with previous results showing that more attentional resources are necessary for synchronizing with more complex stimuli, as reflected in behavioral and neurophysiological processes. 5 , 6 , 7 , 8 , 9 Interestingly, these differences in attention allocation were not symmetrical; the effect was larger for slower metronomes ( d = 0.90) than faster metronomes ( d = 0.66). This asymmetry fits with previous studies where synchronization tends to be worse for slower tempi than faster tempi. 40 , 41 , 42 This may be an effect of expertise, since the asymmetry seems to arise in nonmusicians more than in musicians who are more flexible in their ability to synchronize, including to slower tempi. 41 Why this asymmetry exists, however, is still unclear. The precision of timing processing decreases with larger intervals (Weber’s law), 43 leading to increased tapping variability and asynchronies at slower rates. 44 , 45 The increased pupil size when tapping at slow rates may therefore reveal cognitive and attentional resources needed to overcome the natural tendency to tap with less precision and accuracy at slow tempi, as predicted by Weber’s law. Section title: Discussion Educational score: 4.091473579406738 Domain: biomedical Document type: Study Language: en The oscillatory pupil activity results demonstrate that synchronous movements result in greater attentional entrainment. While most participants generally displayed a modest degree of entrainment even while listening, this entrainment was greater while synchronizing their taps to the metronomes. This is consistent with theories of active sensing and embodied cognition where action plays a central role in cognitive processes like attention. 46 , 47 , 48 , 49 Contrary to our second hypothesis, there was no interaction with tempo, indicating that this motor effect is persistent across the range of tempi. Given participants’ successful behavioral synchronization at all tempi, this is rather unsurprising in hindsight, especially since all of our participants’ metronome rates fell well within the ecological range that bounds human rhythm perception and production. 50 , 51 Thus, it seems that attentional deployment is more entrained for successful behavioral synchronization relative to passive listening. Section title: Discussion Educational score: 4.260881423950195 Domain: biomedical Document type: Study Language: en Taken together, these results paint a more nuanced picture of dynamical systems theories of attention. As dynamical systems theories predict, synchronizing beyond one’s preferred tempo does seem to require more attention as evidenced by our evoked pupil dilation effects. However, our oscillatory pupillary activity results seem to contradict dynamical systems accounts based on resonance properties that would predict worse pupillary entrainment at tempi faster and slower than one’s SMT. It is possible that a reduction of pupillary entrainment would be observed at larger deviations from one’s SMT than the ones used in this study; this should be addressed in future experiments to test whether pupil oscillations are flexible or relatively rigid resonators that only entrain to a narrow set of frequencies. Entrained pupil dilations may also represent a form of active sensing, i.e., a top-down sampling routine that sharpens sensory representations of the environment. 47 , 52 , 53 , 54 Under this view, the locus coeruleus may proactively release norepinephrine, dilating the pupils, to maximize the encoding of sensory information at attended moments in time, specifically those coinciding with our own movements, regardless of the motor rate. Section title: Discussion Educational score: 4.163957595825195 Domain: biomedical Document type: Study Language: en Our study demonstrates the usefulness of considering participants’ comfortable rates in experimental settings where tempo is manipulated because there could be latent yet systematic differences in the attentional demands among subjects. This would be particularly important to consider in experiments where the complexity or difficulty of tracking the rhythm is of interest. For instance, there are well-known interactions between rhythmic complexity and tempo in both perception and synchronization 55 , 56 ; these interactions may partially reflect the distance from participants’ SMT in addition to differences stemming from the physical stimulus rates. These considerations may be even more important outside of the lab, particularly in clinical settings for movement disorders. For instance, if existing rhythmic- and movement-based interventions for Parkinson’s disease are titrated to each individual’s preferred rate, these treatments could be less attentionally demanding to adhere to which could in turn boost retention. Our pupillary entrainment results may similarly have clinical implications for attention and developmental disorders. For example, both children and adults with attention deficit hyperactivity disorder (ADHD) display more variable tapping performance so pupillometry could provide a cheap, non-invasive biomarker for diagnosis alongside tapping behavior. 57 , 58 , 59 Moreover, children with dyslexia have been found to have faster SMTs and more variable sensorimotor synchronization, indicating that pupillary biomarkers may exist for them as well. 60 , 61 , 62 Additionally, because attention was better entrained while synchronizing, rhythmic interventions may offer a promising avenue for treating attention disorders. Section title: Conclusion Educational score: 4.050660610198975 Domain: biomedical Document type: Study Language: en We provide evidence that the pupil signal can capture variations in attentional demands when synchronizing at different tempi. Consistent with modern theories of rhythmic attention such as dynamical systems and active sensing, we observed pupil oscillations aligned to the frequency of the perceived metronomes, with stronger alignment when movement coordination was required. These results thus demonstrate that pupillary activity is a useful measure of both attentional demands and attention allocation over time, providing opportunities for scientists and clinicians alike. Section title: Limitations of the study Educational score: 4.120933532714844 Domain: biomedical Document type: Study Language: en One limitation of this work is that our analysis to control for purely motor (rather than synchronization) effects was limited to participants’ SMT tempo. It’s possible that moving without metronome cues at rates faster or slower than their comfortable rate could elicit results similar to our synchronization condition. However, were we to implement a movement only condition at the manipulated tempi, we would introduce a secondary task demand of moving at a faster or slower rate in addition to simply moving at a comfortable rate. This increased cognitive demand would induce larger pupil sizes, and thus confound a pure motor effect. Future work could investigate this possibility more directly using a synchronization-continuation paradigm where participants synchronize to a metronome cue and then continue tapping at that tempo after the metronome stops. That said, the continuation window would have to be long enough (e.g., longer than ∼4–10 s) for the pupil size to restabilize after the orienting response dilation from the metronome stopping, but not so long that the participants’ tapping rate drifts to a new tempo. Another limitation is that the influence of sex and gender was not tested in this study. The sample size was not sufficient to address this question. However, we do not expect that gender or sex would influence the results, as no studies on SMT and synchronization reported any effects from these factors. 63 Section title: Lead contact Educational score: 1.0286712646484375 Domain: other Document type: Other Language: en Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Connor Spiech ( connorrichard.spiech@concordia.ca ). Section title: Materials availability Educational score: 1.1638790369033813 Domain: biomedical Document type: Other Language: en This study did not generate new materials. Section title: Data and code availability Educational score: 1.0466415882110596 Domain: other Document type: Other Language: en • The data has been deposited at OSF: https://osf.io/7us96/?view_only=be3f4fd8cb1448129483295144b8f805 and is publicly available as of the date of publication. Accession numbers are listed in the key resources table . • Code has been deposited at OSF: https://osf.io/7us96/?view_only=be3f4fd8cb1448129483295144b8f805 and is publicly available as of the date of publication. Accession numbers are listed in the key resources table . • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Section title: Acknowledgments Educational score: 0.9198054671287537 Domain: other Document type: Other Language: en We thank the Joint Conference of the 17th International Conference on Music Perception and Cognition (ICMPC) and the 7th Conference of the Asia-Pacific Society for the Cognitive Sciences of Music (APSCOM) for publishing an article presenting preliminary results of this study and nominating the authors for a Young Researchers Award. Section title: Acknowledgments Educational score: 1.0287703275680542 Domain: other Document type: Other Language: en This work was supported by an Arnold Bentley New Initiatives Fund from the 10.13039/100014693 Society for Education, Music and Psychology Research awarded to Valentin Bégel and Connor Spiech as well as by the 10.13039/501100005416 Research Council of Norway through its Centres of Excellence scheme, project number 262762 and 10.13039/501100005366 Universitetet i Oslo . Section title: Author contributions Educational score: 0.9243959188461304 Domain: other Document type: Other Language: en Author V.B. conceived the study and all authors contributed to the design of the experiment. Authors M.H. and V.B. collected the data while authors V.B. and C.S. analyzed the tapping and pupillometry data, respectively. Visualizations of the data were generated by authors C.S. and M.H. with feedback from V.B. All authors contributed equally to the drafting and revision of the manuscript. Authors V.B. and C.S. acquired funding. Section title: Declaration of interests Educational score: 0.8786384463310242 Domain: other Document type: Other Language: en The authors declare no competing interests. Section title: Key resources table Educational score: 1.2845474481582642 Domain: biomedical Document type: Other Language: en REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data Open Science Framework Center for Open Science https://www.cos.io/ https://osf.io/7us96/?view_only=be3f4fd8cb1448129483295144b8f805 Software and algorithms MATLAB 2019a Mathworks, Inc, MA, USA https://www.mathworks.com/products/matlab.html R 4.4.0 The R Project for Statistical Computing https://www.r-project.org/ Reaper v. 6.45 Cockos Inc., NY, USA https://www.reaper.fm/ Section title: Experimental model and study participant details Educational score: 2.526212215423584 Domain: biomedical Document type: Study Language: en Twenty-five healthy adults gave informed consent to participate in the study in accordance with the ethical approval granted by the Department of Psychology’s internal review board for research ethics at the University of Oslo . Participants were compensated with a 150 NOK (∼€15) gift card for their time. Two participants were discarded because their average SMT or average pupil size were outside the normal range of the sample (i.e., differing from the median of the sample by more than three standard deviations). The final sample of participants was thus composed of 23 participants (10 men, 13 women) with an average age of 28.22 years (range: 20–36, SD = 4.03 years). Section title: Equipment and stimuli Educational score: 4.0616350173950195 Domain: biomedical Document type: Study Language: en Tapping data were collected using a Roland V-drums snare. The auditory stimuli consisted of metronome sequences composed of 60 cues (using a woodblock sample) created with MATLAB 2019a. They were played at a comfortable volume from two Genelec speakers using custom scripts in MATLAB. The auditory signal and the Midi signal were recorded by a Roland V-drums Sound Module TD17 connected to a Windows computer using Reaper v. 6.45 (Cockos Inc., NY, USA). Pupil size was continuously recorded at 500 Hz with an EyeLink Portable Duo and a chin rest 70 cm from the eye-tracker in a well-lit room. Section title: Procedure Educational score: 4.111028671264648 Domain: biomedical Document type: Study Language: en The experiment started with participants’ spontaneous motor tempi being recorded. To familiarize them with the procedure, the participant completed a short practice trial where they produced a sequence of ten taps at an uncued rate. Once they understood the task, they completed three experimental trials consisting of 60 taps. Each participant’s SMT was then calculated as the mean intertap interval (ITI, in ms) across the three SMT trials after the first ten taps were discarded from each trial. This value was used to create the metronomes; one metronome matched their SMT (SMT condition), one was 20% faster (Faster condition), and one was 20% slower (Slower condition). At the start of the main experiment, participants completed a five-point (cross-shaped) calibration procedure for the eye-tracker. Subsequently, one of the three metronome sequences were played, selected pseudo-randomly such that all three tempi were played before being randomly shuffled again. Participants were instructed to fixate within a black ring presented in the center of a gray background while either passively listening to the metronome or synchronizing their taps to the metronome with their dominant index finger. Participants completed nine trials of either listening or tapping before completing nine more trials of the other for a total of 18 trials with the task order counterbalanced across subjects. Thus, the experiment followed a within-subject design with Tempo (SMT, Faster, Slower) and Task (Listen, Tap) as factors with three trials per condition. The study design is schematized in Figure 5 below. Section title: Quantification and statistical analysis Educational score: 4.113559246063232 Domain: biomedical Document type: Study Language: en Each finger tap was matched with the nearest metronome cue. Signed asynchrony values were calculated as the participant’s tap onset time minus the auditory cued onset time (in ms). A negative asynchrony value indicates that the tap occurred before the cue while a positive value indicates that the tap occurred after the cue time. The first four taps of each sequence were also discarded to allow for tempo stabilization as is standard procedure. Some taps (i.e., when participants tapped too softly) were not captured by the midi touchpad. Therefore, a minimum of forty taps was necessary to analyze a trial; ten trials (out of 225, 4.4%) were discarded because there were fewer than 40 recorded taps. Finally, outliers, defined as taps with signed asynchronies of more than three standard deviations from the mean signed asynchrony in a trial, were discarded (fewer than 0.5% of the taps in total). There was at least one trial per condition for each participant after these data cleaning procedure. Section title: Quantification and statistical analysis Educational score: 4.114400863647461 Domain: biomedical Document type: Study Language: en In order to compare the magnitude and variability of the asynchronies across tempi conditions, circular statistics 64 , 65 were conducted on the synchronization data using the MATLAB Circular Statistics Toolbox. 66 The interval between metronome cues was represented on a 360° circular scale, and individual taps were represented as unit vectors with a given angle. A mean resultant vector R was calculated based on each individual tap in a trial. The length of the resultant vector, between zero and one, indicates synchronization consistency (i.e., the reciprocal of variability; the closer to one, the less variable the performance). The angle of the vector (in degrees) represents the relative phase, an index of synchronization accuracy. Negative angles indicate that participants tapped before metronome cue. Section title: Quantification and statistical analysis Educational score: 4.17570686340332 Domain: biomedical Document type: Study Language: en Pupil data were exported offline from Eyelink DataViewer 67 for preprocessing with custom R scripts using functions from the “pupillometry” and “gazeR” packages in R. 68 , 69 First, the timeseries for each participant’s right pupil was read in and locked to the stimuli onsets. Blinks were removed along with their preceding and succeeding 100 ms to exclude artifacts arising from partial occlusions of the pupil by the eyelid during a blink. Each trial was then smoothed using a 4 ms moving average and gaps smaller than 1000 ms were interpolated using cubic splines. Afterward, each trial was baseline-corrected by subtracting the median pupil value recorded during the 1000 ms of silence preceding stimuli onsets to remove random fluctuations in pupil size between trials. 24 At this point, surviving artifacts were removed in a tripartite fashion: trials with more than 33% of missing data were discarded, samples containing rapid pupil size changes were detected and eliminated using the median absolute deviation of the dilation speed time series and a constant of 16 as suggested by Kret & Sjak-Shie, 70 and visual inspection of a histogram of all remaining pupil sizes as recommended by Mathôt and colleagues. 71 Each subject’s cleaned pupil size was averaged over the entire trial. Section title: Quantification and statistical analysis Educational score: 4.163747787475586 Domain: biomedical Document type: Study Language: en To investigate pupillary entrainment, additional processing was needed. First, the data from the first four taps needed to be discarded because of the orienting responses elicited by the metronome onset. Next, each trial for each subject was high-pass filtered with a third order Butterworth filter at 0.05 Hz to remove the signal drift, and then the data were z-scored according to the preprocessing steps employed and validated in past research. 30 , 31 The time series for the remaining 56 taps were grouped into 14 four-tap phrases and timelocked to the start of each phrase for greater statistical power. Fourier series at the frequency of the metronome in each condition were extracted from each phrase in each trial using a fast Fourier transform. 72 Three neighboring frequency bins were kept as well to control for spectral leakage as done in past work. 73 As in previous studies, 74 , 75 pupillary phase coherence was calculated by dividing the Fourier coefficients by their absolute values to get the phase angles, summing the resulting phase angles, and then dividing them by the total number of phrases per trial, and then taking the absolute value of the complex mean. The complex mean was then normalized, so that it can be compared across tempi (between zero and one, the closer to one, the less variable the performance). Section title: Quantification and statistical analysis Educational score: 4.0823974609375 Domain: biomedical Document type: Study Language: en Synchronization (Vector Length, Vector Direction) and pupillometry (Pupil Size, Phase Coherence) indices were used as the dependent variables in a three (Faster, SMT, Slower) by two (Listen, Tap) repeated measures ANOVA. Following a significant interaction, Welch’s paired sample t-tests were conducted and corrected for multiple comparisons using the false discovery rate method. 76 In all figures, significance is denoted with a single asterisk for p < 0.05, a double asterisk for p < 0.01, and a triple asterisk for p < 0.001. | Review | biomedical | en | 0.999996 |
PMC11699398 | Section title: Introduction Educational score: 1.1040595769882202 Domain: other Document type: Other Language: en Stellar streams form when globular clusters or dwarf galaxies are tidally stripped. There are more than 100 streams discovered in the Milky Way; see, e.g., A. Bonaca & A. M. Price-Whelan , T. S. Li et al. , N. Shipp et al. , and references therein. Among them, the GD-1 stream is one of the longest and coldest streams , and it has been used to constrain the Milky Way's gravitational potential . The GD-1 stream has rich structural properties, such as the gaps and spur , suggesting that it has been perturbed through interactions with a substructure in the Milky Way. Section title: Introduction Educational score: 3.3584487438201904 Domain: other Document type: Study Language: en In particular, A. Bonaca et al. demonstrated that the perturber must be surprisingly dense to account for the spur and gap features in the GD-1 stream. Assuming a Hernquist density profile, the perturber's mass is estimated to be in the range of 10 5.5 –10 8 M ⊙ , with a scale radius of ≲20 pc; recent encounters within the last 1 Gyr are favored. The perturber is significantly denser than the subhalos predicted in the standard cold dark matter (CDM) model, at the ~3 σ level. Thus, even if the perturber were a known satellite galaxy of the Milky Way, its unusually high density would remain puzzling. Furthermore, none of the known globular clusters can match the orbit of the inferred perturber . Section title: Introduction Educational score: 3.814973831176758 Domain: biomedical Document type: Study Language: en In this work, we assume that the GD-1 perturber is a dark matter subhalo and explore its formation in within the framework of self-interacting dark matter (SIDM); see S. Tulin & H.-B. Yu and S. Adhikari et al. for reviews and references therein. The gravothermal evolution of an SIDM halo occurs in two sequential phases. In the core-forming phase, dark matter self-interactions transport heat inward, resulting in a shallow density core, while in the core-collapsing phase, heat transfer reverses, leading to a higher central density than in the CDM counterpart . Notably, SIDM models with large cross sections could explain the high density of the strong lensing perturber for SDSSJ0946+1006 and the low density of the Crater II satellite galaxy , both challenging CDM. It is intriguing to explore the SIDM scenario to account for the high density of the GD-1 perturber. Section title: Introduction Educational score: 4.153055191040039 Domain: biomedical Document type: Study Language: en We will analyze progenitors of CDM subhalos from a zoom-in cosmological simulation of a Milky Way analog from D. Yang et al. and E. O. Nadler et al. and explicitly show that their inner densities are systematically lower than those inferred for the GD-1 perturber. We then take one of the progenitor halos, with a mass of ~10 8 M ⊙ , and evolve it in the tidal field of the Milky Way, including both halo and stellar components. For a self-interacting cross section in the range σ / m = 30–100 cm 2 g −1 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}10\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 10 km s − 1 , the SIDM halo enters the collapse phase within 3–6 Gyr while evolving in the tidal field. By the final snapshot, its enclosed mass within the inner 10 pc is increased by more than 1 order of magnitude compared to its CDM counterpart, making it consistent with the high density of the GD-1 perturber. Additionally, we will discuss future investigations aimed at further improvement. Section title: Introduction Educational score: 1.5099126100540161 Domain: other Document type: Other Language: en The rest of this Letter is organized as follows: In Section 2 , we discuss the properties of CDM halos in the cosmological zoom-in simulation of a Milky Way analog. In Section 3 , we introduce the setup of our N -body simulations. In Section 4 , we present the properties of our simulated SIDM and CDM subhalos and compare them with the GD-1 perturber. In Section 5 , we discuss future investigations for further improvement and conclude. In Appendix A , we present the SIDM simulation of an isolated halo for testing numerical artifacts that could lead to violation of energy conservation. In Appendix B , we show the convergence test. Section title: CDM Halos of a Milky Way Analog Educational score: 3.4624216556549072 Domain: other Document type: Study Language: en We first present progenitor halos from a cosmological zoom-in CDM-only simulation of a Milky Way analog , with initial conditions drawn from the suite in Y.-Y. Mao et al. . This simulated system includes a main halo with a mass of 1.14 ×10 12 M ⊙ h −1 ≈ 1.6 × 10 12 M ⊙ ( h = 0.7) and a Large Magellanic Cloud analog. The simulation has a particle mass of 4 ×10 4 M ⊙ h −1 , a Plummer-equivalent softening length of ϵ = 0.08 kpc h −1 , and a spline length of ℓ = 2.8 ϵ =0.22 kpc h −1 , the characteristic length scale of the smoothing kernel used to calculate gravitational forces between particles . Section title: CDM Halos of a Milky Way Analog Educational score: 4.255626678466797 Domain: biomedical Document type: Study Language: en We select subhalos of the main halo with the virial mass larger than 10 8 M ⊙ h −1 at z = 0 and then identify their progenitors at infall. With the mass cut, there will be at least 2500 simulation particles for each progenitor halo so that we can accurately reconstruct its density profile. The radial resolution of the cosmological simulation ℓ ≈ 0.3 kpc is more than 1 order of magnitude larger than the radial scale relevant for the GD-1 perturber. To overcome this resolution limit, we fit each progenitor halo with a truncated Navarro–Frenk–White (NFW) profile for the region r > 0.3 kpc: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}\rho (r)=\frac{{\rho }_{s}}{\left(r/{r}_{s}\right){\left(1+r/{r}_{s}\right)}^{2}}\times \frac{\exp (-r/{r}_{{\mathrm{cut}}})}{{(1+{r}_{s}/{r}_{{\mathrm{cut}}})}^{0.3}},\end{eqnarray*}\end{document} ρ ( r ) = ρ s r / r s 1 + r / r s 2 × exp ( − r / r cut ) ( 1 + r s / r cut ) 0.3 , where ρ s and r s are the scale density and radius, respectively, and r cut is the truncation radius due to tidal stripping. We determine the three parameters for each progenitor at infall, achieving excellent overall fit quality. The truncated NFW profile is then extrapolated inward to compute the total enclosed mass within r = 10 pc. Additionally, we confirm that many progenitor halos can be well-fitted with the standard NFW profile, while some exhibit density profiles slightly steeper than r −3 in the outer regions. For these cases, the standard NFW fit may introduce bias and overestimate the central density. However, the truncated NFW profile provides a significantly better fit. Section title: CDM Halos of a Milky Way Analog Educational score: 3.7205817699432373 Domain: biomedical Document type: Study Language: en Figure 1 (left) shows the density profiles for the 125 progenitor halos at infall (blue). We also present the fit to one of the progenitors (black), which will be used as the initial condition for our SIDM simulations; see the detailed comparison in the inset panel. For this halo, the standard NFW profile provides a good fit. The simulated density profile is flattened for r ≲ 0.3 kpc due to the resolution limit. However, we expect that the NFW profile provides a good approximation for extrapolating the density inward before the halo undergoes significant tidal stripping. Section title: CDM Halos of a Milky Way Analog Educational score: 4.117746353149414 Domain: biomedical Document type: Study Language: en Figure 1 (middle) shows the enclosed mass within 10 pc versus virial mass of the progenitor halos at infall. For comparison, we include a reference case from the viable parameter region of the GD-1 perturber in A. Bonaca et al. 2019 : a Hernquist scale radius of r H = 15 pc and a total mass of M = 4.6 × 10 5 M ⊙ , which approximately corresponds to a substructure with the minimum density required to explain the spur and gap features of the GD-1 stream. For this reference case, the enclosed mass within 10 pc is ≈7.4 × 10 4 M ⊙ , as denoted by the horizontal line in the middle panel. We see that none of the CDM progenitor halos are sufficiently dense to be the perturber, and this conclusion holds when comparing the enclosed mass within r = 15 pc. The inner density of these CDM halos would further decrease as they evolve within the Milky Way's tidal field. The progenitor CDM halos shown in Figure 1 correspond to subhalos with masses >10 8 M ⊙ h −1 at z = 0. We plan to relax this mass threshold and examine halos with lower masses. Based on the resolution limit in the cosmological CDM simulation , we expect to reconstruct the density profiles of progenitors for subhalos with masses a few times 10 7 M ⊙ h −1 using the truncated NFW profile in Equation ( 1 ). A more detailed investigation will be deferred to future work. Section title: CDM Halos of a Milky Way Analog Educational score: 4.129834175109863 Domain: biomedical Document type: Study Language: en For the CDM progenitor halos, we estimate the timescale of gravothermal collapse in SIDM : \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{t}_{c}=\frac{150}{C}\frac{1}{{r}_{s}{\rho }_{s}\left({\sigma }_{{\mathrm{eff}}}/m\right)}\frac{1}{\sqrt{4\pi G{\rho }_{s}}},\end{eqnarray*}\end{document} t c = 150 C 1 r s ρ s σ eff / m 1 4 π G ρ s , where C = 0.75 is a numerical factor and σ eff is the effective cross section . For simplicity, we assume a constant cross section in this work. Figure 1 (right) shows the collapse time versus virial mass for the progenitors, where we have taken σ / m = 50 cm 2 g −1 . About one-third of the halos are expected to collapse within 10 Gyr. Since tidal stripping could speed up the onset of the collapse , we expect that more halos would be in the collapse phase after they evolve in the tidal field, and their overall mass would be reduced as well. Section title: Simulation Setup Educational score: 1.6231119632720947 Domain: other Document type: Other Language: en In this section, we introduce our simulation setup, including the initial halo density profile, the SIDM cross section, orbital parameters, and the gravitational potential model of the Milky Way. Section title: The Initial Halo Density Profile and Cross Section Educational score: 4.137448787689209 Domain: biomedical Document type: Study Language: en We choose the CDM progenitor with the earliest infall time among the five halos that have t c < 10 Gyr, and its density profile is shown in Figure 1 (left, black). For this halo, the fitted NFW parameters are ρ s = 7.5 × 10 7 M ⊙ kpc −3 and r s = 0.50 kpc. The maximum circular velocity and the associated radius are \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}=14.8\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max = 14.8 km s − 1 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${r}_{{\mathrm{\max }}}=1.1\,{\mathrm{kpc}}$\end{document} r max = 1.1 kpc , respectively. We use the public code SpherIC to generate the initial condition, and the total halo mass is M = 3.25 × 10 8 M ⊙ . The simulation has a particle mass of 32.5 M ⊙ , a total number of 10 7 particles, and a softening length of ϵ = 2 pc. We use the public N -body code GADGET-2 implemented with an SIDM module from D. Yang et al. , which follows the algorithm in A. Robertson et al. with small modifications. Section title: The Initial Halo Density Profile and Cross Section Educational score: 4.210319995880127 Domain: biomedical Document type: Study Language: en As indicated in Figure 1 (right), the halo would collapse within 10 Gyr for σ / m = 50 cm 2 g −1 even if it is isolated. In our N -body simulations, we consider three values, σ / m = 30 cm 2 g −1 (SIDM30), 50 cm 2 g −1 (SIDM50), and 100 cm 2 g −1 (SIDM100), to explore a wide range of cross sections. A viable SIDM model should exhibit a velocity-dependent cross section that is large at low velocities while decreasing toward high velocities to evade constraints on massive halos around cluster scales ≲0.1 cm 2 g −1 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}1000\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 1000 km s − 1 . Nevertheless, for a specific halo, we can use a constant effective cross section to characterize its gravothermal evolution . In our case, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}$\end{document} V max decreases from ~15 to 7 km s −1 due to tidal mass loss. Thus, the σ / m values we consider can be regard as effective cross sections for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}10\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 10 km s − 1 on average, which overall align with SIDM models proposed to explain diverse dark matter distributions in galaxies . Section title: The Milky Way Model Educational score: 4.3606438636779785 Domain: biomedical Document type: Study Language: en The Milky Way is modeled as a static potential that contains three main components. 1. A spherical NFW halo: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{{\mathrm{\Phi }}}_{\,\mathrm{DM}\,}(r)=-4\pi G{\rho }_{s}{r}_{s}^{3}\frac{\mathrm{ln}\left(1+r/{r}_{s}\right)}{r},\end{eqnarray*}\end{document} Φ DM ( r ) = − 4 π G ρ s r s 3 ln 1 + r / r s r , with ρ s = 8.54 × 10 6 M ⊙ kpc −3 and r s = 19.6 kpc. G is the Newton constant. 2. A spherical stellar bulge with a Hernquist profile : \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{{\mathrm{\Phi }}}_{{\mathrm{b}}}(r)=-\frac{G{M}_{{\mathrm{b}}}}{{r}_{{\mathrm{H}}}+r},\end{eqnarray*}\end{document} Φ b ( r ) = − G M b r H + r , with M b = 9.23 × 10 9 M ⊙ and r H = 1.3 kpc. 3. Two stellar disks and two gas disks with an axisymmetric Miyamoto–Nagai profile : \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{eqnarray*}{{\mathrm{\Phi }}}_{{\mathrm{}}}(R,z)=-\frac{G{M}_{{\mathrm{d}}}}{{\left[{R}^{2}+{\left({a}_{{\mathrm{d}}}+\sqrt{{z}^{2}+{b}_{{\mathrm{d}}}^{2}}\right)}^{2}\right]}^{1/2}}.\end{eqnarray*}\end{document} Φ ( R , z ) = − G M d R 2 + a d + z 2 + b d 2 2 1 / 2 . The parameters for each disk are as follows. Thin stellar disk: M d = 3.52 × 10 10 M ⊙ , a d = 2.50 kpc, and b d = 0.3 kpc; thick stellar disk: M d = 1.05 × 10 10 M ⊙ , a d = 3.02 kpc, and b d = 0.9 kpc; thin gas disk: M d =1.2 × 10 9 M ⊙ , a d = 1.5 kpc, and b d = 0.045 kpc; and thick gas disk: M d = 1.1 × 10 10 M ⊙ , a d = 7.0 kpc, and b d = 0.085 kpc. Section title: The Milky Way Model Educational score: 4.179800987243652 Domain: biomedical Document type: Study Language: en These parameters are motivated by the Milky Way mass model in P. J. McMillan . Note that the stellar and disk density profiles in P. J. McMillan use exponential functions, which are challenging to implement in controlled N -body simulations due to the lack of analytical expressions for their corresponding potentials. Nevertheless, we have verified that the difference in the total potential remains within 2% in the regions with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\sqrt{{R}^{2}+{z}^{2}}> 10\,{\mathrm{kpc}}$\end{document} R 2 + z 2 > 10 kpc , which are most relevant for our simulated subhalo. Since the host halo is treated as a static potential, we neglect dark matter particle scatterings between the host halo and the subhalo. This approximation is well justified for velocity-dependent SIDM models with σ / m ≲ 1 cm 2 g −1 at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}200\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 200 km s − 1 . Section title: Orbital Parameters Educational score: 4.212336540222168 Domain: biomedical Document type: Study Language: en A. Bonaca et al. found that the best-fit orbit of GD-1 has a pericenter of r peri = 13.8 kpc and an apocenter of r apo = 22.3 kpc, while the orbit of its perturber remains highly uncertain. For our simulation, we adopt an orbit with r peri = 17 kpc and r apo = 142 kpc, with the simulated subhalo undergoing five pericenter passages over 10 Gyr. Although we do not aim to explicitly model the encounter event, at t ≈ 10 Gyr, the simulated subhalo's coordinates are R.A. = 21 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mathop{.}\limits^{\unicode{x000b0}}$\end{document} . ° 5 and decl. = −7 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\mathop{.}\limits^{\unicode{x000b0}}$\end{document} . ° 9, consistent with the inferred position range of the present-day GD-1 perturber . While this orbit differs from that of the progenitor halo selected from the cosmological merger tree , it remains typical for many subhalos in the simulation. We emphasize that gravothermal collapse is intrinsic to SIDM halos, and the overall properties of our simulated perturber are robust regardless of the specific orbit chosen. Section title: Results Educational score: 4.167745113372803 Domain: biomedical Document type: Study Language: en Figure 2 (left) shows the evolution of the enclosed mass within the inner r = 10 pc for CDM (blue), SIDM30 (amber), SIDM50 (orange), and SIDM100 (pink) subhalos. For CDM, the inner mass decreases monotonically due to tidal stripping. In contrast, for SIDM, the mass initially decreases sharply due to core expansion, followed by an increase as core collapse occurs. By t ≈ 10 Gyr, the inner mass of the SIDM subhalos is 1 order of magnitude higher than that of the CDM subhalo, aligning well with the reference Hernquist profile (horizontal line). Additionally, the collapse times are t c ~ 6, 4, and 2 Gyr for SIDM30, SIDM50, and SIDM100, respectively, about a factor of 2 shorter than those estimated using Equation 2 , which is calibrated for isolated halos. In a subhalo, tidal stripping reduces the velocity dispersion of dark matter particles from the intermediate to outer regions as a result of mass loss. Consequently, a negative “temperature” gradient—a necessary condition for the onset of core collapse—is more easily established compared to an isolated halo . Section title: Results Educational score: 3.0959746837615967 Domain: other Document type: Study Language: en In Figure 2 (middle), we show the evolution of the total bound mass for the simulated CDM and SIDM subhalos. Initially, the halo mass is 3.25 × 10 8 M ⊙ and is reduced by 1 order of magnitude by t ≈ 10 Gyr due to tidal stripping. As expected, the total mass loss is more significant as the cross section increases. For SIDM, the final halo mass ranges from 4 × 10 6 to 10 7 M ⊙ , which falls well within the favored mass range of the GD-1 perturber 3 × 10 5 –10 8 M ⊙ . Section title: Results Educational score: 3.755852699279785 Domain: biomedical Document type: Study Language: en Figure 2 (right) shows the corresponding density profiles at t = 10 Gyr for the CDM and SIDM subhalos, along with the initial NFW profile. For comparison, the viable region for the GD-1 perturber (shaded gray), converted from Figure 6 of A. Bonaca et al. , and the reference Hernquist profile (dashed gray) are also shown. Compared to CDM, the density profiles of the SIDM subhalos are significantly steeper and overall consistent with the favored Hernquist profiles from A. Bonaca et al. . This indicates that dark matter self-interactions can both increase central density and accelerate tidal mass loss in the outer regions. Consequently, an SIDM subhalo can become more compact and dense than its CDM counterpart. Section title: Results Educational score: 4.223201274871826 Domain: biomedical Document type: Study Language: en We note that the CDM subhalo has a small density core near the center. This is due to the resolution limit as ℓ = 2.8 ϵ ≈ 5.6 pc, although the simulated subhalo contains more than 6 × 10 5 simulation particles at t = 10 Gyr. We fit the density profile using the analytical function \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\rho (r)={\rho }_{{\mathrm{cut}}}\exp (-r/{r}_{{\mathrm{cut}}}){r}_{{\mathrm{cut}}}/r$\end{document} ρ ( r ) = ρ cut exp ( − r / r cut ) r cut / r from R. Errani & J. F. Navarro , which is proposed to model a tidally stripped CDM halo. With ρ cut ≈ 1.2 × 10 8 M ⊙ kpc −3 and r cut ≈ 0.22 kpc, we find a good fit for the region r ≳ 10 pc; see Figure 2 (right, dotted blue). The fitted function has a cusp ρ ( r ) ∝ r −1 near the center, and it provides a correction to the core due to the resolution limit. For the fitted profile, the enclosed mass within 10 pc is 1.6 × 10 4 M ⊙ . However, even with this correction, the CDM subhalo remains insufficiently dense to explain the high density of the GD-1 perturber. Section title: Results Educational score: 4.00177001953125 Domain: biomedical Document type: Study Language: en In Figure 2 (right), we also present the density profile of the globular cluster NGC 2419 (cyan), modeled using the King profile from H. Baumgardt et al. . Interestingly, this profile closely resembles the density profile of the SIDM30 subhalo within 30 pc. This similarity is not coincidental, as the formation of globular clusters follows the same mechanism as the collapse of SIDM halos. This suggests that distinguishing between SIDM and globular cluster scenarios in explaining the GD-1 perturbation could be challenging. However, NGC 2419 itself cannot be the GD-1 perturber, as its orbit does not align with the perturbation . If the perturbation is caused by an undetected globular cluster that emits light, it could be identified in future astronomical surveys. Section title: Results Educational score: 3.2808098793029785 Domain: other Document type: Study Language: en Furthermore, narrowing down the favored parameter space in the mass–size plane for the perturber would help us distinguish the two scenarios. For instance, if the perturber's mass is further constrained to the range 10 7 –10 8 M ⊙ , the SIDM scenario would be favored, as globular clusters typically have masses below a few times 10 6 M ⊙ . Another intriguing possibility is that the perturber is an SIDM substructure hosting stars, as we will discuss later. It may have undergone significant tidal stripping, resulting in an ultrafaint dwarf with mass and structural properties similar to those of a massive globular cluster . Confirming this scenario would require detecting a stellar counterpart at the inferred location of the perturber. Distinguishing between these possibilities will require dedicated observational campaigns and detailed modeling efforts, making this an exciting avenue for future research. Section title: Discussions and Conclusion Educational score: 3.9559900760650635 Domain: biomedical Document type: Study Language: en The inner density profiles of our simulated SIDM subhalos ( r ≲ 10 pc) could be underestimated due to numerical issues in N -body simulations when the halo is deeply collapsed . Specially, numerical artifacts introduce additional “energy” that heats the simulated halo, slowing down or even preventing further increases in inner density; see M. S. Fischer et al. for discussions about potential causes. As shown in Figure 2 (left), for the SIDM subhalos, the enclosed mass within inner 10 pc stalls after t ≈ 4.5–6.5 Gyr, suggesting that they may suffer from the artificial heating effect. To further test this, we conducted an isolated simulation without the tidal field for the same initial NFW profile and σ / m = 50 cm 2 g −1 . Since the isolated halo experiences no tidal mass loss or heating, its total energy can be computed straightforwardly. See Appendix A for details on the isolated simulation and comparison with the subhalos. Section title: Discussions and Conclusion Educational score: 4.173301696777344 Domain: biomedical Document type: Study Language: en Indeed, we find that the energy increases when the isolated halo enters the deep collapse phase, corresponding to a Knudsen number of Kn ≈ 0.4 within 10 pc, i.e., the ratio of the mean free path to the gravitational height . For the SIDM subhalos, the stalling behavior occurs when their Kn values reach 0.3–0.6. In comparison, the total energy of the simulated SIDM halo in M. S. Fischer et al. starts to increase when Kn reaches 0.1. Even at Kn = 0.01 energy conservation violation is at the 1.5% level, better than our simulation. This is likely because M. S. Fischer et al. adopted a more accurate criterion for the gravity computations while at a higher computational cost. Since the artificial heating effect leads to an underestimation of the inner density profile for a collapsed SIDM halo, our results are conservative in this regard. Nevertheless, it will be important to further improve the SIDM prediction as future measurements of the GD-1 stream could narrow down the viable parameter space of the perturber . Section title: Discussions and Conclusion Educational score: 3.638481378555298 Domain: biomedical Document type: Study Language: en When modeling the Milky Way, we used static potentials for both halo and stars, calibrated with present-day measurements. Simulations show that Milky Way–like systems could grow significantly over the last ~6 Gyr due to mergers and accretion . If these effects were incorporated, our simulated subhalo would experience weaker tidal stripping in the early stages. However, we note that this is degenerate with the orbital parameters; similar results can be achieved by lowering the pericenter if a weaker potential is adopted at early times. In Appendix A , we will see that even for an isolated halo, the SIDM50 case can still collapse to the viable parameter region. Additionally, encounters between the GD-1 stream and the perturber are likely to have occurred within the last 1 Gyr , and hence the growth history of the Milky Way may not directly impact the inference of the perturber's properties. Section title: Discussions and Conclusion Educational score: 3.329848527908325 Domain: other Document type: Study Language: en The subhalo we used to demonstrate the SIDM scenario for the GD-1 perturber has an infall mass of ≈3 × 10 8 M ⊙ . Interestingly, this is near the upper limit on the peak mass of subhalos that host currently observed satellite galaxies in the Milky Way . Thus, it remains an open question whether the perturber is a truly dark substructure, devoid of a galaxy. To further investigate detectability, we conducted additional simulations for the CDM and SIDM50 cases with live stellar particles, assuming a Plummer stellar profile with a scale radius of 0.3 kpc and a total mass of 3.2 × 10 4 M ⊙ , motivated by hydrodynamical simulations of the Local Group . At t = 10 Gyr, the bound stellar masses are 1.8 × 10 4 and 1.3 × 10 4 M ⊙ for the CDM and SIDM50 cases, respectively, with the latter also exhibiting a steeper stellar density profile toward the central regions. These substructures fall into the category of ultrafaint dwarf galaxies and could potentially be detected in the near future through observations, e.g., with the Rubin Observatory . Section title: Discussions and Conclusion Educational score: 3.380737066268921 Domain: other Document type: Study Language: en More work is needed along these lines. For instance, the stellar–halo mass relation becomes increasingly steep in the ultrafaint regime and exhibits significant scatter , which must be taken into account. Additionally, since the Rubin Observatory can only detect objects in the southern hemisphere, it would be crucial to assess Rubin's sky coverage in conjunction with the orbital information of the GD-1 perturber from A. Bonaca et al. . We leave these investigations for future work. Furthermore, our scenario should also apply to smaller infall masses below ~10 8 M ⊙ . Indeed, for SIDM models with large velocity-dependent cross sections, the population of core-collapsing (sub)halos increases as the mass decreases . Thus, stellar streams like GD-1 can probe both population and density profile of core-collapsing subhalos even below the mass threshold for galaxy formation. Section title: Discussions and Conclusion Educational score: 3.2252326011657715 Domain: biomedical Document type: Study Language: en We used the Hernquist profile for the GD-1 perturber from A. Bonaca et al. as a reference to assess the simulated subhalos. It would be intriguing to take the SIDM subhalo and directly model its encounter with GD-1, incorporating the influence of the Large Magellanic Cloud . We could use the parametric model to generate a population of collapsed SIDM subhalos in Milky Way analogs. To overcome the numerical issues in N -body simulations of core-collapsing halos, we may complement them with the semianalytical fluid model to better capture the dynamics in the the central regions . Section title: Discussions and Conclusion Educational score: 4.152914047241211 Domain: biomedical Document type: Study Language: en In summary, we have conducted controlled N -body simulations and shown that a core-collapsed SIDM halo could explain the high density of the GD-1 stellar stream perturber. For progenitor halos from the cosmological simulation of a Milky Way analog, the required self-interacting cross section σ / m ≳ 30 cm 2 g −1 for ~10 8 M ⊙ halos with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${V}_{{\mathrm{\max }}}\unicode{x0007E}10\,{\mathrm{km}}\,{{\mathrm{s}}}^{-1}$\end{document} V max ~ 10 km s − 1 . Dark matter self-interactions can both increase inner density and accelerate tidal mass loss in the outer regions, producing a compact and dense perturber to explain the spur and gap features of the GD-1 stream. Our findings demonstrate that stellar streams provide a novel probe into the self-interacting nature of dark matter. We have also outlined future investigations to further improve this promising approach. | Other | other | en | 0.999997 |
PMC11699422 | Section title: Background Educational score: 3.698218584060669 Domain: biomedical Document type: Other Language: en Medical imaging is an essential tool, crucial in diagnosis and treatment planning in the medical field. Various approaches, such as Magnetic resonance imaging (MRI), computed tomography (CT), digital mammography, X-ray, ultrasound, positron emission tomography (PET), and digital pathology, are used to generate images that aid in a variety of tasks such as pathology localization, the study of anatomical structure, treatment planning, and even computer-integrated surgery, among others [ , , ]. Section title: Background Educational score: 3.8966140747070312 Domain: biomedical Document type: Other Language: en Humans have traditionally performed medical image analysis and interpretation for several years. However, with the rapid advancement of artificial intelligence (AI), the medical field increasingly embraces computer-assisted tools as a resource to solving diagnostic problems with precision and efficiency. This allows for real-time prediction of various diseases and in-depth examination of different treatment options , avoiding limitations such as inter-observer and intra-observer variability and error resulting from significant variations in pathologies and the possible exhaustion of human experts . Section title: Background Educational score: 3.8117382526397705 Domain: biomedical Document type: Study Language: en The bibliometric analysis allows for uncovering emerging trends and patterns in the scientific knowledge of a specific domain in a quantitative approach , by combining disciplines such as information science, mathematics, and statistics . Deep learning application in medical image analysis has been significantly exploited in recent years due to its current relevance. Many bibliometric analysis works have focused on various fields of research including the usage of artificial intelligence in healthcare , medical image segmentation , the application of deep learning in the medical field , medical data mining research , and machine learning-based disease diagnosis . To the best of our knowledge, bibliometric and qualitative analysis research has not been published on applying object detection algorithms in the medical field. Section title: Research problem and aim Educational score: 3.9185121059417725 Domain: biomedical Document type: Study Language: en In this work, we propose to conduct a comprehensive quantitative and qualitative analysis of the literature related to applying deep learning object detectors in medical imaging and to reveal potential trends in this field. Our contributions include. ⁃ A bibliometric analysis of the application of deep learning object detectors was conducted using two of the most widely used databases: Scopus and the Web of Science (WoS). ⁃ The reporting of several relevant quantitative indicators, such as annual publication analysis, research area analysis, journal publication analysis, publications by country and author, and keyword analysis. Section title: Research problem and aim Educational score: 4.072760105133057 Domain: biomedical Document type: Study Language: en By conducting and employing bibliometric analysis, we aim to evaluate the overall status of the application of object detection in the medical field and identify possible divergences among distinct screening methods and anatomical areas. Furthermore, our work also reflects on. • The publications trends on this topic, and concretely the growth trajectory of object detection applications in medical imaging. • The most influential authors, i.e., the most prolific and leading contributors concerning object detection in medical field research. • The international collaborations existing in this field. • The most highly cited papers and the topics they cover. • The emerging research themes in this topic, how those themes evolved over time, and the ones that gained the most attention in recent years. • The academic journals representing the main outlets for the research on object detection in the medical field. • The algorithms, screening methods, and anatomical areas under study that experienced the most significant growth in terms of publications. • The data sets used, their availability, the sample size of these data sets, whether external or internal data sets are used in the different studies, and the nature of the study (retrospective or prospective). Section title: Research problem and aim Educational score: 1.7191206216812134 Domain: biomedical Document type: Other Language: en This research will allow researchers to gain a deeper understanding of the field's development and status and guide future research by identifying potential research gaps and collaborative efforts within the research community. Section title: Research problem and aim Educational score: 2.8806357383728027 Domain: biomedical Document type: Study Language: en The top publications were filtered based on annual citation rates and analyzed in greater depth in the qualitative analysis. Additionally, we aim to assess the frequency at which different algorithms are applied within the identified articles, and to identify the imaging modalities and the anatomical application areas where object detectors are used with more incidence. Section title: Research problem and aim Educational score: 3.6495633125305176 Domain: biomedical Document type: Study Language: en By assessing the frequency at which different algorithms are applied, we aim to understand the diversity of approaches used in object detection for medical imaging, giving insights into the popularity and effectiveness of various algorithms. The occurrence analysis of the algorithms allows us to identify which approaches are commonly employed, which may lead to better performance, and which areas might require further research. Section title: Research problem and aim Educational score: 1.9364824295043945 Domain: biomedical Document type: Other Language: en By identifying the imaging modalities and anatomical regions, we can draw some conclusions concerning areas where future research is needed and identify the related specific challenges and requirements. Section title: Research problem and aim Educational score: 2.477529764175415 Domain: biomedical Document type: Other Language: en The combination of the quantitative analysis with the qualitative approach provides a holistic view of the state of the art in the field. We consider this knowledge valuable for further research, guiding future studies, and supporting decision-making for the development and implementation of object detection techniques in medical settings. Section title: Research problem and aim Educational score: 2.038299322128296 Domain: biomedical Document type: Study Language: en To the best of our knowledge, there have been no prior studies conducted exclusively on the topic of object detectors in medical imaging. We acknowledge that some studies proposed the application of deep learning techniques in the medical field, but focused mainly on classification problems and segmentation problems. Section title: Research problem and aim Educational score: 3.5992839336395264 Domain: biomedical Document type: Study Language: en The structure of this work is as follows: in the first section, a brief introduction, background to the problem, and purpose of this work are provided. The following section outlines the research methodology, data collection procedures, bibliometric tools, and screening methods. The third section presents a quantitative and descriptive view supported by a bibliometric analysis of the filtered information. In the fourth section, a qualitative analysis of the most cited papers is conducted, and an exploratory analysis is performed on the incidence of different object detectors applied and the medical fields where object detection is more prevalent. In the final section, the conclusions of this study and the overall findings are presented. Section title: Materials and methods Educational score: 2.4803545475006104 Domain: biomedical Document type: Study Language: en We utilized two well-known academic databases to construct a bibliometric analysis that is complemented by a qualitative analysis. The Web of Science (WoS) Core Collection provides researchers with a substantial amount of pertinent publications dating back to 1900, including six Citation Indexes such as the Science Citation Index Expanded (SCIE), in addition to journal citation reports (JCR) and essential science indicators (ESI). The second database is Scopus, which encompasses peer-reviewed journals in the life, physical, social, and health sciences. Section title: Materials and methods Educational score: 3.535513162612915 Domain: biomedical Document type: Study Language: en The framework for gathering and filtering data was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The query search, conducted on January 20th, 2023, was centered around three main topics that align with our purpose, excluding the concept of segmentation on keywords or titles, and limiting the results to articles written in English, as depicted in the Supplementary File 1. Those results comprise papers up until the end of 2022. Section title: Materials and methods Educational score: 2.318284511566162 Domain: biomedical Document type: Study Language: en The number of records retrieved from the WoS was 673, and 760 from Scopus. Following the PRISMA methodology, as illustrated in Fig. 1 , duplicates were identified, and exclusion criteria were applied. Fig. 1 Data collection and filtering process for object detection bibliometric analysis, following PRISMA methodology. Fig. 1 Section title: Materials and methods Educational score: 3.954326868057251 Domain: biomedical Document type: Study Language: en Removing duplicated articles resulted in a database of 951 reports that underwent a screening process for the title and abstract for enhanced filtering. Of those, 390 were excluded, 197 were included in the final analysis, and 364 were subjected to a comprehensive report screening as their titles and abstracts were inconclusive. Of the latter, 250 articles were rejected as they were unrelated to object detection; the remaining were added to the final inclusion list. This resulted in a total of 311 articles pertaining to the topic of interest. Those 311 papers are the object of quantitative analysis. From those, papers with an annual citation rate lower than five (n = 231) were removed, and the remaining (n = 80) were further analyzed following a qualitative approach. Section title: Materials and methods Educational score: 1.4398072957992554 Domain: biomedical Document type: Other Language: en The tools used during this analysis were VosViewer version 1.6.18, ScientoPy version 2.1.0, with additional exploration carried out using Python. The application of these advanced analytical tools enabled a thorough examination of the literature and provided valuable insights into the field's current state. Section title: Materials and methods Educational score: 2.646310806274414 Domain: biomedical Document type: Other Language: en The bibliometric analysis is a valuable procedure to gather the overall state-of-the-art of a specific field of research, allowing the reader to obtain information concisely and identify relevant patterns and trends for further research in the field . VosViewer is a software tool able to create and explore maps based on network data , being able to analyze efficiently large amounts of text data, as is the case of scientific texts and academic literature . On the other hand, ScientoPy is a scientometric tool capable of identifying topic trends efficiently, being able to manage and process datasets from the two main bibliographic databases, which are used in this study, namely Clarivate Web of Science (WoS) and Scopus . Section title: Quantitative analysis Educational score: 1.8950754404067993 Domain: biomedical Document type: Study Language: en In this section, a quantitative analysis was undertaken on 311 documents. This analysis was developed into five distinct perspectives, including an annual publication analysis, research area analysis, journal publication analysis, country publication analysis, author-publication analysis, and keyword analysis. Section title: Annual publication analysis Educational score: 1.8243204355239868 Domain: biomedical Document type: Other Language: en According to the data available in WoS and Scopus, 311 publications focused on object detection in medical imaging. Section title: Annual publication analysis Educational score: 1.8727357387542725 Domain: biomedical Document type: Other Language: en The number of articles on the subject is the most basic indicator of publications trends. Fig. 2 shows that the first publications in the field date from 2018, with a consistent upward trend in the number of publications thereafter. Fig. 2 Growth of literature related to object detection in medical imaging until 2022. Fig. 2 Section title: Annual publication analysis Educational score: 1.160561442375183 Domain: other Document type: Other Language: en In 2018, only thirteen papers were published in the area. In 2019, more than twice as many articles as the previous year were published, and in 2022 the number of publications was around ten times higher than the first year. This change reflects the growing acceptance of this topic in the research community. Section title: Annual publication analysis Educational score: 4.060147285461426 Domain: biomedical Document type: Study Language: en The citations of these 311 publications were analyzed as a whole, as shown in Table 1 . In total, those articles were cited 5052 times, and only 5.79 % (n = 18) of the publications have more than 50 citations. On the other hand, 77.81 % (n = 242) of the total publications have at least one citation. Table 1 Article papers and citations per year of object detection in medical imaging field. a Table 1 Year TP TC AC Total publications with citations ≥50 ≥20 ≥10 ≥5 ≥1 2018 13 1062 81.69 6 9 12 13 13 2019 28 799 28.54 6 14 22 23 27 2020 57 2358 41.37 6 18 36 47 57 2021 78 593 7.60 0 7 19 42 73 2022 135 240 1.78 0 0 2 14 72 % 100.00 % 5,79 % 15.43 % 29.26 % 44.69 % 77.81 % a TP stands for total papers, TC for total citations, and AC for average article citations. An analysis of the number of publications according to their quantity of citations (higher or equal to 50,20,10,5 and 1) is performed for each year. Section title: Annual publication analysis Educational score: 1.4141677618026733 Domain: other Document type: Other Language: en With the emergence of this field in 2018, the total number of publications in that year was significantly low. However, the average number of citations for those articles stands at a significant value of around 82 citations per publication. Despite a considerable decrease to an average of 28 citations per publication in 2019, the publications in 2020 achieved a peak of 41 average citations per year. This, combined with the increase in the number of publications in recent years, demonstrates the potential growth in this field. Section title: Research area analysis Educational score: 1.9583076238632202 Domain: biomedical Document type: Study Language: en According to Scopus, and based on their subject area categories and classifications , the selected publications are associated with 17 distinct fields, where publications can be included in more than one area. In total, 656 links have been established between the 311 publications and the 17 research areas. As expressed in Fig. 3 , the treemap clearly illustrates that most of the publications are included in Computer Science, Medicine, and Engineering, with 152, 139, and 107 records, respectively. Fig. 3 Treemap with the distribution of publications across various research areas provided by Scopus. Fig. 3 Section title: Research area analysis Educational score: 2.473078727722168 Domain: biomedical Document type: Other Language: en When viewed from a broader medical perspective, the areas of "Medicine" (n = 139), "Biochemistry, Genetics and Molecular Biology" (n = 58), "Health professions" (n = 33), "Dentistry" (n = 12), "Neuroscience" (n = 7) and "Immunology and Microbiology" (n = 8) correspond to approximately 39,30 % of the total associations. Section title: Research area analysis Educational score: 1.1254091262817383 Domain: other Document type: Other Language: en From an engineering-oriented perspective, "Computer Science" (n = 152) and "Engineering" (n = 107) account for 39,60 % of the total links. Section title: Research area analysis Educational score: 1.8334649801254272 Domain: biomedical Document type: Other Language: en In a multidisciplinary view, publications from "Materials Science" (n = 32), "Multidisciplinary" (n = 24), "Mathematics" (n = 25), "Physics and Astronomy" (n = 22), "Chemistry" (n = 13), "Chemical Engineering" (n = 12), "Agricultural and Biological Sciences" (n = 4), "Environmental Sciences" (n = 4), account for around 20,80 % of the total number of links. Two publications were not available on Scopus and were classified as "Others". Section title: Source analysis Educational score: 2.191991090774536 Domain: biomedical Document type: Study Language: en The 311 publications obtained were published in 161 journals from different fields of study. There were 111 journals (68.94 %) with only one publication associated, followed by 17 journals (10.60 %) with two articles published. Notably, only two journals have more than ten publications, namely IEEE Access (n = 17) and Scientific Reports (n = 13). All journals with five or more publications, as shown in Supplementary file 2, account for 33.12 % of the total publications and belong to the first quartile, excluding Diagnostics and Applied Sciences (Switzerland) Journals, which belong to the second quartile. Section title: Source analysis Educational score: 1.357410192489624 Domain: biomedical Document type: Other Language: en Among these journals, those with the highest average number of citations based on the total number of publications were "Computers in Biology and Medicine", with an average of 159 citations; Scientific Reports, with approximately 53 citations on average; and PLoS One, with an average of 24 citations. Section title: Countries publication analysis Educational score: 1.8714570999145508 Domain: biomedical Document type: Study Language: en In this analysis, each publication has been linked to the countries with which each author is associated. Since each publication can have more than one author, and an author can be associated with more than one country, a paper can be associated with one or further countries. However, if a paper has two authors from the same country, we only considered it once to avoid duplicating the same article in the same country. Section title: Countries publication analysis Educational score: 1.6456671953201294 Domain: other Document type: Other Language: en At the time of research, 51 countries have published at least one paper. Table 2 shows countries with more than one hundred citations sorted by the number of citations. Table 2 Countries with a higher number of citations. a Table 2 Rank Country P C AC 1 Japan 36 1791 49.75 2 United Kingdom 12 1465 122.08 3 Turkey 14 1451 103.64 4 Taiwan 17 1404 82.59 5 Singapore 2 1328 664.00 6 China 124 1273 10.27 7 United States 48 828 17.25 8 Hungary 1 364 364.00 9 South Korea 22 356 16.18 10 Australia 10 119 11.90 11 Germany 9 105 11.67 12 Hong Kong 6 116 19.33 a P stands for the number of publications, C for the number of Citations, AC is the average number of citations per publication. Section title: Countries publication analysis Educational score: 1.0210161209106445 Domain: other Document type: Other Language: en Japan stands as the country with more citations associated with its publications, followed by the United Kingdom and Turkey . However, regarding the average number of citations, Singapore is at the front with 664 citations per publication, Hungary in second with 364, and the United Kingdom stands in third place with around 122 publications. Section title: Countries publication analysis Educational score: 1.5191084146499634 Domain: other Document type: Other Language: en Fig. 4 illustrates the top ten most productive countries, with a supplementary analysis of co-authorship, which indicates collaboration among authors from different countries. China is the leading country in this field, with 124 publications, followed by the United States with 48 publications and Japan with 36 publications. The United Kingdom displays the highest percentage of affiliations in terms of international collaboration, with all of its articles resulting from international partnerships. In contrast, Australia has only one out of ten papers with international collaboration. In absolute terms, China has published 86 papers with international collaboration out of 124 total papers, the United States has published 30 out of 48 papers with international affiliations, and Japan has published 26 papers out of 36. Fig. 4 The top ten most productive countries and their international co-authorship. Fig. 4 Section title: Countries publication analysis Educational score: 1.117467999458313 Domain: other Document type: Other Language: en When we assess the average number of citations of these three leaders in terms of publications, China presents a value of 10.27, the United States has 17.25, and Japan has 49.75, representing the 25th place, 15th place, and 7th place, respectively, among the 51 countries available. Section title: Authors publication analysis Educational score: 1.7899411916732788 Domain: biomedical Document type: Study Language: en In the set of 311 publications chosen, the author analysis was done through the SCOPUS ID of the authors. This approach was adopted to prevent the association of papers for authors with the same name, even if they are not the same researcher. Section title: Authors publication analysis Educational score: 1.7170382738113403 Domain: other Document type: Study Language: en From this analysis, it was found that there were a total of 1870 related authors. Of those, 1752 authors were associated with only one paper, while only eight are associated with four or more papers, as seen in Table 3 (which lists the most productive authors in the field). Table 3 The most productive authors. a Table 3 Rank Author Country P C 1 Ariji, Eiichiro Japan 7 195 2 Aniji, Yoshiko Japan 7 195 3 Katsumata, Akitoshi Japan 6 182 4 Fukuda, Motoki Japan 5 165 5 Liang, Yixiong China 5 81 6 Fujita, Hiroshi Japan 5 176 7 Muramatsu, Chisako Japan 4 118 8 Kuwada, Chiaki Japan 4 107 a Authors with four or more publications in the field. P stands for the number of papers, and C for the number of citations on those papers. Section title: Authors publication analysis Educational score: 1.1714755296707153 Domain: other Document type: Other Language: en Of the 1870 related authors, nine have three publications, 100 have two publications, and the remaining have just one single publication. Notably, all the most productive authors (authors with four or more publications) belong to Japan or China. Section title: Authors publication analysis Educational score: 1.5068408250808716 Domain: other Document type: Other Language: en With regard to the number of citations, out of all the authors, 32 are cited more than 100 times, 15 have received more than 200 citations, and 401 authors have no citation associated with their work. Table 4 reveals the identities of all authors with more than 200 citations. Table 4 Authors with more than two hundred citations associated. a Table 4 Rank Author Country P C 1 Baloglu, Ulas Baran United Kingdom 1 1306 2 Ozturk, Tulin Turkey 1 1306 3 Rajendra Acharya U. Singapore, Taiwan, Japan 1 1306 4 Talo, Muhammed Turkey 1 1306 5 Yildirim, Eylul Azra Turkey 1 1306 6 Yildirim, Ozal Turkey 1 1306 7 Csabai, István Hungary 1 364 8 Horváth, Anna Hungary 1 364 9 Pollnar, Péter Hungary 1 364 10 Ribli, Dezso Hungary 1 364 11 Unger, Zsuzsa Hungary 1 364 12 Lu, Le United States 3 242 13 Yan, Ke United States 3 240 14 Wang, Xiaosong United States 1 228 15 Summers, Ronald M. United States 1 228 a The authors with more than 200 citations are reported with their country, the number of publications associated to them, and the total number of citations. Section title: Authors publication analysis Educational score: 1.3111212253570557 Domain: other Document type: Other Language: en As shown in Table 4 , 14 out of the 15 authors with 200 or more citations associated have published only one paper on the field, and only two are associated with the publication of three papers, namely Le Lu and Ke Yan from the United States. Section title: Keyword analysis Educational score: 2.916342258453369 Domain: biomedical Document type: Study Language: en To gain insight into the central focus of the research, the commonly utilized words in the keyword section of the 311 selected articles were also evaluated. The analysis was conducted using the software ScientoPy. The word cloud in Fig. 5 illustrates the most frequently employed terms of the 311 selected articles. It is evident, as their frequency determines the size of the concepts in a word cloud, that concepts such as deep learning (n = 138), object detection (n = 84), convolutional neural networks (n = 54), and artificial intelligence (n = 34) are among the most frequently used terms. Fig. 5 Wordcloud with keyword analysis. The size of a word or concept corresponds to the frequency of occurrence. Fig. 5 Section title: Keyword analysis Educational score: 1.531617522239685 Domain: other Document type: Other Language: en Concerning the algorithms used, Faster R-CNN appears in 19 articles as a keyword, and the YOLO algorithm (regardless of the version) is present as a keyword in 31 records. Section title: Keyword analysis Educational score: 2.135565996170044 Domain: biomedical Document type: Study Language: en In Fig. 6 , we report the network of co-occurrence of index keywords provided by VosViewer. In all the 311 articles, we defined the threshold where only keywords with five or more occurrences are shown. From the several keywords represented, Deep Learning appears 181 times, object detection has 123 appearances, and convolutional neural network appears 101 times. Fig. 6 Co-occurrence of index keywords network developed in VosViewer. Only keywords with five or more occurrences are shown. Fig. 6 Section title: Keyword analysis Educational score: 1.754416823387146 Domain: other Document type: Other Language: en Each node in Fig. 6 represents a keyword, and its size indicates the rate of frequency of the corresponding keyword, where the larger the node, the higher the significance of the keyword. The distance between two nodes reflects the strength of the relationship between two keywords. In other words, the closer two nodes are to each other, the higher the frequency of those two keywords appearing together. Section title: Keyword analysis Educational score: 1.7993425130844116 Domain: other Document type: Other Language: en Additionally, the color of the nodes defines the cluster to which each node belongs. VosViewer's clustering approach represents the connections and relations among nodes based on distance and density. The algorithm tries to identify sets of keywords that are prone to co-occur more frequently with each other than with keywords outside the cluster. To ensure visual singularity, each cluster is assigned a unique color. Section title: Keyword analysis Educational score: 2.0280234813690186 Domain: biomedical Document type: Other Language: en From the network, we can verify four main clusters created. The red cluster seems to include articles where the main focus is on the technology used, with deep learning, medical imaging, and convolutional neural networks as the terms with more weight. There is a similar interpretation for the blue cluster with algorithm, neural network, and automation as the most significant keywords. The green cluster seems to point out articles more medically oriented, with human, article, and controlled study as the main keywords. The purple cluster has some similarities with the former cluster, with human, male, and aged as keywords with higher occurrence. Section title: Qualitative analysis Educational score: 2.0844175815582275 Domain: biomedical Document type: Study Language: en The qualitative analysis was based on publications with an annual citation rate greater than 5. This selection resulted in 80 publications, with 30 presenting an annual citation rate greater than 10, as shown in Supplementary File 3. Section title: Qualitative analysis Educational score: 2.5685694217681885 Domain: biomedical Document type: Review Language: en In the papers undergoing a qualitative analysis, it is crucial to discern the incidence of algorithms used, the medical imaging modality most frequently associated with publications concerning object detection, and the anatomical application areas where object detectors are applied. These topics are thoroughly reviewed in the following three subsections. Section title: Used algorithms Educational score: 1.8705042600631714 Domain: biomedical Document type: Study Language: en Concerning the object detection algorithms applied in the 311 publications, and as illustrated in Fig. 7 , several algorithms have been applied as strategies to identify objects of interest in the given images. Among the most common, we should mention Faster R-CNN, YOLO, SSD, DetectNet, RetinaNet, Mask R-CNN, and R-CNN. Fig. 7 Stacked bar plot of the algorithms explored by year. Fig. 7 Section title: Used algorithms Educational score: 1.6559725999832153 Domain: other Document type: Other Language: en R-CNN, proposed in 2014 , was one of the first emerging techniques for object detection using convolutional neural networks. As a two-stage object detection algorithm, R-CNN has a first stage where regions of interest are proposed, while the second phase is focused on detecting the final boxes and classifying them. Section title: Used algorithms Educational score: 2.0377628803253174 Domain: other Document type: Other Language: en The former algorithm's inefficiency was recognized due to its limitations on speed and accuracy. Faster R-CNN is proposed by Ren et al. as an advancement to R-CNN, where, in the first stage, a Region Proposal Network (RPN) is implemented to generate more efficiently the proposals and, in the second phase, Fast R-CNN is implemented to detect the final boxes and classify them. The RPN and the object detection network are trained simultaneously, allowing to reduce the computational cost, increase the training speed, and improve the detection. Section title: Used algorithms Educational score: 1.9760273694992065 Domain: other Document type: Other Language: en Mask R-CNN extends the Faster R-CNN approach by adding the capability to perform instance segmentation at the pixel level to the task of detecting objects of interest. Section title: Used algorithms Educational score: 1.569023609161377 Domain: other Document type: Other Language: en One-stage detectors emerged with Single Shot MultiBox Detector (SSD) and You Only Look Once (YOLO) . These detectors possess a simpler architecture than two-stage detectors and simultaneously locate and classify each object without the need for a region proposal process. The main difference between these two approaches is that the former computes a feature map and combines distinct grids of different sizes to better detect objects of any size. The latter, on the other hand, is faster by defining one single grid where class probabilities are associated with anchor boxes with different scales and aspect ratios. Section title: Used algorithms Educational score: 1.567036509513855 Domain: other Document type: Other Language: en In posterior years, other one-stage approaches appeared, such as RetinaNet and DetectNet . RetinaNet introduces the concept of Focal Loss to the classification where different weights are assigned to hard samples, solving the problem of class imbalance and improving the detection performance. DetectNet is an object detection architecture created by NVIDIA and optimized for real-time object detection. Section title: Used algorithms Educational score: 1.5373562574386597 Domain: other Document type: Other Language: en While two-stage detectors, at the emergence of object detection architectures, tended to yield higher detection accuracy, but also a slower training phase , with the advance and development of one-stage frameworks, the performance of both types appears to be similar, if not better in the latter one. Section title: Used algorithms Educational score: 1.9357844591140747 Domain: other Document type: Study Language: en Faster R-CNN stands as the most frequently used algorithm, with 32.5 % of the publications, followed by the YOLO algorithm (independently of the version) with 30 % of the records, and SSD with 8.75 %. RetinaNet and DetectNet have five publications associated, and the remaining 13 papers (16.25 %) apply other object detectors, as shown in Fig. 7 . Section title: Used algorithms Educational score: 1.9379664659500122 Domain: biomedical Document type: Other Language: en Faster R-CNN was the most widely utilized algorithm during the emergence of object detection in medical imaging. However, since 2019, the algorithm YOLO has gained significance in the field, with more publications in 2021 and 2022 than Faster R-CNN, which was previously the most popular algorithm. This fact reveals that the YOLO algorithm is gradually replacing the position previously held by Faster R-CNN and has gained increased popularity in recent years. Section title: Modalities Educational score: 2.0305097103118896 Domain: biomedical Document type: Other Language: en There are several types of medical imaging modalities. As shown in Fig. 8 , the most frequent modalities include Computed Radiography (CR), Pathological Imaging, Endoscopy, and Computed Tomography (CT). Fig. 8 Stacked barplot of the used image modalities by year. Fig. 8 Section title: Modalities Educational score: 2.3550360202789307 Domain: biomedical Document type: Other Language: en Computed Radiography is a widely accessible and affordable screening method broadly used to detect several pathologies, such as lung diseases and mass screening. As an example, this method is applied to mammography in a low-dose X-ray approach to visualize the breast structure and identify possible breast anomalies . Section title: Modalities Educational score: 3.0887320041656494 Domain: biomedical Document type: Other Language: en Endoscopy allows the visual inspection of an internal organ or tissue in detail . One possible application of endoscopic procedures is to identify colorectal lesions, where techniques such as high-resolution microendoscopy, fluorescence imaging, and enhanced endoscopy are available to improve the detection rate of tumors using endoscopy . Section title: Modalities Educational score: 3.273162364959717 Domain: biomedical Document type: Other Language: en Pathology Imaging uses static images, live streaming of images, and whole slide imaging (WSI) to make pathology diagnoses. The latter allows the digitalization of entire glass slides ,enabling quantitative analyses for the entire landscape of tissue morphology without the need for microscopy . Section title: Modalities Educational score: 3.2741663455963135 Domain: biomedical Document type: Other Language: en Digital photography plays an important role in various specific medical specialties, enabling the identification and diagnosis of potential lesions and diseases in a cost-effective, noninvasive, readily accessible, and real-time manner. The usage of this approach encompasses a wide range of tasks, including the detection of skin cancer, dermoscopy imaging, assessment of oral and dental health, and the diagnosis of hair and nail conditions . Section title: Modalities Educational score: 2.688969850540161 Domain: biomedical Document type: Other Language: en Ultrasound is a widely used medical imaging modality, providing a noninvasive technique for imaging human anatomy , being widely applied in prenatal screening due to its relative safety, low cost, noninvasive nature, and real-time display . Section title: Modalities Educational score: 2.792522668838501 Domain: biomedical Document type: Other Language: en Magnetic Resonance Imaging (MRI) is a noninvasive method for acquiring high-resolution images of any organ in the body. This imaging technique can be applied to different tasks, such as stroke detection, tumorigenesis, and myocardial infarction . Section title: Modalities Educational score: 3.796271562576294 Domain: biomedical Document type: Other Language: en Microscopy images can support quantitative analysis for diagnosing and characterization of several diseases, playing an essential role in computer-aided diagnosis and prognosis . Associated with Histopathology, the field of study of human tissue using a microscope, it allows, through staining, the visualization of specific parts of tissues that enables the identification of different diseases such as kidney cancer, lung cancer, and breast cancer, among others, through nuclei and cell detection . Section title: Modalities Educational score: 2.8310816287994385 Domain: biomedical Document type: Other Language: en Fundus Photography is a non-contact ophthalmologic examination of the eye, considered the most cost-effective imaging modality for screening fundus disease, that allows the visualization of several eye lesions such as of the optic nerve and retina . Section title: Modalities Educational score: 3.566434621810913 Domain: biomedical Document type: Other Language: en Conventional angiography and contrast-enhanced magnetic resonance angiography is regarded as the reference examination for investigation of atherosclerotic lesions of the supra-aortic extracranial vessels, and especially for detecting stenosis of the carotid artery, being the latter one the less invasive and with similar results to the former . Section title: Modalities Educational score: 2.407254219055176 Domain: biomedical Document type: Study Language: en Of the 80 papers analyzed, 25 % utilized CR as the primary image modality, followed closely by Endoscopy with 15 % and pathology imaging with 12.50 % of the publications. Although it is unclear whether there have been significant changes in the type of images used over the years, it is evident an increase in the use of pathological images in 2021 and 2022. Section title: Anatomical application areas Educational score: 2.7308857440948486 Domain: biomedical Document type: Study Language: en Over the past five years, the anatomical application areas where object detection has been applied have been diverse, but digital pathology and microscopy have emerged as the most popular, with a total of 16 publications, as illustrated in Fig. 9 . Closely following in popularity is the detection of abnormalities in abdominal organs, with 15 publications. Fig. 9 Stacked barplot of the anatomical application areas explored by year. Fig. 9 Section title: Anatomical application areas Educational score: 1.8755789995193481 Domain: biomedical Document type: Study Language: en In the following subsections, we will qualitatively analyze the various publications distributed by their specific anatomical application area. Section title: Abdominal organs Educational score: 3.9977829456329346 Domain: biomedical Document type: Study Language: en As shown in Table 5 , on abdominal organs, the research is focused on the colon, gallbladder, liver, stomach, cervix, and uterus. Table 5 Publications in which object detection algorithms are applied to abdominal organ images. a Table 5 Reference C Modality Organ Algorithm-based Task 98 E Colon YOLO Polyp detection 52 E Colon SSD Polyp detection 16 E Colon YOLO Polyp detection 7 E Colon SSD Polyp detection 14 E Colon RefineNet Polyp detection 21 E Gallbladder YOLOv3 Detection of bile duct injury 41 CT Gallbladder YOLOv3 Detection of gall stones 17 US Liver Faster R-CNN Abnormality detection 25 E Stomach Faster R-CNN Detection of gastric lesions 9 E Stomach YOLOv3 Detection of gastric lesions and neoplasm prediction 13 PH Cervix R-CNN Cervical cancer detection 27 PH Cervix Faster R-CNN Cervical cancer detection 18 PH Cervix Faster R-CNN Cervical cancer detection 30 US Uterus YOLOV2 Detection of cardiac structural abnormalities 27 US Uterus Custom Detection of cardiac structural abnormalities a Overview of papers using object detection techniques for detecting abnormalities in abdominal organs, where E stands for endoscopy, CT for computed tomography, PI for pathology image, PH for photography, and US for ultrasound. In the header of the table, C stands for the number of citations. Section title: Abdominal organs Educational score: 4.123088359832764 Domain: biomedical Document type: Study Language: en Five of the selected papers are dedicated to colon polyps detection, using images obtained through endoscopy. Among those publications, YOLO and Faster R-CNN are applied twice, while RefineNet is applied once. In the first study in which YOLO is implemented, an improved framework is developed to detect polyps in a dataset consisting of more than 17,000 frames, yielding a precision of 88.6 % and a recall of 71.6 % at a prediction speed of 6.5 frames per second . In the second study, YOLOv3 and YOLOv4 architectures are applied to two datasets, achieving a precision of 90.6 % and a recall of 91.0 % at a processing speed of 100 frames per second .This last publication combines the YOLO structure with CSPNet to obtain a higher-performance model. In the SSD Implementations, one of the studies is validated in 7077 images, where the model was able to identify correctly 1073 colon polyps out of the existent 1172, with a sensitivity of 90 %, and a processing time of 20 frames per second . In the second implementation, a multiscale pyramidal fusion single-shot multibox detector network is applied, resulting in an mAP of 93.4 % , with a testing speed of 62.5 FPS. RefineNet is also employed for polyp detection and applied to a combination of four public data sets. In this study , the authors compare RefineNet with different algorithms such as YOLO and Faster R-CNN, achieving the best performance among all the tested approaches with an mAP of 73.5 %, while YOLOv3 and SSD achieve the fastest inference with 60 FPS. Section title: Abdominal organs Educational score: 3.8800508975982666 Domain: biomedical Document type: Study Language: en Two studies have been conducted using YOLOv3, with distinct objectives concerning gallbladder organs. In the first one, endoscopic images were used to detect four landmarks to prevent bile duct injury in 23 short videos. The authors of this study recognize the system's suboptimal performance . In contrast, the second study uses 223,846 CT images to detect gall stones, achieving an mAP of 89.5 % for granular and muddy stones . Section title: Abdominal organs Educational score: 3.8186891078948975 Domain: biomedical Document type: Study Language: en Regarding the liver, one study has been conducted in which cysts and primary hepatic carcinoma detection in ultrasound imaging were accomplished using Faster R-CNN with a ResNet backbone. In this publication , the authors achieved an mAP of 60 %, representing an improvement over the results obtained with YOLOv2, which had an mAP of 51 %. Section title: Abdominal organs Educational score: 3.967414140701294 Domain: biomedical Document type: Study Language: en In two different studies, the detection of gastric lesions on endoscopic imaging was approached. In the Faster R-CNN implementation , more than one million images were employed to train and test the system. The authors of this study enhance the significant difference in detection time between the automatic approach and clinicians’ performance. The results are reported by sensitivity values, ranging from 87.2 % to 99.3 %. In the second publication, where YOLOv3 was applied, the data was obtained from over 100,00 patients, and the final model sensitivity ranged between 91.7 % and 96.9 % . Section title: Abdominal organs Educational score: 4.022642135620117 Domain: biomedical Document type: Study Language: en Three of the fifteen papers on abdominal organs are focused on cervical cancer detection, obtained through applying two-stage object detection approaches on photographic images. In a study published in 2022 , a generative adversarial network was developed using an R-CNN to accurately detect small cervical cells and classify them into normal, precancerous, or cancerous. The remaining two publications employed Faster R-CNN. The first study created a system based on 2853 images labeled as high-risk or low-risk, achieving an AUC value of 0.87 . In the second study, an improved version of Faster R-CNN was developed and tested on 2528 images with 5294 lesion regions, achieving an mAP of 80.37 %. This represented better results than those obtained with SSD (mAP = 74.27 %), YOLOv3 (mAP = 77.11 %), and Faster R-CNN (mAP = 75.89 %). Section title: Abdominal organs Educational score: 1.4265408515930176 Domain: other Document type: Study Language: en Out of the fifteen papers identified, seven of them use one-stage object detectors. Section title: Abdominal organs Educational score: 2.2688310146331787 Domain: biomedical Document type: Study Language: en Moreover, we assess additional relevant aspects, including the data sets employed, their magnitude, data accessibility, the integration of external organizational data, and the study's temporal orientation concerning being retrospective or prospective. The outcomes are summarized in Table 1 of Supplementary File 4. Section title: Bones Educational score: 3.9561383724212646 Domain: biomedical Document type: Study Language: en Nine of the eighty studies selected based on the annual citation rate are employed in bone imaging, as exposed in Table 6 . Table 6 Publications in which object detection algorithms are applied to bone images. a Table 6 Reference C Modality Algorithm Task 73 CT Faster R-CNN Detection of distal radius fractures 66 CR DetectNet Detection of mandible fractures 28 CR DetectNet Detection of maxillary sinus lesions 7 US YOLOv3 Detection of vertebras in spine 7 CT Ensemble Detection of wrist fractures 13 CR YOLOv3 Detection of hip dislocation 12 MRI YOLOv3 Detection of Lumbar Disc Herniation 22 CR Faster R-CNN Skeletal bone age assessment 10 CT YOLOv3 Detection of vertebral fractures a Overview of papers using object detection techniques on bones, where CT stands for computed tomography, CR for computed radiography, US for ultrasound and MRI for magnetic resonance imaging. In the table's header, C stands for the number of citations. Section title: Bones Educational score: 3.9992315769195557 Domain: biomedical Document type: Study Language: en On CT imaging, three studies use object detection to locate fractures, namely on the distal radius , on the wrist , and on the vertebras . In the first one, a comparison is made between the automatic approach using a Faster R-CNN, and professional assessment. The authors conclude that the network achieved performances similar to those obtained from orthopedists and superior to radiologists. In a second study, wrist fractures are located with an Ensemble technique based on WBF. Ten base models are tried out, and the highest AP Score was obtained with the ensemble version obtained from the base learners. The authors enhance the potential of ensemble techniques in this field. In the third publication, vertebral fractures are detected using a YOLOv3 architecture, and one remark appointed by the authors is the highest interobserver reliability of the automatic system compared to human observers. Section title: Bones Educational score: 4.0184431076049805 Domain: biomedical Document type: Study Language: en CR imaging is used in object detection studies with different purposes. Two publications, in particular, apply DetectNet , a network developed in DIGITS. The first study employs a model built on 210 training images to locate mandible fractures, achieving a sensitivity of 88 % in the testing data . The second study, also utilizing DIGITS, identifies maxillary sinus lesions, with a sensitivity of 100 % on healthy and inflamed maxillary sinuses and 98 % and 89 % on distinct data sets for cyst maxillary sinus region . Another study trains YOLOv3 to assess the risk of hip dislocation on 1490 radiographs from dislocated cases and 91,094 from non-dislocated cases, achieving a sensitivity of 89 % . A Faster R-CNN is employed to assess skeletal bone age by detecting the ossification centers of the epiphysis and carpal bones , with a performance measured by MAE, achieving values of 0.48 and 0.51 in two tested data sets. Section title: Bones Educational score: 3.201514482498169 Domain: biomedical Document type: Study Language: en On ultrasound imaging, various object detectors, such as SSD, YOLOv3, and YOLOv4, were trained to detect the vertebrae in the spine . While SSD achieved the highest mAP with a value of 90.84 %, YOLOv3 was chosen as the final model due to its short inference time of around 7 FPS, around ten times faster than SSD's. Section title: Bones Educational score: 2.777864694595337 Domain: biomedical Document type: Study Language: en An automatic detection system based on YOLOV3 and MRI was also proposed to assist in the initial lumbar disc herniation exam for lower back pain . In this study, an mAP of 92.4 % was obtained at 550 images with data augmentation. Section title: Bones Educational score: 2.166306495666504 Domain: biomedical Document type: Study Language: en Table 2 of Supplementary File 4 summarizes the data sets used and the type of study applied to bone images. Section title: Brain Educational score: 3.7728922367095947 Domain: biomedical Document type: Study Language: en An MRI is a powerful tool for producing detailed images of the brain, which can be used to detect a wide range of conditions, such as tumors, injuries, strokes, and blood vessel problems. In recent years, some object detection algorithms have been applied to this field to automate the diagnostic process and increase the accuracy of results. Table 7 exposes those studies. Table 7 Publications in which object detection algorithms are applied to brain images. a Table 7 Reference C Modality Algorithm Task 9 MRI YOLOv5 Brain tumor diagnosis 20 MRI YOLOv4 Brain tumor diagnosis 11 MRI Faster R-CNN Stroke lesion detection a Overview of publications that employ object detection techniques on the brain, where MRI stands for magnetic resonance imaging. In the header of the table, C stands for the number of citations. Section title: Brain Educational score: 3.9255635738372803 Domain: biomedical Document type: Study Language: en In the publications considered for qualitative analysis, two studies applied the YOLO architecture to brain tumor diagnosis, while another study applied Faster R-CNN for stroke lesion detection. In the study where YOLOv5 is applied , the authors compared the performance of different algorithm variants on 800 images to brain tumor detection. Unsurprisingly, the larger version of the model achieved the best mAP at 91.2 % using YOLOv5x, while the smaller versions of YOLOv5 (YOLOv5n and YOLOv5s) achieved the worst performances with an mAP of 85.2 % and 87 %. However, the trade-off is that larger versions take more time to train. Section title: Brain Educational score: 4.010619640350342 Domain: biomedical Document type: Study Language: en In a second study for brain tumor diagnosis and detection, a transfer learning approach and fine-tuning techniques were applied to YOLOv4 to build a model on a data set of 3064 MRI scans, which was able to detect three distinct tumors, namely glioma, meningioma, and pituitary. This improved version of YOLO achieved a final mAP of 93.14 % . Section title: Brain Educational score: 4.006074905395508 Domain: biomedical Document type: Study Language: en A third study on Brain MRI scans is focused on stroke lesion detection . In this study, the authors compared the performance of Faster R-CNN, YOLOv3 and SSD, and concluded that SSD performed the best, with the highest mAP (89.77 %). When comparing the inference time, YOLO was the fastest approach, but this advantage was reflected in a significant decrease in the mAP, with a value of 74.9 %. Section title: Brain Educational score: 2.3229215145111084 Domain: biomedical Document type: Study Language: en Table 3 of Supplementary File 4 provides a summary of the data sets employed and the study methodologies applied to brain images. Section title: Breast Educational score: 3.926414728164673 Domain: biomedical Document type: Study Language: en Breast cancer is the most common cancer diagnosed worldwide, with 685,000 deaths associated in 2020, being estimated that in 2040 this value will rise to 1 million . With eight publications associated, as expressed in Table 8 , object detectors in breast imaging focus on detecting lesions. Table 8 Publications in which object detection algorithms are applied in breast images. a Table 8 Reference C Modality Algorithm Task 364 CR Faster R-CNN Breast lesion detection 90 CR YOLO 9000 Breast lesion detection 78 US SSD Breast lesion detection 43 CR Custom Breast lesion detection 61 CR RetinaNet Breast lesion detection 21 MRI RetinaNet Breast lesion detection 6 CR Mask R-CNN Breast lesion detection 10 CR YOLOv3 Breast lesion detection a Overview of publications using object detection techniques for breast lesion detection, where CR stands for computed radiography, US for ultrasound and MRI for magnetic resonance imaging. In the header of the table, C stands for the number of citations. Section title: Breast Educational score: 1.9649702310562134 Domain: biomedical Document type: Other Language: en Various object detection models are applied in these studies, including YOLO, SSD, RetinaNet, Faster R-CNN, Mask R-CNN, and custom-designed networks. The most commonly used imaging modality was CR, but ultrasound images and MRI were also used. Section title: Breast Educational score: 4.059063911437988 Domain: biomedical Document type: Study Language: en Regarding CR imaging, six studies were conducted using different algorithms as a basis. One of the publications applied Faster R-CNN to detect and classify malignant or benign lesions on a mammogram in a data set with 2620 screening exams. The system achieved an AUC of 0.85, with 10 % of the malignant lesions being missed. RetinaNet was tested in seven different data sets in the same year and, according to the authors, outperformed the conventional mass detection models . In 2020, two publications addressed this task. One of these applied YOLO 9000 on two distinct data sets, comprising 600 and 360 images, respectively. 99.17 % of the total cases were predicted correctly on these data sets, with 0.83 % being false detections . In the second case, a custom network was built to address class imbalance for small lesion detection on breast . Another publication used a YOLO-based model on 235 publicly available mammograms and 487 privately collected mammograms, achieving a detection accuracy rate of more than 95 % for the distinct datasets used and an inference time of around 0.55 s per image . In 2022, a Mask R-CNN-based framework was developed and implemented on public and in-house datasets. The model's performance was analyzed only using recall, achieving values of 0.82 for the first data set and 0.87 for the private data set, with an IoU threshold of 0.5 . Section title: Breast Educational score: 3.0283920764923096 Domain: biomedical Document type: Study Language: en In 2019, ultrasound images were applied on distinct object detectors, including Fast R-CNN, Faster R-CNN, YOLO, YOLOv3 and SSD. YOLO, and SSD performed significantly better than the other methods, and SSD was chosen as the most suitable model for this task, achieving an mAP of 96.89 % in a total of 1041 cases . Section title: Breast Educational score: 3.4572131633758545 Domain: biomedical Document type: Study Language: en Regarding MRI imaging, only one publication was included in this qualitative analysis , using RetinaNet. The authors compared the sensitivity, specificity, and AUC between the AI system and human readers, concluding that the former had a better diagnostic performance. Section title: Breast Educational score: 2.286517858505249 Domain: biomedical Document type: Study Language: en Table 4 of Supplementary File 4 summarizes the data sets used and the study methodologies applied to breast images. Section title: Chest Educational score: 3.908874750137329 Domain: biomedical Document type: Study Language: en In thoracic image analysis of both computed radiography and tomography, the detection of pulmonary tuberculosis and COVID-19 were the most commonly addressed applications, as seen in Table 9 . Table 9 Publications in which object detection algorithms are applied in chest images. a Table 9 Reference C Modality Algorithm Task 1306 CR YOLO 9000 COVID-19 detection 9 CT CenterNet Pulmonary tuberculosis detection 7 CR SSD COVID-19 detection 19 CR Faster R-CNN Pulmonary tuberculosis detection a Overview of publications that apply object detection techniques on chest imaging for COVID and pulmonary tuberculosis detection, where CT stands for computer tomography and CR for computed radiography. In the table's header, C stands for the number of citations. Section title: Chest Educational score: 3.882037878036499 Domain: biomedical Document type: Study Language: en SSD and YOLO 9000 are applied to COVID detection on CR imaging. Several backbones have been tested with SSD, including VGG, Residual Network, DarkNet, and DenseNet, and their performance has been compared. DenseNet achieves the highest performance, with a precision of 0.93 and a recall of 0.94 . The most cited publication in our qualitative research focuses on implementing YOLO for COVID detection. The proposed model is developed to provide accurate diagnostics for binary classification (COVID vs No-findings) and multi-class classification, where Pneumonia is added to the former classes. The authors report an accuracy of 98.08 % on the binary problem and 87.02 % for the multi-class problem . Section title: Chest Educational score: 4.11012077331543 Domain: biomedical Document type: Study Language: en In pulmonary tuberculosis detection, a Faster R-CNN adapted model has been applied to a multi-class problem on CT images, which encompasses co-existing cases of exudation, calcification, nodules, miliary tuberculosis, and other related conditions. This model achieves an mAP of 53.74 %, outperforming the original Faster R-CNN model, which reached 22.66 % mAP and surpassing the results obtained with FPN, which achieved 50.96 % mAP . Additionally, CenterNet has also been used on 892 CT scans for pulmonary tuberculosis detection, yielding an mAP of 68 % . Section title: Chest Educational score: 2.1184823513031006 Domain: biomedical Document type: Study Language: en The data presented in Table 5 of Supplementary File 4 defines the data sets employed and the research methodologies specific to the analysis of chest images. Section title: Digestive system organs Educational score: 4.046963214874268 Domain: biomedical Document type: Review Language: en Concerning the digestive system, object detection techniques have been applied to various forms of medical imaging, including esophagus imaging obtained through endoscopy, tongue images obtained through photography, and teeth images obtained through CR imaging, as seen in Table 10 . Table 10 Publications in which object detection algorithms are applied to images of digestive system organs. a Table 10 Reference C Modality Organ Algorithm Task 92 E Esophagus YOLOV2 Early esophageal neoplasia detection 44 E Esophagus SSD Early esophageal adenocarcinoma detection 29 PH Tongue R-CNN Tooth-marked tongue detection 117 CR Teeth Faster R-CNN Tooth detection 58 CR Teeth DetectNet Root fracture detection 24 CR Teeth DetectNet Tooth detection 26 CR Teeth Faster R-CNN Periodontal compromised teeth detection 8 CR Teeth Faster R-CNN Permanent teeth detection 11 CR Teeth Faster R-CNN Dental disease detection 5 CR Teeth Faster R-CNN Marginal bone loss around implants detection a Overview of publications that apply object detection techniques on digestive system organs, where CR stands for computed radiography, E for endoscopy and PH for photography. In the header of the table, C stands for the number of citations. Section title: Digestive system organs Educational score: 4.098100662231445 Domain: biomedical Document type: Study Language: en In particular, YOLOv2 and SSD have been applied to endoscopy imaging, with the former being used to detect early stages of esophageal neoplasia and the latter being used to detect early stages of esophageal adenocarcinoma . In the first study, 916 images were used to train the model, and 458 images were used to test it, achieving an mAP of 75.33 % for narrow bone imaging and 80.19 % for near-focus images at a speed of 45 FPS. In the second study, the performance of SSD and Faster R-CNN were compared in terms of average recall rate (ARR), average precision rate (APR), sensitivity (SE), specificity (SP), and F-measure (FM) using a 5-fold cross-validation method. SSD achieved the highest values with 0.7 on APR, 0.90 in SE, 0.88 in SP, and 0.88 in FM, being surpassed by Faster R-CNN only in ARR with a difference of 0.04. Section title: Digestive system organs Educational score: 3.5752553939819336 Domain: biomedical Document type: Study Language: en In a tongue photography study, the goal was to identify tooth-marked areas on the tongue using a data set containing 641 images, 297 of which were tooth-marked. An adapted version of R-CNN was applied, and the authors reported an average accuracy of 72.7 %, a TPR of 69.1 %, and a TNR of 76.2 % . Section title: Digestive system organs Educational score: 3.8970489501953125 Domain: biomedical Document type: Study Language: en Tooth detection appears to be a prevalent subject in the realm of object detection frameworks in medical imaging. Seven of the 80 studies analyzed were dedicated to tooth detection and related tasks, and the Faster R-CNN algorithm was applied in five of these studies. In the first study , the model achieved a precision and recall rate of over 90 %, with a mean average precision (mAP) of 91 %. These values were only attainable after implementing several postprocessing procedures. Section title: Digestive system organs Educational score: 3.8851115703582764 Domain: biomedical Document type: Study Language: en In another study, Faster R-CNN was employed for Periodontal compromised teeth detection , resulting in an accuracy rate of 80 %, a positive predictive rate of 81 %, a sensitivity of 84 %, a specificity of 88 %, and a F-measure of 81 %. The authors of this study concluded that the system exhibited satisfactory detection abilities. Section title: Digestive system organs Educational score: 4.01175594329834 Domain: biomedical Document type: Study Language: en Faster R-CNN was also applied to permanent teeth detection in a study where the authors reported results of 0.99 for recall and precision. Additionally, the algorithm was used in another work to detect dental diseases such as decay, periapical periodontitis, and periodontitis at varying levels of severity. The results showed that decay and periapical periodontitis lesions were detected with precision, recall, and average precision values of less than 0.25 for mild levels, while moderate and severe levels had values ranging from 0.2 to 0.3 and 0.5–0.6, respectively. Section title: Digestive system organs Educational score: 4.032930850982666 Domain: biomedical Document type: Study Language: en In the more recent study where Faster R-CNN was applied, the goal was to detect Marginal bone loss around dental implants . The system was evaluated using a dataset of 1670 images and was assessed in terms of sensitivity, specificity, diagnostic error rate, omission diagnostic rate, and positive predictive value. Kappa statistics were also compared between the system and dental clinicians, and the authors concluded that there was a high level of agreement between the automatic system and the clinicians. Section title: Digestive system organs Educational score: 3.93424129486084 Domain: biomedical Document type: Study Language: en On DetectNet, two studies are implemented, both applied to CR imaging. In one of these studies, the primary objective was the detection of root fractures . The study employed 300 images, containing a total of 330 root fractures. Out of these, 267 were successfully detected by the automatic approach, while twenty were falsely detected, resulting in a recall of 0.75, a precision of 0.93, and an F measure of 0.83. In a second study , DetectNet was applied to tooth detection. However, the authors acknowledged several limitations in their study, such as the small data set used, which was composed of only 75 training cases and 25 test cases. Section title: Digestive system organs Educational score: 2.374567985534668 Domain: biomedical Document type: Study Language: en Table 6 of Supplementary File 4 summarizes the data sets used and the type of study applied to images of digestive system organs. Section title: Digital pathology and microscopy Educational score: 4.069112777709961 Domain: biomedical Document type: Review Language: en The growing availability of large-scale gigapixel whole-slide images of tissue specimens has made digital pathology and microscopy a highly popular application area for deep learning techniques . These imaging modalities have been applied to various purposes, such as cell detection and malaria detection, among others, as seen in Table 11 . Table 11 Publications in which object detection algorithms are applied to digital pathology and microscopy images. a Table 11 Reference C Modality Organ Algorithm Task 93 PI Blood Faster R-CNN White blood cells detection 37 PI Tissue Faster R-CNN Glomerular detection in multistained human renal tissue 27 M Others Faster R-CNN Human intestinal organoid detection 16 M DNA Faster R-CNN Detection of DNA damage 29 PI Cervical cells YOLOv3 Cervical cell recognition 31 M Blood YOLO Malaria detection 73 PI Blood SSD Peripheral leukocyte recognition 53 PI Blood YOLO Blood cells detection 18 PI Blood YOLOv4 Blast cell detection 15 PI Blood RetinaNet Malaria detection 9 PI Brain tissue YOLOv3 Alzheimer's diagnose 22 PI Blood R-CNN Cells detection 15 M Others Faster R-CNN Detection of cellular organelles 12 PI Liver and Kidney tissue RetinaNet Diatom detection 24 M Others RetinaNet Pulmonary Hemosiderophages detection 8 M Others YOLOv4 Sperm detection a Overview of publications that apply object detection techniques on digital pathology and microscopy imaging, where PI stands for pathological imaging and M for microscopy imaging. In the header of the table, C stands for the number of citations. Section title: Digital pathology and microscopy Educational score: 4.189416885375977 Domain: biomedical Document type: Study Language: en The YOLO algorithm is regarded as the most prevalent among these studies, with six out of sixteen publications devoted to its application. In particular, three studies have explored the use of YOLO in the analysis of blood samples, specifically for detecting blood cells , blast cells , and malaria . In the first study, YOLO was trained and tested using 364 images with red blood cells, white blood cells, and platelets. The results showed an mAP of 82.58 % using the VGG16 backbone. Another study compared YOLOv3 with other popular models, including R-CNN, Fast R-CNN, Faster R-CNN, and SSD. Ultimately, the Faster R-CNN model arose as the winner with an mAP of 71 % and an inference time of 609 ms per image with VGG16. A third study on blood smears applied 14,700 images of peripheral leukocytes with eleven distinct categories, resulting in an mAP of 93.10 % for SSD and an inference time of 53 ms per image. Although YOLOv3 also performed well, it achieved only 92.10 % mAP. The blast cell detection study applied YOLOv4 on blood smears to aid in early leukaemia diagnosis and achieved an mAP of 95.57 % with an inference value of 50 FPS. In the malaria detection study with microscopic images, YOLOv3 and YOLOv4 were compared, with the last being the superior model, achieving an mAP of 96.32 % and an inference rate of 29.60 frames per second. Another study applied RetinaNet to detect malaria in 169 microscopic images for train and 130 for validation, with results reported in terms of sensitivity (0.92), specificity (0.90), accuracy (0.91), positive predicted value (0.92) and negative predicted value (0.90). Section title: Digital pathology and microscopy Educational score: 3.774824857711792 Domain: biomedical Document type: Study Language: en The YOLO object detector has also been used for cervical cell recognition and sperm detection . In the first study, YOLOv3 was applied to cervical cytology screening and achieved an mAP of 63.4 %, with 95.7 % of sensitivity and 67.8 % of specificity, on a data set of 12,909 images split into ten distinct categories. Section title: Digital pathology and microscopy Educational score: 2.5233852863311768 Domain: biomedical Document type: Study Language: en In the sperm study, with 125,000 objects annotated, YOLOv4 and other models such as YOLOv3, SSD, RetinaNet, and Faster R-CNN were compared, with YOLOv4 and SSD achieving the highest mAPs of 40.50 % and 41.98 %, respectively. However, YOLOv4 was selected due to a higher AP on the class impurity. Section title: Digital pathology and microscopy Educational score: 3.705867052078247 Domain: biomedical Document type: Study Language: en The last study on YOLO was on Alzheimer's diagnosis . The YOLOv3 was trained to detect five tau lesion types, including neuronal inclusions, neuritic plaques, tufted astrocytes, astrocytic plaques, and coiled bodies, on 2522 immunostained slides images of the motor cortex, achieving a maximum mAP of 74.4 %. Section title: Digital pathology and microscopy Educational score: 4.007204055786133 Domain: biomedical Document type: Study Language: en The Faster R-CNN algorithm was the second most used among the studies, appearing in five of the sixteen works available. In the study of glomerular detection in multistained human renal tissue , Faster R-CNN was used to automate the task and was trained on 33,000 images. The results were reported regarding recall, precision, and F measure for four different classes. Detection of human intestinal organoids was performed using Faster R-CNN on a data set of 1750 image patches with 14,242 ground truths, resulting in an mAP of 80 %. The authors argue that the quality of the automatic detection was similar to human capability but substantially faster. Another study, examining DNA damage detection , reported an mAP of 74 % with Faster R-CNN, while SSD and YOLOv3 achieved mAPs of 22 % and 30 %, respectively. The detection of cellular organelles was also considered using Faster R-CNN, with results reported solely for classification purposes. Section title: Digital pathology and microscopy Educational score: 4.034157752990723 Domain: biomedical Document type: Study Language: en The RetinaNet algorithm was used in three studies. Excluding the malaria study also reported in this subsection, this algorithm was used on liver and kidney tissue for diatom detection , and to detect Pulmonary Hemosiderophages . The first study obtained an average precision of 0.82 and an average recall of 0.88. In the second study, a RetinaNet was trained on seventeen completely annotated cytology whole slide images (WSI) containing 78,047 hemosiderophages and achieved an mAP of 66 % for the five classes approached, exceeding human expert concordance. Section title: Digital pathology and microscopy Educational score: 2.697073459625244 Domain: biomedical Document type: Study Language: en The R-CNN algorithm was only applied in one of the studies for cell detection , with a dataset of 600 training images and 100 testing images, and achieved an mAP of 82 %. Section title: Digital pathology and microscopy Educational score: 2.5317323207855225 Domain: biomedical Document type: Study Language: en Table 7 of Supplementary File 4 provides a summary of the data sets employed and the study methodologies applied to digital pathology and microscopy images. Section title: Eye Educational score: 2.166146755218506 Domain: biomedical Document type: Other Language: en The field of ophthalmologic imaging has experienced significant growth in recent years, but it is only in recent times that deep learning techniques have been harnessed for eye image analysis, with a majority of the studies centering around color fundus imaging . Section title: Eye Educational score: 3.5877206325531006 Domain: biomedical Document type: Study Language: en The qualitative analysis of the eighty studies revealed that only two of them have been applied to eye imaging, as expressed in Table 12 . Table 12 Publications in which object detection algorithms are applied to eye images. a Table 12 Reference C Modality Algorithm Task 29 FP Faster R-CNN Diabetes-Based Eye Disease Detection 12 FP YOLOv3 Lesion detection a Overview of publications that apply object detection techniques on eye imaging, where FP stands for fundus photography. In the header of the table, C stands for the number of citations. Section title: Eye Educational score: 4.004279613494873 Domain: biomedical Document type: Study Language: en The first study used FRCNN, a Keras implementation of Faster R-CNN, to identify diabetes-based eye diseases, including diabetic retinopathy, diabetic macular edema, and glaucoma . The authors achieved an mAP of 94 % with the proposed model, outperforming two other approaches, SSPnet and R-CNN, which achieved mAPs of 85 % and 89 %, respectively. In the second study , the authors used YOLOv3 to identify lesions in eye images. The results were reported in terms of average precision (0.08 and 0.52), average recall (0.86 and 0.91), and F1 Score (0.16 and 0.66) for both the number of lesions and the number of images analyzed. Section title: Eye Educational score: 2.1545674800872803 Domain: biomedical Document type: Study Language: en Table 8 of Supplementary File 4 outlines the data sets used and the type of study applied to eye images. Section title: Other anatomical application areas Educational score: 3.882751703262329 Domain: biomedical Document type: Review Language: en This final subsection lists papers that address multiple applications, where the organ is not unique, and diverse applications that were not included in the previous subsections, as seen in Table 13 . Table 13 Publications in which object detection algorithms are applied to various topics. a Table 13 Reference C Modality Organ Algorithm Task 38 PH Foot Faster R-CNN Diabetic foot ulcers detection 37 PH Skin Mask R-CNN Melanoma detection 19 PH Skin Faster R-CNN Scalp health diagnosis 98 US Thyroid Faster R-CNN Thyroid papillary cancer detection 27 A Vessels Custom Intracranial aneurysm detection 10 CT Lymph nodes DetectNet Cervical lymph nodes detection 228 CT Several Faster R-CNN Lesion detection 54 CT Several Faster R-CNN Organ localization 20 US Several RFCN Region detection in Ultrasounds 25 E Several Fast R-CNN Wireless capsule endoscopy abnormal pattern detection 12 CT Several Mask R-CNN Lesion detection 22 E Several YOLO Upper gastrointestinal disease 26 MRI Several SSD Tuberculous and pyogenic spondylitis diagnosis a Overview of publications that apply object detection techniques with various purposes, where PH stands for photography, US for ultrasound, A for angiography, CT for computed tomography, E for endoscopy and MRI for magnetic resonance imaging. In the header of the table, C stands for the number of citations. Section title: Other anatomical application areas Educational score: 4.020745754241943 Domain: biomedical Document type: Study Language: en Seven of the thirteen studies analyzed in this subsection focus on object detection without specifying a particular anatomical area. For the lesion detection task, two studies have been carried out. The first study uses an algorithm based on Faster R-CNN and a dataset that was compiled with 23,735 lesions from 32,120 CT scans. The results show a sensitivity rate of 81.1 %, with five false positives per image. The second study proposes a universal lesion detection in CT imaging. The data set was created by merging images from data sets related to the liver, lymph nodes, and the entire body, resulting in a total of over 30,000 lesions. The average sensitivity of the proposed model was reported to be 47.6 %. Section title: Other anatomical application areas Educational score: 3.420290470123291 Domain: biomedical Document type: Study Language: en Lesions in various gastrointestinal regions, including the esophagus, stomach, pylorus, duodenum, and cardia, can be detected in gastroscopic images using YOLOV3 . The authors reported an mAP of 58.10 %. Additionally, this performance was compared against the of SSD and RetinaNet performances. Section title: Other anatomical application areas Educational score: 3.1571075916290283 Domain: biomedical Document type: Study Language: en Organ localization is the focus of a study where Faster R-CNN is used in CT imaging. In this work, the authors combined two datasets, one containing images of eleven body organs and the other containing images of twelve head organs, achieving an mAP of 73.01 % for the former and 84.78 % for the latter. Section title: Other anatomical application areas Educational score: 4.036813259124756 Domain: biomedical Document type: Study Language: en A study on region detection in ultrasounds employs Faster R-CNN, and its performance is compared against SSD and RFCN. This study diagnoses various diseases, including gallbladder stones and polyps, hydronephrosis, kidney stones, renal cysts, hemangiomas, and fatty liver. RFCN emerged as the top performer of the three models with an mAP of 78.7 %, followed closely by SSD with 76.7 % and Faster R-CNN with 75.4 %. Section title: Other anatomical application areas Educational score: 2.7699759006500244 Domain: biomedical Document type: Study Language: en Another study uses a Fast R-CNN based model on wireless capsule endoscopy abnormal pattern detection on more than 7000 annotated images. The final proposal achieved an mAP of 72.3 % . Section title: Other anatomical application areas Educational score: 3.882965087890625 Domain: biomedical Document type: Study Language: en In medical imaging, tuberculous and pyogenic spondylitis diagnosis is carried out using an SSD approach on MRI scans . The results of this study show that the model achieved an AUC of 0.80, with a sensitivity of 85 % and a specificity of 67.9 %. Section title: Other anatomical application areas Educational score: 3.3676111698150635 Domain: biomedical Document type: Study Language: en Additionally, the skin is also the subject of research in automatic detection systems. A study was conducted on melanoma detection using Mask R-CNN , to identify potential skin lesion areas and crop those areas for further analysis by a classifier algorithm. Another study analyzed scalp health and analyzed four distinct scalp hair symptoms . The results showed that Faster R-CNN achieved an mAP of 91.75 %, while SSD achieved an mAP of 87.16 %. Section title: Other anatomical application areas Educational score: 4.077913284301758 Domain: biomedical Document type: Study Language: en The detection of diabetic foot ulcers is assessed using Faster R-CNN on a photographic imaging dataset consisting of 2000 training images, 200 validation images, and 2000 testing images . In this study, the authors compare the performance of different algorithms in terms of mAP and other metrics, with Faster R-CNN achieving an mAP of 69.40 %, YOLOv3 an mAP of 65.60 %, Cascade DetNet an mAP of 63.94 %, YOLOv5 an mAP of 62.94 %, and EfficientDet an mAP of 56.94 %. Section title: Other anatomical application areas Educational score: 3.565962076187134 Domain: biomedical Document type: Study Language: en In CT imaging for patients with oral cancers, a DetectNet is utilized for cervical lymph node detection . The sensitivity for metastatic lymph nodes was identified as 73 %, and for non-metastatic lymph nodes, a sensitivity of 52.5 % was observed. Section title: Other anatomical application areas Educational score: 3.6560304164886475 Domain: biomedical Document type: Study Language: en For intracranial aneurysm detection on digital subtraction angiography, a custom object detection network is employed on vessel imaging . The proposed architecture attained an accuracy of 93 % and an AUC of 0.942. In this study, the proposed network is also compared to YOLOv3 and RetinaNet, both with lower performance results. Section title: Other anatomical application areas Educational score: 2.0374953746795654 Domain: biomedical Document type: Study Language: en Table 9 of Supplementary File 4 defines the data sets employed and the type of study applied to images of various topics. Section title: Principal results Educational score: 4.030577659606934 Domain: biomedical Document type: Study Language: en This study conducted a thorough bibliometric analysis of the relevant studies in the rapidly growing field of the application of object detection in medical image analysis. A PRISMA methodology was applied to identify 311 relevant publications in this field. Our research findings led us to understand that this is a recent area of study but one in which research is steadily increasing and has the potential to grow. Most of the publications were included in the research areas of Medicine, Computer Science, and Engineering and were published in top journals belonging to the first quartile. China, the United States, and Japan were the most productive countries, with the United States being the country with the highest level of international cooperation among these three countries. The majority of the authors with the highest number of citations were associated with only one published paper, and only six authors had five or more publications associated. In the authors’ keyword analysis, it was clear that concepts such as object detection, deep learning, artificial intelligence, and convolutional neural networks were prevalent, while in index keyword analysis, medical concepts were introduced with higher relevance. Section title: Principal results Educational score: 3.92108154296875 Domain: biomedical Document type: Study Language: en Despite the emergence of deep learning object detection methods in 2014, medical imaging only adopted this approach in 2018, when the algorithms had evolved and demonstrated improved performance capabilities, which is a high demand in the medical field. Regardless of its relatively late arrival in the field, the introduction of object detectors in medical imaging has been modest, and only thirteen studies were published in 2018. Nonetheless, our research findings indicate this is a rapidly developing study area. Four years later, in 2022, the number of publications has increased by more than ten times, demonstrating the significant growth of the field. Additionally, it can be understood that while the establishment of object detectors in the field was initially cautious, it quickly expanded into various medical imaging techniques and a diversity of anatomical application domains. Section title: Principal results Educational score: 3.9877965450286865 Domain: biomedical Document type: Review Language: en Taking into account the results obtained in the quantitative approach, several insights can be obtained concerning the application of object detection algorithms in medical imaging, namely. • This is a promising field of future research, weakly explored due to the late arrival of these techniques in the medical field. With the expansion and development of new and optimized frameworks of object detection, it is possible nowadays to obtain models with high performance, and with the demand of real-time detection needed for some specific fields of medicine, such as in endoscopies. • The publications in this area have a bi-discipline nature, being almost equally distributed between Medicine-related and Computer Science fields. This dual can bring some advantages, such as comprehensive solutions and interdisciplinary insights if research is done in cross-domain collaboration. When this collaboration is not applied, there is frequently a lack of consensus regarding the terminology used in both approaches, particularly on how to evaluate the performance of those models in a manner that facilitates comparison across studies and enables the identification of best approaches and procedures for specific cases and data sets. • China, the United States, and Japan stand as the most productive countries in this area, with the United States having the highest level of international cooperation among these countries. • When comparing the authors' keywords analysis with the index keyword analysis, the former has a prevalence of keywords related to the algorithms themselves, such as deep learning, object detection, and medical field. In the latter analysis, medical concepts were found to be of higher relevance, suggesting the growing interest in applying advanced computational techniques to solve medical challenges, with a high number of those articles published on journals medicine- oriented. Section title: Principal results Educational score: 3.9564361572265625 Domain: biomedical Document type: Study Language: en Of the 311 papers that were analyzed, 80 of them underwent a rigorous qualitative analysis from three distinct perspectives: the algorithms utilized, the imaging modalities employed, and the anatomical areas of application. The analysis results revealed that most of the publications employed Faster R-CNN and YOLO (regardless of their specific variant) in their research, indicating that both one-stage and two-stage object detectors play a significant role, depending on the trade-off between performance and training speed. For instance, in applications where real-time predictions are a demand, such as endoscopic exams, the authors prefer one-stage algorithms because they can make faster inferences on new images. It is clear, however, that there is no consensus on the best algorithm to use since its selection depends on several factors, such as the quantity of data available, the high need for precision versus the increased demand for inference speed, and even the particular characteristics of the objects of interest in the problem at hand. Section title: Principal results Educational score: 1.3596752882003784 Domain: other Document type: Other Language: en Additionally, the YOLO algorithm has gained relevance in recent years, with the various evolutions and developments across its multiple versions. However, there is still no consensus on the best algorithm to use, reinforcing the No Free Lunch Theorem concept, which states that all optimization algorithms perform identically on average when evaluated over the space of all possible problems. The strategy for one model surpassing another relies on the personalization and optimization of the algorithm for the particular task. Section title: Principal results Educational score: 3.554302453994751 Domain: biomedical Document type: Review Language: en With regards to medical imaging modalities, object detection algorithms have been applied to a wide variety of imaging techniques, including CR scans, pathology images, and endoscopic imaging, which are among the most commonly used, and this diversity is also reflected in the broad scope of anatomical areas to which these automatic systems have been applied. Nevertheless, object detection is also applied to CT scans, photography, MRI, ultrasound, fundus photo, and angiography imaging. Section title: Principal results Educational score: 3.418802499771118 Domain: biomedical Document type: Review Language: en Regarding the anatomical applications of object detection algorithms, digital pathology and microscopy imaging of tissues and blood smears are the most commonly explored. However, other areas are also frequently studied in this field, such as abdominal organs focusing on colon polyp detection, digestive system organs emphasizing tooth-related tasks, bone imaging for fracture identification, and breast CT scans for breast lesion detection. Section title: Principal results Educational score: 1.8739370107650757 Domain: biomedical Document type: Study Language: en In relation to the data sets employed in the various studies, a total of 125 data sets were used across the 80 studies under evaluation. Among these, 53 of them were classified as private, 63 as public, and nine were exclusively made available by the authors upon request. Section title: Principal results Educational score: 2.589002847671509 Domain: biomedical Document type: Study Language: en On average, these data sets contained approximately 17,111 samples. However, when comparing the sample sizes based on data availability, private data sets had a notably higher average of 23,619 samples, whereas public data sets averaged only 9773 samples. Looking at the median values, 50 % of private data sets contained at least 921 images, whereas public data sets had a significantly lower median of only 700 images. Section title: Principal results Educational score: 2.0702459812164307 Domain: biomedical Document type: Study Language: en Of the 125 data sets, the minimum number of samples available was seven, and the data set with the highest number of images contained 1,024,039 samples. Section title: Principal results Educational score: 3.950244188308716 Domain: biomedical Document type: Study Language: en In the studies evaluated, we also examined the proportion of samples used for each partition. As good practice, datasets should be divided into three non-overlapping partitions: train (to train the model), validation (to tune the hyperparameters of the model) and testing (to evaluate the model's performance on unseen data and assess generalization) . Several studies follow this best practice, often splitting the dataset into two or three partitions or using external datasets (an approach that is also effective in preventing overfitting and ensuring that the test set remains independent). Although this important detail is considered in several studies, it is not consistently applied, and the proportions of each partition vary significantly, indicating a lack of standardization. Section title: Principal results Educational score: 2.8615922927856445 Domain: biomedical Document type: Study Language: en Additionally, further aspects and techniques should be considered, particularly associated with the number of samples available in each study. In many studies, the small size of the dataset highlights the need to apply sampling techniques, such as k-fold cross-validation, to maximize the use of the available data and ensure more reliable evaluations . Section title: Principal results Educational score: 1.85872220993042 Domain: biomedical Document type: Study Language: en When considering whether the data sets were external or internal, out of those 125 data sets, 71 of them were categorized as external, while the remaining data were classified as internal. Section title: Principal results Educational score: 2.619051218032837 Domain: biomedical Document type: Study Language: en Regarding the type of study applied, the majority of the studies adopted a retrospective approach, encompassing 70 out of the 80 studies. Only three studies were prospective, and two exhibited a mixed nature. In five of the 80 studies, it was not clear whether the study was retrospective or not. Section title: Principal results Educational score: 1.8101669549942017 Domain: biomedical Document type: Study Language: en Some of the public data sets were used more than once in distinct studies, namely BCCD (n = 2), CBIS-DDSM (n = 2), DDSM (n = 3), DeepLesion (n = 2), Etis-Larib (n = 2) and INBreast (n = 5). Section title: Principal results Educational score: 3.8908369541168213 Domain: biomedical Document type: Study Language: en Based on these findings, it becomes evident that the research field could significantly benefit from increased public availability of data or making data accessible upon request. Such an initiative would consequently lead to improved performance in the models utilized within these studies. Furthermore, an evident gap exists in the lack of prospective studies in the conducted research. Encouraging prospective studies is crucial for comprehending the long-term effectiveness of the developed approaches and for collecting data with the specific purpose of implementing these techniques. This approach allows researchers to exercise control over the data collection methods, ensuring data quality, reliability, and minimizing bias. Moreover, the collected data can be customized to address specific research questions in development. Such an emphasis on customization can foster further improvements and innovations in object detection techniques applied to medical imaging. Section title: Principal results Educational score: 4.124387741088867 Domain: biomedical Document type: Review Language: en It is also important to note that some studies highlight the high capability of these automatic systems in detecting the objects of interest, sometimes with equal or higher performance than human specialists. • The choice of an object detection algorithm is, in most cases, independent of the task itself and the screening method. As an example, Faster R-CNN is applied to the detection of gastric lesions , abnormality detection on the liver , detection of distal radius fractures , stroke lesion detection , pulmonary tuberculosis detection , among others. Exceptions are made when those tasks demand predictions at a faster rate or in a real-time manner, such as in endoscopies, where one-stage algorithms are preferred, such as YOLO , SSD and RefineNet , in colon polyp detection. • There is no consensus on the best algorithm to use, as it depends on various factors, including available data, precision needs, and inference speed requirements. • Due to the bi-discipline nature of the publications mentioned above, the lack of agreement on the metrics used leads to a difficult task, if not impossible, to compare different approaches even when the same data set is used. This gap reveals the need in the research field to define ways of evaluating object detection results in an agreed manner, independently of the academic nature of the authors. Taking as an example the chest organ studies approached in this work, where pulmonary conditions such as COVID and tuberculosis are detected, some authors use precision and recall to evaluate their results on the object detection framework , while others focused on accuracy , and others on mAP , only in four distinct articles. In other tasks, other metrics are used, such as specificity , AUC ,F-measure , among others. • YOLO framework has gained importance during time, with prior versions of YOLO being associated with lower performance; however, latter versions achieve similar if not superior performance to two-stage algorithms, indicating that one-stage approaches tend to be preferred in the future. In 2018, only one study applied YOLO , and four studies implement Faster R-CNN . In 2022, YOLO studies were in the number of 6 , while the Faster R-CNN algorithm was only applied in two studies . This continuous advancement in algorithms reflects the evolving nature of the field and, consequently, the necessity of ongoing research and ad-hoc optimization for specific tasks. • Independently of the screening methods, object detection procedures can show satisfactory results, if not better equal or even higher than human specialists, as defended in Refs. , showing the versatility and potential impact of these systems in several medical fields. Section title: Principal results Educational score: 2.170414686203003 Domain: biomedical Document type: Other Language: en Considering these findings, it is reasonable to conclude that object detection in medical imaging is a rapidly developing research area, and this work can have a significant impact in helping to understand the direction of further research in this field. Section title: Limitations Educational score: 4.0247015953063965 Domain: biomedical Document type: Study Language: en Based on two widely used academic databases, the quantitative and qualitative analysis performed in this study has employed the PRISMA methodology to screen for relevant articles. However, due to the bi-disciplinary nature of the field of study, combining both medicine and computer science, it is apparent that each discipline utilizes distinct concepts and terminologies to address similar issues. One of the primary challenges in this study was to evaluate the performance of various models across different tasks. There is a lack of consensus among researchers on which metrics should be used to make such comparisons, with some advocating for the use of commonly accepted metrics such as Average Precision (AP) and mean Average Precision (mAP) , while others prefer to utilize alternative metrics such as sensitivity, specificity, and accuracy. Section title: Limitations Educational score: 4.0230889320373535 Domain: biomedical Document type: Study Language: en The search strategy used in this study was focused on the title, abstract, and keywords of the publications. However, it became evident that many medical-oriented studies did not include AI concepts as a primary topic, while computer science-oriented works did not focus on medical concepts. To capture a more comprehensive set of relevant publications, future research should consider adjusting the search queries to account for the different terminology used in both disciplines. Furthermore, a more extensive comparison between the developed work should be addressed, including important details such as the data sets used, the metrics used to evaluate the models and their corresponding performance, and the remarks of the most relevant publications. Additionally, the search was limited to articles and focused on English-language publications, which may have resulted in a biased selection. This filtering may have missed relevant studies conducted in different formats and languages. Furthermore, the quantitative analysis can be further explored, including topics such as subgroup analysis for different countries. Considering these limitations, future research should aim to broaden the search scope, exploring a wider range of relevant studies in various formats and languages. Section title: Limitations Educational score: 3.7544562816619873 Domain: biomedical Document type: Study Language: en Concerning the models and the data exposed in the publications under analysis, we acknowledge the significance of addressing safety and privacy concerns. While assuming that the journals publishing the analyzed studies adhere to strict procedures that take into account those concerns, our study did not comprehensively explore the potential safety and privacy implications associated with each individual study. This concern is of utmost importance and should be highlighted, taking into account the possible implications that these models can lead when applied in real-world situations. The guidelines and future work that this particular study can induce should always be accompanied by a thorough understanding of the safety and privacy characteristics of the baseline publications. The tools and techniques developed and investigated in these studies should be viewed as supplementary procedures able to reinforce the medical expertise rather than substitutes for it. Section title: CRediT authorship contribution statement Educational score: 1.0064617395401 Domain: other Document type: Other Language: en Carina Albuquerque: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Roberto Henriques: Writing – review & editing, Validation, Supervision, Conceptualization. Mauro Castelli: Writing – review & editing, Validation, Supervision, Conceptualization. Section title: Data availability Educational score: 1.1044012308120728 Domain: other Document type: Other Language: en Data sharing is not applicable to this article as no data sets were generated or analyzed during this study. Section title: Declaration of generative AI in scientific writing Educational score: 0.8821380734443665 Domain: other Document type: Other Language: en The authors declare no usage of generative AI in scientific writing. Section title: Declaration of competing interest Educational score: 0.9818207025527954 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Other | biomedical | en | 0.999997 |
PMC11699423 | Section title: Introduction Educational score: 3.331671714782715 Domain: biomedical Document type: Other Language: en Medicinal plants have been an essential component of conventional health care around the world for ages since Ayurveda . According to these natural resources provide a wide range of chemicals that are bioactive with a variety of therapeutic potential. Tinospora cordifolia , often called Amrita or Guduchi, holds a great significance in Ayurveda owing to the presence of various secondary metabolites . Giloy leaves have historically been utilized to cure a variety of ailments, such as diabetes, rheumatism, jaundice, and skin diseases . Because of its historical use and the increasing popularity of natural therapies, scientists are looking into the possible therapeutic uses of giloy leaves and the underlying mechanisms behind their medicinal action . Section title: Introduction Educational score: 1.6399253606796265 Domain: biomedical Document type: Other Language: en Extract of the giloy leaves helps to alleviate the digestive ailments such as acidosis, gastroenteritis, parasitic infections, lack of appetite, stomach discomfort, extreme thirst, and nausea, as well as liver-related issues like hepatitis . Section title: Introduction Educational score: 2.1579642295837402 Domain: biomedical Document type: Other Language: en The latest study indicates that giloy leaves immunomodulatory capabilities may be associated with its numerous potential uses. These characteristics may strengthen the immunological response within the body, providing a natural means of promoting general well-being . Despite having a wide range of uses in traditional medicine, the immunomodulatory effects of giloy leaves is less known . Section title: Introduction Educational score: 4.0325398445129395 Domain: biomedical Document type: Review Language: en This review aims to reduce this gap by thoroughly analyzing immunomodulatory potential of giloy leaves. The research findings about its capacity to regulate the immune system, with particular attention to important pathways such as NF-κB signalling, inflammatory regulation, induction of apoptosis, phagocytosis stimulation, and interleukin modulation. Giloy leaves have been used in the creation of functional food products by comprehending these processes. Through the integration of contemporary scientific findings with traditional wisdom, our goal is to provide insight into the immunomodulatory mechanisms of Giloy leaves and their potential to enhance overall health and well-being. Then, by utilizing this knowledge, innovative giloy leaves-based functional food products can be made, bridging the gap between conventional wisdom and current medical developments. Section title: Identifying relevant research Educational score: 4.036944389343262 Domain: biomedical Document type: Study Language: en A systematic search of significant electronic databases such as PubMed, Scopus, and Google Scholar has been done to thoroughly examine the immunomodulatory activities of Giloy ( Tinospora cordifolia ) leaves. The search strategy included various terms that addressed multiple aspects of immune system regulation and prospective uses. Terms such as “ Tinospora cordifolia ” or “Giloy leaves” were used here, along with terms on general immunomodulatory effects, cellular mechanisms of the immune response, and product development, such as “cytokines,” “macrophages,” and “natural killer cells,” as well as terms related to immune system function and anti-inflammatory properties. This comprehensive strategy intended to collect all relevant data on how Tinospora cordifolia affects the immune system and to investigate the potential use of giloy leaves in the development of future functional food products. Section title: Inclusion and exclusion approach Educational score: 3.9552927017211914 Domain: biomedical Document type: Review Language: en The review process involves a two-tiered selection procedure with a primary focus on research that clarifies the mechanisms of action by which giloy ( Tinospora cordifolia ) leaves affect the immune system. The featured research examines how Tinospora cordifolia affects immune cells or function in animal models or in vitro (lab-based), is published in scholarly journals that undergo peer review, and provides clear evidence of Tinospora cordifolia's action mechanisms on immunological function, including the involvement of specific bioactive chemicals extracted from its leaves. Section title: Inclusion and exclusion approach Educational score: 2.873206377029419 Domain: biomedical Document type: Study Language: en The research will focus on the utilization of Tinospora cordifolia in value-added products, particularly in functional foods, supplements, and other immunomodulatory products. Only studies that are published in reputable journals focused on product development or peer-reviewed scientific journals were considered. These studies must provide evidence of the product's effectiveness and safety. Research that does not meet these criteria or focuses on different plant species was excluded. Section title: Taxonomic position of Tinospora cordifolia Educational score: 3.740023374557495 Domain: biomedical Document type: Study Language: en Tinospora cordifolia is a member of the Menispermaceae family, which is part of the Ranunculales order in the Magnoliopsida class (commonly known as dicots) of the plant world. This classification includes it amid a wide range of blooming plants, while the Menispermaceae family is best recognized for its climbing vines and bushes. Tinospora cordifolia , often known as Guduchi or Gurjo, is an ancient medicinal plant from the Menispermaceae family of moonseeds . The Menispermaceae family is abundant in tropical lowland environments, with 70 genera and 450 species. Tinospora is one of the most common genera in the Menispermaceae family, with around 15 different species . Tinospora cordifolia is extensively widespread in Tropical India and may reach elevations of 1000 feet in South Asia, Indonesia, Philippines, Thailand, Myanmar, China, and Sri Lanka . Ayurvedic and folk medicine systems frequently employ this compound, which may be found in a wide range of soil types (from acidic to alkaline) and only needs a small amount of soil moisture . Section title: Plant description Educational score: 1.6633362770080566 Domain: other Document type: Other Language: en Tinospora cordifolia , is a big, glabrous, perennial, deciduous vine with papery bark and juicy branches that spread widely. It is extensively distributed in Sri Lanka, Myanmar, and India . Guduchi is indigenous to India's tropical areas, where it can survive in temperatures between 25 and 45 °C at elevations of up to 500 m. The leaves are heart-shaped, straightforward, and have a deep, vivid green hue. They have a broadly elliptical lamina that is 10–12 cm long and 8–15 cm broad, and they are alternating, stipulate, and whole . Section title: Plant description Educational score: 1.6598553657531738 Domain: other Document type: Other Language: en The veins of leaves are multicostate and reticulated. The stems' surface has a completely studded appearance of warty tubercles. The skin's surface displays longitudinal fissures, with stems that have a diameter of 3–8 mm and a length of 3–5 cm. Large lenticels that resemble rosettes grow out of the succulent bark, which also includes deep clefts and spots . The bark can be either grey or creamy white in hue. The branches give rise to aerial roots that are long and thread-like branches are either light greyish brown in hue or long and dingy white. Section title: Plant description Educational score: 1.8956410884857178 Domain: other Document type: Other Language: en Grown on auxiliary and terminal racemes, the tiny, greenish-yellow blooms are unisexual. Female flowers often form single inflorescences, while male flowers are grouped . Six sepals total, arranged freely in two sets of three each on each flower. In addition, six free, oval, membrane petals are smaller than the sepals.: Fruits develop in the winter (November) while flowers blossom in the summer (March to June) . Fruits have a fleshy, orange-red hue and are composed of one to three ovoid, smooth droplets arranged in an aggregate on a stout stalk that has a subterminal-style scar. The moonseed family (Menispermaceae) is characterized by its bent seeds and embryo. Different decorations may be seen on the endocarp, or inner layer of the fruit wall . Section title: Phytochemistry and nutritional composition Educational score: 4.031098365783691 Domain: biomedical Document type: Study Language: en Giloy leaves are a potent source of nutrients and other essential elements. It was found that dehydrated giloy leaves contained high levels of calcium, protein, iron, crude fiber and ash as depicted in Table 1 . All parts of Tinospora cordifolia , including leaves, stems, fruits, and roots, are used as functional foods. Tinospora cordifolia leaves are rich sources of nutrients, essential macronutrients, and micronutrients as shown in Table 1 . Table 1 Nutrient content of Tinospora cordifolia leaves per 100g . Table 1 Nutrients Fresh Dehydrated References Moisture % 31.36 9.64 [ , , ] Ash % 2.3 5.880 Carbohydrates (g) 3.34 7.53 Protein (g) 2.30 5.23 Fat (g) 0.36 1.05 Fibre (g) 11.321 52.295 Iron (g) 5.87 22.55 Calcium (g) 85.247 210 Vitamin C (mg) 56 16 Beta Carotene (μg) 303.7 428.5 Energy (Kcal) 88.64 240 Section title: Phytochemistry and nutritional composition Educational score: 2.3395018577575684 Domain: biomedical Document type: Other Language: en Giloy leaves are a potent source of bioactive compounds such as Alkaloids, Glycosides, Diterpenoids, and steroids as represented in Fig. 1 . Fig. 1 Bioactive components of Tinospora cordifolia (Giloy) leaves. Fig. 1 Section title: Immunomodulatory activity of giloy leaves Educational score: 4.242825508117676 Domain: biomedical Document type: Study Language: en The significant immunomodulatory qualities of Tinospora cordifolia leaves are attributed to a plethora of bioactive substances . These active ingredients include alkaloids (found primarily in the stem) and steroids (beta-sitosterol, d-sitosterol, and g-sitosterol), as well as glycosides (18-norclerodane glycosides, furanoid diterpene glycosides, and tinocordiside) and palmatine D, choline D, tinosporine, magnoflorine, tetrahydropalmatine, and isocolumbin) . Additional components found in its aerial sections include diterpenoid lactones (furanolactone, tinosporon, and columbin) as well as many other substances such cordifolioside A, 11-hydroxymuskatone, N-methyl-2-pyrrolidone, octacosanol, heptacosanol, and nonacosan-15-one . Interestingly, the alkaloid palmatine is also present in the root. RR1, a polysaccharide extracted from the leaves, has been found to possess immune stimulatory properties and has the potential to be used as an adjuvant to boost immunity . Additionally, the various bioactive ingredients present in giloy leaves affect the immune system by increasing cytokine synthesis, enhancing immune cell mitogenicity, and activating specialized immune cells like B cells and macrophages, which can trigger targeted immune responses , . Further research into the exact mechanisms of these active ingredients will help to clarify the immunomodulatory potential of giloy leaves. The various mechanisms through which bioactive compounds of leaves help in boosting the immunomodulatory properties of the body have been illustrated in Fig. 2 . Fig. .2 Immunomodulatory effects of giloy leaves. Fig. .2 Section title: Cellular and molecular mechanism of immunomodulation Educational score: 4.3732805252075195 Domain: biomedical Document type: Study Language: en Giloy leaf extract has a high concentration of bioactive components such as alkaloids, glycosides, and terpenoids, contributing to its well-known immunomodulatory activities. The leaf extract constitutes bioactive components that stimulate a variety of immune cells, including natural killer (NK) cells, B cells, and T cells, resulting in the generation of favourable immuno-stimulating cytokines [ , , ]. In addition, among HIV-positive patients, giloy leaves lower total leucocyte, neutrophil, and eosinophil counts . These immuno-stimulating effects are linked to several bioactive chemical compounds found in the leaf, including 11-hydroxymuskatone, N-methyl-2-pyrrolidone, cordifolioside A, magnoflorine, tinocordioside, and Synringin . These compounds specifically target immune cells present inside the human body such as B lymphocytes and macrophages, implying a regulated activation of the immune response . This increases haemoglobin levels and polymorphonuclear leukocytes, indicating an overall increase in immune response . These compounds are well known for their ability to promote a greater immune response by enhancing the proliferation and differentiation of lymphocytes. Thus, it increases total WBC, bone marrow cellularity and alpha esterase-positive cells in the marrow of bone which leads to an increase in humoral immune response. Some of the bioactive components appear to considerably raise IgG antibody levels, which are important components of the immunological response for complimentary inhibition of the pathway . Section title: Inflammation and immunity boosting Educational score: 4.0766215324401855 Domain: biomedical Document type: Review Language: en Inflammation is an immune system response that is necessary for healing, but it can be harmful if it is severe or chronic. Herbal medicines such as giloy leaves have long been utilized to treat inflammation, however, worries about potential side effects have been highlighted . It's important to note that while giloy ( Tinospora cordifolia ) has been used in Ayurvedic medicine for a long time, it should be used with caution due to potential negative effects, especially when taken in large amounts. Research indicates that exceeding the recommended dosage can lead to gastrointestinal issues such as nausea, upset stomach, and constipation . Some studies conducted by researchers exhibits potential hepatotoxic effects due to Giloy's influence on liver enzymes and metabolism . While the blood-sugar-lowering properties of giloy can be beneficial for diabetics, excessive use or concurrent use with blood-sugar-lowering medications can increase the risk of hypoglycemia (low blood sugar) . Finally, allergic reactions are a possibility, particularly for individuals sensitive to plants of the Menispermaceae family . These reactions may include skin rashes and swelling. Giloy leaf extract intake has been associated with the activation of phagocytic cells that aid in wound healing, as well as the acceleration of skin regeneration when applied topically to wounds and bruises. Giloy leaves' immunomodulatory properties, which have been explained by their bioactive chemical constituents such as alkaloids, glycosides, terpenoids, and polysaccharides, which contribute to their capacity to modulate immune responses and decrease inflammation [ , , ]. Section title: Inflammation and immunity boosting Educational score: 4.200625896453857 Domain: biomedical Document type: Study Language: en Tinospora cordifolia leaf extract encompasses bioactive chemical compounds that help to reduce inflammation and decrease immunological responses. The leaf extract contains a variety of metabolites, some of which have anti-inflammatory and antioxidant activities . A molecule known as 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8dione was discovered as a potential dual inhibitor of COX enzymes, which is critical for treating inflammation. This chemical compound has been demonstrated high binding energies against COX 1 and COX 2, showing its potential as a drug like molecule for inflammatory therapy . In addition, T. cordifolia leaf extract has been studied for in-vitro anti-inflammatory action and shown to decrease protein denaturation, a fundamental process in inflammation. These findings indicate that the chemicals in Tinospora cordifolia leaf extract produce anti-inflammatory actions and modify immunological responses by inhibiting COX and suppressing protein denaturation ,. Compounds in giloy leaves decrease inflammatory mediators, inhibit enzyme activity such as COX and alter immune cell function. Giloy leaves have the potential anti-inflammatory properties through a variety of pathways as shown in Fig. 3 about Inflammation and its action mechanism. More research will be required to determine their clinical benefits in controlling inflammatory diseases. Fig. 3 Lipopolysaccharide-induced inflammation and its attenuation by giloy leaf components through NF-kB pathway. Fig. 3 Section title: Modulation of NF-κB pathway Educational score: 4.144284248352051 Domain: biomedical Document type: Study Language: en Giloy leaves anti-inflammatory mechanisms that have been studied. According to studies giloy leaf extract prevented lipopolysaccharide (LPS)-stimulated macrophages from releasing prostaglandin E2 (PGE2) and nitric oxide (NO) . Another study reported that giloy leaf extract reduced the expression of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), two pro inflammatory cytokines in LPS-stimulated macrophages , . Studies demonstrated by that giloy leaf extract suppressed the activation of the transcription factor-kappa B (NF-κB) signalling pathway, which is crucial for the inflammatory response . Wilkinson showed that giloy leaf extract inhibited the activity of an enzyme produced by the human body cyclooxygenase-2 (COX-2), which plays a role in PGE2 synthesis . Section title: Modulation of NF-κB pathway Educational score: 3.934723138809204 Domain: biomedical Document type: Study Language: en These studies suggest that giloy leaves have multiple anti-inflammatory mechanisms, including the inhibition of NO and PGE2 production, the reduction of pro-inflammatory cytokine expression, and the suppression of NF-κB and COX-2 activity. These mechanisms make giloy leaves a promising natural anti-inflammatory agent with potential uses in the treatment of a variety of inflammatory conditions, which has been illustrated in Fig. 3 . Section title: Regulation of apoptosis in immune cells Educational score: 4.462732315063477 Domain: biomedical Document type: Study Language: en Apoptosis is a process which helps in regulating the population of immune cells. For instance, after an immune response, excess immune cells may undergo apoptosis to prevent overactivity of the immune system. Apoptosis is used as a mechanism to eliminate infected cells . When a cell is infected with a virus or bacteria and gets damaged or destroyed beyond repair, it can trigger apoptosis to prevent the spread of the infection , . Some of the bioactive compounds of giloy leaves such as aporphine, magnoflorine, palmatine, tinocordiside, and cordifolioside -A, have demonstrated potential as anti-cancer agents . They induce apoptosis and inhibit the growth of cancer cells, suggesting their significance in cancer therapy. Cancer cells undergo apoptosis, a process of planned cell death, when exposed to giloy leaf extract. It triggers nuclear condensation, the production of apoptotic bodies, and the activation of the essential apoptotic enzyme caspase-3 . It also causes apoptosis in cancer cells by suppressing anti-apoptotic genes like Bcl-2 and upregulating pro-apoptotic genes like Bax (Bcl-2 associated X protein). Giloy leaf extract effectively inhibits cancer cell development by interfering with the G1 phase of the cell cycle , . Cell division and DNA replication depend on this stage. Giloy leaf extract stops the cell cycle at this point, which stops cancer cells from proliferating and growing. Nitric oxide (NO) generation is enhanced by the stimulation of immune cells, specifically macrophages, by giloy leaf extract. NO causes cancer cells to become cytotoxic, leading to their destruction , . Section title: Antioxidant Educational score: 4.154384613037109 Domain: biomedical Document type: Study Language: en Giloy leaves contain several polyphenolic chemicals, including tannins, magnoflorine, jatrorrhizine, tembetarine, tinosporine, isocolumbin, palmatine, and tetrahydropalmatine. These substances are good candidates for antioxidant therapy because of their demonstrated potent antioxidant properties . The giloy leaves extract's antioxidant capacity employs a range of in vitro tests. The extract exhibited noteworthy efficacy against DPPH, hydroxyl, and nitric oxide radicals, as demonstrated by the obtained results. The existence of energy-producing polyphenolic compounds that fight off free radicals and stop oxidative damage is the cause of this waste activity , . The antioxidant activity of giloy leaf extracts in human lymphocytes against oxidative stress - driven through DNA damage. Studies have indicated that these extracts can mitigate DNA damage resulting from exposure to hydrogen peroxide, which is a strong inducer of oxidative stress . Section title: Antioxidant Educational score: 4.003392696380615 Domain: biomedical Document type: Study Language: en This protection is due to the antioxidant capabilities of giloy leaves polyphenols, which remove free radicals and prevent DNA oxidation . The effect of giloy leaf extract on oxidative stress and antioxidant state in rats with experimental liver injury . Studies indicate that these extracts can enhance Antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase as well as lower oxidative stress markers like malondialdehyde . According to these results, giloy leaf extract may shield the liver from oxidative stress and liver damage . The majority of the research indicates that the polyphenols present in giloy leaves have significant antioxidant properties and can be used to treat a variety of oxidative disorders associated with stress using antioxidant-based therapy. Section title: Interleukin modulation Educational score: 4.124162673950195 Domain: biomedical Document type: Study Language: en The immune-modulatory function of Tinospora cordifolia is associated with its bioactive compounds such as terpenes, glycosides, alkaloids, steroids, flavonoids, and polysaccharides . IL-17, Th17 Cells, and the JAK-STAT Pathway interplay is crucial for understanding the potential immunomodulatory activity of Giloy leaf extract ( Tinospora Cordiofolia ). Proinflammatory cytokines like interleukin-17 (IL-17) are released by Th17 cells among other immune cells . It is essential for protecting the body from infections, especially those caused by fungus and extracellular bacteria. Studies have shown that Autoimmune diseases and chronic inflammatory illnesses have been associated with elevated or dysregulated IL-17 generation . Section title: Interleukin modulation Educational score: 4.379013538360596 Domain: biomedical Document type: Study Language: en T helper 17 (Th17) cells are a subset of CD4 + T lymphocytes that primarily secrete IL-17. Although they may contribute to autoimmune disorders, they are necessary for immune-mediated defence from external infections . The differentiation and activation of diverse immune cells, including Th17 cells, are facilitated by the JAK-STAT pathway, a crucial signalling cascade . It involves the phosphorylation of Janus kinases (JAKs) by cytokines attaching to particular cell surface receptors. Following JAK activation, STAT proteins (Signal Transducers and Activators of Transcription) relocate to the nucleus and regulate the expression of genes required for Th17 development and synthesis of IL17 . Section title: Interleukin modulation Educational score: 4.117612838745117 Domain: biomedical Document type: Study Language: en Studies have shown that Tinospora cordifolia extract can reduce the amount of IL-17 generating cells in CD4 + T cells developed under Th17-polarizing circumstances suggesting an immunomodulatory effect . Another study discovered that the water-soluble extract of Tinospora cordifolia improves macrophage phagocytic capabilities and significantly increases nitric oxide generation by stimulating splenocytes and macrophages at a dose of 1 mg/kg Research found that 100 μg/ml of Tinospora cordifolia extract caused 90 % cytotoxicity in B16F10 murine melanoma cells after 72 h of treatment . Section title: Interleukin modulation Educational score: 4.0708208084106445 Domain: biomedical Document type: Study Language: en Another research identified a polysaccharide from Tinospora cordifolia, RR1, which has been demonstrated to have innate immune stimulatory capabilities and can offer adjuvant-like action in the formation of a Th1-type immune response to an antigen , . To put it in simple terms, Tinospora cordifolia activates the immune system by influencing cytokine synthesis, mitogenicity, and immune-effector cell activation, as well as enhancing phagocytic power and nitric oxide generation in macrophages . The specific mechanism of interleukin modulation by which Tinospora cordifolia extract produces these benefits is unknown and will require more investigation and study. Section title: Application of value-added products Educational score: 4.177431583404541 Domain: biomedical Document type: Study Language: en Research on the uses of giloy ( Tinospora cordifolia) leaf extract in various value-added products has revealed its significant immune-boosting properties and health benefits. Giloy extracts have been incorporated in different forms such as powder, fresh leaves, and stem juice to enhance the nutritional value and taste of the products which has been shown in detail in Table 2 , Table 3 . For instance, a combination of giloy leaf powder, besan, wheat flour, and adusa leaves was utilized to produce biscuits and sev, resulting in nutrient-rich products confirmed through tests like DPPH and Folin-Ciocalteu . The phytochemicals present in giloy have been found to have a positive impact on the immune system, leading to increased consumption of homemade kadha (a herbal concoction) containing fresh giloy leaves and other ingredients during the COVID-19 pandemic . Moreover, products like value-added cookies made with giloy and tulsi powders, herbal squash made with giloy leaf extract and pineapple juice, and herbal lassi made with giloy stem juice have displayed enhanced nutritional properties . Table 2 Immunomodulatory giloy leaves-based value-added products. Table 2 S. No. Objective of study Value added Product Overview of Study Reference 1. Evaluation of Herbal Leaves for the Development of Value-Added Food Product Biscuit and Sev In this study, the giloy leaves were used to make value-added products such as biscuits and sev. The greatest method to add medicinal plants' nutritional advantages to humans' everyday diets to help fight degenerative illnesses is to provide value to food items by including them. It shows that the nutrients in these products can help in the fight against diseases and improve immunity. 2. Consumption of natural products and Ayurvedic decoctions “Kadha” as immunity-boosting measures during the spread of COVID-19 in Delhi Ayurvedic Decoction “Kadha” This study aimed to explore the prevalence of consumption of natural products and Ayurvedic decoctions “kadha” as immunity-boosting measures during the initial phase of the COVID-19 pandemic. Around 540 responses were taken through an online survey which shows the usage of immunity-boosting measures and Ayurvedic decoctions “kadha” among the adult residents of Delhi belonging to different age groups was effective. 3. Development of Giloy leaves-based Herbal Squash incorporated with Pineapple Giloy leaves based Herbal Pineapple Squash This research work was an effort in the direction of producing value-added instant beverages. Due to the functional and nutritional goodness of both plants, it can be further exploited in the development of healthy beverage products to develop giloy leaves-based herbal squash incorporated with pineapple and to determine its physical and biochemical properties. 4. Development of value-added cookies supplemented with giloy leaves and Tulsi powder Cookies with giloy leaves and tulsi powder In this study, giloy leaves powder was utilized in place of whole wheat flour to create herbal biscuits. When preparing herbal cookies, whole wheat flour partially replaces the powdered tulsi leaves and giloy leaves stems. Table 3 Traditional Giloy leaves Based Value-added Products. Table 3 S. No. Objective of study Value-added Product Overview of Study Reference 1. Development of herbal lassi using giloy leaves stem juice Herbal lassi Based on the current study, giloy leaves may be utilized to successfully make herbal lassi. Based on microbiological characteristics, the produced lassi was deemed safe for consumption. Natural antioxidants, such as giloy leaves, effectively lower the risk of heart disease, cancer, and many inflammatory processes, and have a positive impact on cardiovascular illnesses. It also strengthens immunity. 2. Development of Antioxidant-Rich Herbal Tea Bags Herbal Tea Bags with Giloy leaves This study was conducted to develop herbal tea bags using giloy leaves and different herbs which can provide antioxidant properties, boost immunity and also enhance the efficacy of white blood cells which helps in fighting against infections and bacteria-causing diseases. 3. Value Addition and Fortification in NonCentrifugal Sugar (Jaggery): A Potential Source of Functional and Nutraceutical Foods Jaggery fortified with giloy leaves. Jaggery is classified as a nutraceutical since it contains a range of vital amino acids, antioxidants, phenolics, minerals (calcium, phosphorus, iron), and vitamins. Jaggery is a more natural source of nutrients for health benefits and might be utilized as a healthier nutritional option for white sugar. Giloy leaves are incorporated as a health-supporting herb to generate even superior antioxidant, detoxifier, digestive, and immune booster products with potential functional and nutraceutical value. 4. Formulation of Herbal Candies Containing Giloy Leaves Satva: A Nutritious and Palatable Herbal Confectionery Option Herbal Candies Containing Giloy leaves The addition of giloy leaves satva in confections shows potential for improving immunity as it fights free radicals and boosts the body's mechanisms for defence against infections. It offers an intriguing discipline of study and innovation, providing a natural and pleasant method for immunomodulation 5. Sensory Evaluation of Laddu Enriched with Giloy leaves ( Tinospora Cordifolia) - Iron-Rich Powder Laddu Enriched with Giloy leaves The purpose of this study is to formulate and establish a standard giloy leaves laddu for people with anaemia and to determine its level of acceptability. The goal is to make a healthy laddu and put giloy leaves powder into it. 6. A study on incorporation of giloy leaves for the development of shelf-stable goat milk-based functional beverage Goat milk incorporated with giloy leaves By combining debittered giloy leaf juice with goat milk, a study was carried out to develop a shelf-stable giloy leaves goat milk beverage. The product profile was examined in depth, including its proximate composition, bioactive qualities, sensory, rheological, and structural characteristics. The addition of giloy leaves in this creates a functional beverage with a longer shelf life that targets health issues and boosts immunity. Section title: Application of value-added products Educational score: 2.558490753173828 Domain: biomedical Document type: Other Language: en The research has shown that giloy can be used in a plethora of products. The sensory qualities of giloy leaves, tulsi, and ginger herbal tea bags were optimised . Additionally, herbal candies made from giloy stems juice and sugar offer a nutritious and nutrient-dense option . Fortified jaggery with giloy leaves powder has shown enhanced nutritional profiles, and shelf-stable goat milk enriched with giloy stem juice has demonstrated extended shelf life and stability . Furthermore, giloy powder has been used in giloy laddus, where it is combined with other components . These diverse uses highlight Giloy's leaf extract potential to improve the nutritional value and health-promoting properties of food items. Section title: Comparison between traditional and modern value-added products Educational score: 2.0358636379241943 Domain: biomedical Document type: Other Language: en While traditional methods do not allow any combination of other ingredients, modern methods often combine giloy leaves with other herbs to enhance its effect for specific health concerns like stress, skin health, joint pain etc. Quality assurance varies in both traditional and modern value-added products, with modern manufacturing practices there is more focus on quality control and standardized formulations to ensure consistency and quality control in Giloy leaves-based products. Section title: Comparison between traditional and modern value-added products Educational score: 3.452991008758545 Domain: biomedical Document type: Study Language: en Different Varieties of modern value-added products are available nowadays, while traditional value-added products are very limited as shown in Table 3 , Table 4 . Table 3 , Table 4 illustrate the contrast between traditional and current applications of giloy leaves. Table 4 displays the Giloy-based products available in the market, while Table 3 outlines traditional uses. There are various value-added products in today’s modern era like capsules, juice, giloy leaves powder, giloy leaves extract and many more while traditional value-added products were limited to most consumers where churan, and kadha (juice blend) are common among all. Modern products are fortified with different vitamins and minerals to enhance the nutritional value of the product while traditional products are not fortified and produced as it is consumed. Modern giloy leaves products are widely available in pharmacies, local stores and online platforms, and are greatly accessible to consumers worldwide, which is not the same in the case of traditional value-added products. Modern giloy leaves products offer a convenient form of consumption, making it easier for individuals to consume and incorporate into their daily routines plus they are less time-consuming. Modern giloy leaves products have a high shelf life as they are preservatives with chemicals like sodium benzoate and potassium sorbate etc. or with various modern preserving methods. Traditional giloy leaves products typically use natural and herbal ingredients plus no chemicals were used resulting in less shelf life of the product. Traditional value-added products are made by methods that are passed down from generation to generation. Which involves manual labour, the use of natural ingredients and traditional equipment, which helps in preparing the perfect desired product. In Modern value-added products, advanced technologies and machines are used for production which works on standardized methods. Automatic machines, standard measurements, and quality control techniques are very common for preparing required products. Table 4 Commercially giloy leaves based on value-added products. Table 4 Product Name Brand Name Health Claims Ingredients Reference Capsules/Tablets rowhead 1. Guduchi Immunity Wellness Himalaya ● Strengthens Immunity ● Fights infections ● Supports detoxification ● Helps in increasing the performance of white blood cells Giloy leaves Stem Extract https://www. amazon. in/HimalayaWellness-Herbs-Guduchi-Immunity/dp/B00B8ROM1M 2. Giloy leaves Immunity Booster Zandu ● Protection Against Infections & Immunity Booster. ● Promotes Liver Health ● Stress Relief ● Anti-oxidant Compounds for Good Skin Health. Giloy leaves Stem Extract https://www. amazon. in/ZanduGuduchi-herbs-immunitywellness/dp/B07V6TPRQ8 3. Guduchi (Giloy leaves) Ghanbati Baidyanath ● Boost Immunity ● Reduces anxiety and improves mental strength. ● Rich in antioxidants and has anti-allergic, antifungal & anti-bacterial benefits. ● Maintain strength and vitality ● Aids pitta disorders & improves digestion. Giloy leaves Stem Extract. Ashwagandha Amla Gorakhmundi https://www. amazon. in/Baidyanath-Guduchi-Giloy leaves-GhanBati/dp/B08CY425YB 4. Divya Giloy leaves Ghanvati Patanjali ● Immune boosting ● Antioxidant properties and anti-inflammatory effects. ● Provide liver support ● Promote respiratory health. Giloy leaves Stem Extract https://www. amazon. com/Patanjali-Giloy leaves-Ghan-Vati-Tablets/dp/B07HWTJ2G3 5. Jiva Giloy leaves Jiva ● Boosts Immunity. ● Helps fight respiratory problems. ● Helps reduce joint pain. ● Improve digestion. ● Helps in detoxification. Giloy leaves Stem Extract https://www. amazon. in/Jiva-Giloy leaves-Capsule-Respiatory-Detoxification/dp/B093KTSXX8 Powder rowhead 1. Organic Giloy leaves/Guduchi Stem Powder CARMEL ORGANICS ● Boosting the immune system. ● Improving digestion. ● Reducing stress and anxiety. ● Controls blood sugar level. Giloy leaves stem powder https://www. amazon. in/CertifiedAmruthavalli-Tinospora-cordifolia-Preservative/dp/B08CHGD429 2. Natural Organic Guduchi Powder/Giloy leaves Powder MY HERB ●Aids in detoxification, increases antibodies, and stimulates vitality. ●Relieves stress and anxiety while also replenishing the body. ●Combats illnesses related to respiration. Demonstrates anti-inflammatory and antiarthritic activities Giloy leaves stem powder https://www. amazon. in/GuduchiPowder-Tinospora-cordifoliaMetabolism/dp/B08MTZPL1C 3. Giloy leaves Satva Baidyanath ●Useful in treating burning sensation in hands & feet, headache, metallic taste in mouth & excessive thirst. ●Enhances immunity of the body. Giloy leaves extract powder https://www. amazon. in/Baidyanath-Giloy leaves-Satwa-Pack-140/dp/B08KT8VB8P Teas Moringa & Giloy leaves Green Tea with Lemongrass Care ●Support to build an Immune system. ●Make bones and joints healthy. ●Help in losing weight. ●Enhance metabolism. Moringa Giloy leaves Licorice Ginger Lemongrass https://www. amazon. in/Moringa-Lemongrass-Immunity-Management-Ayurvedic/dp/B0836MJH7G 2. Giloy leaves Immunity Tea Jiva ●Effective immunity booster. ●Improve skin condition and reduce acne and pimples. ●Helps with seasonal colds and coughs. ●Helps to improve overall health & well-being. Giloy leaves stem powder https://www. amazon. in/Jiva-Giloy leaves-150gm-Pack-Immune-Tea/dp/B085S4ZFZQ 3. Giloy leaves Tea One Herb ●Immunity booster. ●Improves digestion. ●Helps reduce skin infections. ●Relieves chronic fever. ●Help regulate blood sugar levels. ●Boosts metabolism and aids weight management. Giloy leaves stem powder https://www. amazon. in/One-Herb-Ultimate-Immunity-Infections/dp/B08VNLYKTW 4. Ayurvedic Herbs Giloy leaves Green Herbal Tea Rishtpusht ●Immunity booster. ●Helps maintain a healthy heart and nervous system. ●Fight allergies and common infections. ●It improves blood circulation and eliminates harmful substances from the body. Giloy leaves Satva, Dry ginger, Clove, Cardamom, Lemon Basil, Green tea, Cumin seed, Sugar. https://www.amazon.in/Rishtpusht-Ayurvedic-Immunity-Booster-Natural/dp/B091YTDSK8 Juice rowhead 1. Giloy leaves juice Dabur ●Natural immunity booster ●Antioxidant properties ●Good for liver and skin health ●Natural detoxifier Giloy leaves extract https://www. amazon. in/DABUR-Giloy leaves-Neem-JuiceTulsi/dp/B087DJ9L3K 2. Giloy leaves tulsi juice. Vansaar ●Cleans and detox the gut ●Boost immunity & fight against common cough cold ●Antioxidant ●Anti-microbial ●Anti-bacterial ●Anti-inflammatory Giloy leaves stem Tulsi leaves https://www. amazon. in/Enhancing benefits-Handpicked-all-round-Immunity/dp/B07CYYPFFR 3. Giloy leaves juice Baidyanath ●Boost immunity ●Rich source of antioxidant ●Helps maintain a healthy heart and nervous system Giloy leaves extract https://www. amazon. in/Baidyanath-Boost-Immunity-Natural-Giloy leaves/dp/B087LTSC5M 4. Wild tulsi and giloy leaves juice Krishna’s herbal & ayurveda ●Helps regulate blood sugar levels. ●Boost metabolism & improve digestion ●Stimulates insulin secretion ●Helps manage weight Giloy leaves, Jamun, Bel Patra, Amla, Methi, Karela, Kutki, Vijaysar, Tulsi, Gudmaar Neem https://www. amazon. in/Krishnas-Herbal-Ayurveda-GeloyTulsi/dp/B07PLYBMMB 5. Giloy leaves vital AVG health organics ●Immunity booster ●Anti-pyretic ●Reduces inflammation & pain in the body ●Glucose metabolism Giloy leaves Ashwagandha Basil Amla berry https://www. amazon. in/AVGHealth-Organics-Ashwagandha-Premium/dp/B08CYCPTV6 Section title: Comparison between traditional and modern value-added products Educational score: 1.815172553062439 Domain: other Document type: Other Language: en These methods are often more precise and accurate as compared to traditional techniques which require a large amount of labor work. Traditional value-added products are higher in cost as compared to modern products as they have higher production costs which include labor-intensive processes, limited production scale, and the use of expensive and premium quality ingredients. As a result, they are often highly-priced. Modern products benefit from modern techniques, machines, high-scale production processes, and standardized measurement of ingredients. Which often leads to lower production costs and reasonable pricing. Section title: Comparison between traditional and modern value-added products Educational score: 1.3349570035934448 Domain: other Document type: Other Language: en Traditional value-added products also lack innovation as they prioritize traditional or hereditary methods which lack creativity and innovation, resulting in passing down the methods and recipes without any modifications. In the case of modern value-added products modifications are done with time, and new researches are done resulting in new recipes, new flavours, new techniques etc. Traditional production methods have limitations in organizing due to their reliance on manual labour and different techniques. Increasing production volumes is also challenging without compromising quality and effectiveness. Section title: Comparison between traditional and modern value-added products Educational score: 1.2064106464385986 Domain: other Document type: Other Language: en While Modern production methods are highly organized due to automation, Standardization and efficient manufacturing processes. Companies can easily adjust production volumes to meet different demands without sacrificing the quality and effectiveness of the product. Traditional products often target markets or consumers seeking authenticity, and local produce. They may be available through speciality stores or directly from the production area. Traditional products use less marketing while Modern products appeal to a broader consumer base due to their convenience, and wider distribution channels. They are often spread in supermarkets, online platforms, and large retail chains. Section title: Safety and toxicity Educational score: 3.7526981830596924 Domain: biomedical Document type: Review Language: en Tinospora cordifolia , also known as Giloy, is a revered medicinal plant in the Indian Ayurvedic tradition, renowned for its diverse therapeutic applications . However, recent studies have raised concerns about the potential hepatotoxic effects associated with the consumption of giloy leaves powder. The traditional use of giloy in Ayurveda has been well-established, with the plant being recognized for its anti-pyretic, anti-inflammatory, and hepatoprotective properties. However, a growing body of evidence suggests that prolonged or excessive consumption of Giloy leaves powder may lead to liver damage, a phenomenon known as hepatotoxicity. Section title: Safety and toxicity Educational score: 4.29325008392334 Domain: biomedical Document type: Study Language: en Several in vitro and animal studies have investigated the potential hepatotoxic effects of giloy. Researchers have observed that exposure to giloy extracts can result in increased lipid peroxidation, lactate dehydrogenase release, and a decline in glutathione-S-transferase activity, all of which are indicative of cellular stress and potential liver injury . Alkaloids (berberine, palmatine, and jatrorrhizine) and sinapic acid are believed to contribute to its hepatoprotective effects. Berberine, for instance, has been shown to reduce inflammation by inhibiting TNF-α-mediated proinflammatory pathways and nitrosative stress by suppressing iNOS activity . Beyond its hepatoprotective properties, Tinospora cordifolia exhibits a broad spectrum of biological activities, including anticancer, anti-inflammatory, antimicrobial, and antioxidant effects. It is generally considered safe at doses up to 2000 mg/kg. The pharmacological actions of Tinospora cordifolia are attributed to a diverse array of phytochemicals, including polyphenols, alkaloids, steroids, terpenoids, and glycosides . Hepatotoxicity, or liver damage, can be caused by a variety of factors, including the ingestion of toxic substances, overdose of medications, and even certain herbal preparations . Section title: Future prospective Educational score: 4.384851932525635 Domain: biomedical Document type: Review Language: en Although information on the immunomodulatory qualities of giloy leaves is growing, there is still a significant lack of understanding about the precise processes behind these effects. The precise molecular pathways and cellular interactions underlying the observed antioxidant activity, inflammation modulation, Th17 cell regulation, inhibition of the NF-κB pathway, interleukin modulation, and immunosuppressive and white blood cell-enhancing properties remain unclear. To understand the full therapeutic potential of giloy leaves, it is imperative to close this gap between data and intricate processes. Therefore, future research on several areas should be done including important signalling pathways like NF-κB, which have previously been connected to giloy leaves effects, should be the focus of research. These processes have to be clearly understood. Targeted therapies need a clearer comprehension of the molecular interactions that components of giloy leaves have with these pathways. Extensive research is required to determine the particular pathways through which giloy leaves constituents interact with other types of immune modulatory cells, such as Th17 cells, macrophages, and natural killer cells. Having a thorough understanding of how giloy leaves affect these cells' activation, differentiation, and function would help develop therapeutic applications. Investigate the possible benefits that giloy leaves may have when combined with other widely consumed foods that are high in vitamins, minerals, or antioxidants. This can help with dietary suggestions for enhancing immune function and for nutraceutical development. Examine the impacts of food matrices and determine which food ingredients can improve the bioavailability of the bioactive chemicals found in giloy leaves. This information can help formulate products in a way that maximizes health benefits. Contrast the immunomodulatory properties of giloy leaves with known immunomodulatory medications or natural compounds. This analysis can shed light on the distinct processes of giloy leaves and discover potential synergies in combination therapy. Through establishing a connection between current data and comprehensive research, scientists can fully realize the potential of giloy leaves as a medicinal agent. This all-encompassing strategy will open the door for focused therapies and enhanced formulations for particular immune-related ailments. In Future researchers should prioritize the development of standardized protocols for giloy leaves to ensure consistent quality and efficacy. Further research is necessary for negotiating the regulatory environment for giloy-based applications in order to commercialize the product. Section title: Conclusion Educational score: 3.8706648349761963 Domain: biomedical Document type: Study Language: en In conclusion, the study we did on the immunomodulatory properties of giloy leaves states that they have many health benefits such as boosting immunity, regulating apoptosis in immune cells and acting as antioxidants. They have active compounds such as alkaloids, polysaccharides, flavonoids, and terpenes present in them that help in immune cell activation which stops cell damage and immune cells like macrophages and B cells which help in inflammation reduction and stop cancer cells from increasing. Giloy leaves modulate interleukins and Th17 cells which helps in protection against infections and increasing immunity. Section title: Conclusion Educational score: 2.6496639251708984 Domain: biomedical Document type: Other Language: en Many modern products are now available in the market that are adapted, formulated and fortified from traditional uses of giloy leaves which can help in treating immunity-related disorders and other diseases. However, for better application and usage of this, further research and studies are required to understand its mechanisms which further helps in understanding its effects, applications and how they work with other compounds and treatments to enhance its use. Giloy leaves contain active ingredients that may be responsible for these benefits. Convenient options to include Giloy into daily routines are provided by modern products. It is crucial to recognize that there may be interactions between giloy leaves and specific drugs. Therefore, we advise speaking with a healthcare professional before incorporating giloy-based products into daily routine to ensure safe and effective use, particularly if you are taking any medications. Section title: Conclusion Educational score: 2.16033673286438 Domain: biomedical Document type: Other Language: en Overall, giloy leaves are said to be a natural immunomodulatory agent having many other properties such as antioxidant, hepatoprotective, anti-inflammatory and many more which required the researchers to go further to understand its properties fully. Section title: CRediT authorship contribution statement Educational score: 0.9205523729324341 Domain: other Document type: Other Language: en Jyoti Singh: Writing – review & editing, Writing – original draft, Conceptualization. Etika Saxena: Writing – original draft. Anjali Raj Chaudhary: Writing – review & editing. Mandeep Kaur: Writing – review & editing. Molly Salotra: Writing – original draft, Supervision. Prasad Rasane: Writing – review & editing, Writing – original draft. Sawinder Kaur: Writing – review & editing, Supervision. Sezai Ercisli: Writing – review & editing, Methodology, Investigation. Melekber Sulusoglu Durul: Writing – review & editing, Supervision, Formal analysis. Mehmet Ramazan Bozhuyuk: Writing – review & editing, Resources. Ahmet Hakan Urusan: Writing – review & editing, Software. Riaz Ullah: Writing – review & editing, Resources. Section title: Ethics declaration Educational score: 1.5258761644363403 Domain: biomedical Document type: Other Language: en This study did not require informed consent or review and approval by an ethical committee because it was a literature analysis that solely used data from published studies and did not involve any direct experimentation or studies on living beings. Section title: Data availability statement Educational score: 0.9718513488769531 Domain: other Document type: Other Language: en The research reported in the paper did not involve the utilization of any data. This article's accompanying data has not been added to any publicly accessible databases. Section title: Funding Educational score: 1.0467324256896973 Domain: other Document type: Other Language: en This study received no explicit financing from public, commercial, or non-profit organizations. Section title: Declaration of competing interest Educational score: 0.9818206429481506 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Other | biomedical | en | 0.999995 |
PMC11699432 | Section title: Introduction Educational score: 4.021886825561523 Domain: biomedical Document type: Review Language: en Malaria is a parasitic disease transmitted by Anopheles mosquitoes. In 2022, malaria caused 249 million cases and 608,000 deaths, with a high burden in Africa particularly for children under 5 years and pregnant women . The World Health Organization has put in place a global technical strategy targeting the reduction of malaria incidences and mortality rates by 90 % by 2030 with vector control as a cornerstone approach to reaching said target . Common malaria vector control strategies include the use of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) . However, malaria-transmitting mosquitoes, have developed resistance to public health insecticides , and selective pressure has shifted their biting behaviors to spaces, times, and alternative blood sources not protected by LLINs or IRS including feeding outdoors, early in the evening, and upon peri-domestic cattle . Section title: Introduction Educational score: 3.875563859939575 Domain: biomedical Document type: Other Language: en Ivermectin is an antiparasitic drug used in livestock and humans that works by blocking signal transmission in nervous systems, opening chloride channels causing hyperpolarization that results in flaccid paralysis and death in insects . In mammals, the drug has an excellent safety profile . Since ivermectin is effective against mosquitoes (when ingested via blood meal), but safe for mammals, it is under evaluation for mass drug administration (MDA) in humans and livestock as a potential complementary strategy to reduce malaria transmission [ , , , ]. Section title: Introduction Educational score: 3.3087992668151855 Domain: biomedical Document type: Study Language: en About 80 to 90 % of administered ivermectin and its metabolites in cattle are excreted in feces . Dung fauna such as flies, dung beetles, termites, and others feed on excrement and help decompose pats from livestock and return nutrients to soils . Previous studies in temperate settings have demonstrated that ivermectin and its metabolites affect dung fauna, causing delayed dung degradation , which could cause decreased pasture productivity and economic losses . Section title: Introduction Educational score: 3.3775687217712402 Domain: biomedical Document type: Study Language: en In contrast to temperate settings, the environmental effects of ivermectin and its metabolites in tropical settings are understudied . It is unclear if differences in conditions such as higher sun light exposure and different climate pattern at lower latitudes could mitigate the negative impact of ivermectin on dung fauna. If ivermectin MDA in cattle is determined to be effective for malaria control, it will be mostly applied in the tropical countries, making it important to understand how ivermectin treatments in cattle will affect dung fauna and degradation in tropical settings where malaria is prevalent. Section title: Introduction Educational score: 3.740208864212036 Domain: biomedical Document type: Study Language: en Here we studied the impact of ivermectin on dung fauna and degradation in a tropical dry savanna setting in southeastern Tanzania. Specifically, we conducted qualitative observations of dung fauna colonization, and quantified termite colonization, wet weight, water content, dry weight, organic matter, and larval abundance in dung. These data will support the assessment of the environmental impacts of the drug when used for MDA in cattle for malaria vector control. Section title: Materials & methods Educational score: 4.092047214508057 Domain: biomedical Document type: Study Language: en We conducted the study in Sagamaganga village (8°3′50.352″ S, 36°47′46.254″ E), located in Kilombero District within Kilombero Valley, Morogoro Region, southeastern Tanzania. This valley has a wet season from February to June and a dry season from July to January with average annual temperatures between 20 °C and 32 °C . The inhabitants in this valley are involved in human land use activities including livestock husbandry (e.g. cattle). However, the fieldwork was conducted during the dry season from July 2022 to October 2022 but monitored until March 2023. We selected 22 Tanzanian short horn zebu cattle from a herd that fed and grazed in the same area. These were allocated 1:1 to the control and ivermectin treatment groups using a random computer-generated sequence in R. Cattle ranged in age from 1 to 4 years, weighed between 90 and 351 kg, and had not been exposed to antiparasitic drugs in the preceding three months. A veterinarian checked the cattle before the study, qualifying them as physiologically mature. Similar studies have used cattle of different age ranges . We had 11 cattle (8 males and 3 females) in the control group and 11 ivermectin treated cattle (7 males and 4 females). The control group had weight of (186 ± 93.5) kg and age (2.0 ± 1.1) years while the ivermectin group weighed (148 ± 45.7) kg and aged (1.5 ± 0.5) years. We employed a randomized controlled experimental design to evaluate ivermectin's effect on dung degradation and fauna. Section title: Materials & methods Educational score: 2.770634889602661 Domain: biomedical Document type: Study Language: en We used IVOMEC® which is 1 % ivermectin injectable formulation (1 ml = 10 mg of active ingredient) commonly used in most rural villages for routine deworming of livestock including cattle. We administered ivermectin (200 μg/kg body weight) subcutaneously to 11 cattle on 8th July 2022. The cattle owners provided consent for the treatment and agreed to respect withdrawal periods for milking and/or slaughter. All procedures were conducted according to the local veterinary practices and approved by the Ifakara Health Institute Institutional Review Board , and the National Institute for Medical Research in Tanzania . The National Institute for Medical Research in Tanzania also granted permission to publish the research findings ( Ref.No.BD .242/437/01B/61). Section title: Dung degradation study Educational score: 2.888094425201416 Domain: biomedical Document type: Study Language: en For the dung degradation study, on each day, 22 replicate pats were prepared for each treatment group, making a total of 44 pats per day (22 control and 22 treated) and 220 pats in total for 5 collection days. This was achieved by collecting two fresh dung pats daily per treatment from each of the 22 cattle, mixing the two pats from each individual in the grazing field, and then homogenizing the mixture to create 1 kg samples. These homogenized pats were then randomly distributed in a designated field separate from the grazing area. We placed pats 2 m apart on a plastic net (8–10 mm mesh) to facilitate retrieval and covered them with a chicken wire mesh cage to exclude birds but not insects. Section title: Dung degradation study Educational score: 3.009854555130005 Domain: biomedical Document type: Study Language: en On days 15, 30 and 45 in the field, we first measured wet weight of each entire pat, and then randomly scooped 10 g from its surface for further analysis of organic matter content. These 10 g sub-samples were sent to the Ifakara Health Institute laboratory, where they were refrigerated for 5 to 7 days at 4 °C until further analysis. 110 treatment and 110 control samples were collected on those specific days. Termite infestation observations were also done on day 15 and 30 for each pat. Section title: Dung degradation study Educational score: 3.9275472164154053 Domain: biomedical Document type: Study Language: en For processing, 10 g sub-samples were homogenized by grinding them with a mortar and pestle. We put the 10 g sub-samples in a crucible which we previously weighed to the nearest 0.01 g. We then dried the sub-samples in the oven for 6 h at 110 °C to remove all moisture content . After drying, the sub-samples in the crucibles were cooled in a desiccator for 30 min before weighing to prevent moisture absorption. We calculated the percentage dry matter as the remaining weight of a sub-sample after drying . Section title: Dung degradation study Educational score: 3.3783600330352783 Domain: biomedical Document type: Study Language: en We determined organic matter in the dung pat using the loss on ignition method also known as ashing which helps separate mineral content (ash) from organic constituents that are susceptible to decomposition by: heating the oven dried samples from the previous step (sub samples heated at 110 °C that were cooled and weighed) in a pre-heated furnace at 500 °C overnight (12h) and then cooled them for 30 min before weighing. We then calculated organic matter content by determining percentages from the differences in weight of subsamples at 500 °C and at 110 °C . Section title: Dung degradation study Educational score: 2.5206663608551025 Domain: biomedical Document type: Study Language: en Percent LOI ( ploi ) was calculated as: Section title: Dung degradation study Educational score: 2.2883875370025635 Domain: biomedical Document type: Other Language: cy ploi = 100 × dwc 110 − dwc 500 dw 110 Section title: Dung degradation study Educational score: 2.2770912647247314 Domain: biomedical Document type: Other Language: en where dwc 110is the crucible and sample weight at 110 °C, dwc 500 is the crucible and sample. Section title: Dung degradation study Educational score: 2.367478370666504 Domain: biomedical Document type: Other Language: en Weight at 500 °C, and dwc 110is the original dry weight of the sample as obtained from the previous step. Section title: Dung degradation study Educational score: 2.410818338394165 Domain: biomedical Document type: Study Language: en From the sub-sample organic matter that we obtained above, we extrapolated to the full pat to obtain the total organic matter in the individual pats by multiplying wet weight of the sub-samples and the lost organic matter. Section title: Dung degradation study Educational score: 2.5617191791534424 Domain: biomedical Document type: Other Language: en Total organic matter = wet weight × ( dwc 110 − dwc 500 ) / 10 . Section title: Dung degradation study Educational score: 2.2202036380767822 Domain: biomedical Document type: Other Language: en where dwc 500 is the crucible and sample weight at 500 °C and dwc 500 is the crucible and. Section title: Dung degradation study Educational score: 2.1698126792907715 Domain: biomedical Document type: Other Language: en sample weight at 110 °C. Section title: Qualitative insect observations in the field Educational score: 4.009578704833984 Domain: biomedical Document type: Study Language: en We used standard methods to assess dung insect larval abundance [ , ]. In this case fresh dung for this experiment was collected on the same dates from the control and treated animals and counts performed on larvae. For the larval study, on day 3, 10 and 29 after ivermectin administration, we haphazardly collected dung from ivermectin and untreated cattle as they were deposited in the field. We then mixed the individual pats thoroughly. From the collected pats, we made 1 kg standardized treated and control composite pats ( N = 10 each) and placed them on a wire mesh in the field to aid recovery. Following a 1 day field exposure period, we collected pats and placed them in an emergence cage to recover insects . We recorded visitations of arthropods to the dung and documented beetle larval counts for the larval study and termite presence in the two treatments for the degradation study. Termites were only observed and recorded in the pats that were used in the dung degradation study. Section title: Insect abundance under semi-field dung conditions Educational score: 2.7525744438171387 Domain: biomedical Document type: Study Language: en Due to the hot temperature in the field, dung pats dried out quickly and we could not record adult insect emergence. To try and obtain insect data we modified the experiment to a semi-field system. Therefore, we enriched the samples with a second set of 10 (5 treated and 5 control) random pats on day 1, 2, 3 and 5 after administration and left the pats out to be colonized by insects for one day on a mesh and then transferred them to containers housed in a semi-field system that had a screen house enclosure. We counted and recorded the number of larvae found in dung pats on a weekly basis and observed their development until the pats dried up in the semi-field system. Section title: Statistical analysis Educational score: 4.098065376281738 Domain: biomedical Document type: Study Language: en We used R version 4.2.1 for all statistical analyses. To account for the longitudinal nature of our data with repeated measures of individual pats, we used mixed models to study the effect of ivermectin treatment on wet weight, water content, dry weight, organic matter, termite infestation and larval abundance. In these models with the three degradation outcomes as response variables, we added random intercepts for individual dung pats, and we added treatment × time interactions as fixed effects . Similarly, we used Poisson mixed models to study effects the ivermectin treatment on larval abundance. Here, we used larval counts as our response variable and accounted for the source of the data (sets 1 and 2, see above) with random intercepts and treatment, scaled days and scaled days squared as fixed effects. The latter was done to account for the initial increase and then decrease of new larvae emerging. All mixed models were implemented using the lme4 package . We used the emmeans package to extract estimated marginal means and compute standard errors and confidence intervals . Section title: Statistical analysis Educational score: 2.175949811935425 Domain: biomedical Document type: Study Language: en For the dung degradation study, all 220 pats (samples) were measured. Pat loss occurred on day 15 for 2 samples (one control and one ivermectin treated pat) due to termite infestation and on day 30 for 3 ivermectin samples because of oven malfunctioning. Because these failures were in a small part of the sample (i.e., < 5 %), and because they were also largely balanced between treatments, these pats were treated as missing in the data set. Section title: Statistical analysis Educational score: 1.719887375831604 Domain: biomedical Document type: Study Language: en To determine whether termite infestation affected the degradation results or not, we conducted a sensitivity analysis excluding any termite infested pats. The results were comparable to the full analysis set, and we therefore only show the results from the full analysis set. Section title: Qualitative observations of timing and presence of fauna Educational score: 2.452022075653076 Domain: other Document type: Study Language: en In the first few hours after dung was deposited in the field, we observed Diptera, mainly Calliphoridae and Muscidae. While dung pats formed a dried crust on the upper side, they remained moist on the underside where they were in contact with soil. Colonization was followed by Coleoptera mainly Scarabaeidae and Staphylinidae. These were present until pats dried 2 to 7 days after they were deposited in the field, and then they were followed by Isoptera. Hymenoptera (family of Formicidae, mostly safari ants Dorylus sp.), Collembola and Acari were also observed on the dry pats. Section title: Qualitative observations of timing and presence of fauna Educational score: 2.2326314449310303 Domain: biomedical Document type: Study Language: en As the pats dried, they became less attractive to most arthropods. Qualitatively, ivermectin treated pats also lost moisture more slowly as compared to control pats. Overall, we observed fewer Coleoptera larvae in ivermectin pats. Section title: Ivermectin treated pats were more likely to be infested with termites Educational score: 4.143221855163574 Domain: biomedical Document type: Study Language: en Ivermectin treated pats were significantly more likely to be infested by termites on day 15 (odds ratio: 23.2 (3.9 to 136.2); df = ∞; z = −3.48; p < 0.001), but not on day 30 (odds ratio: 4.7 (0.9 to 23.3); df = ∞; z = −1.89; p = 0.059; see Fig. 1 ). The point estimate for the probability of termite infestation in ivermectin pats decreased from 15 to 30 days ( p = 0.484), while the reverse occurred in control pats, though neither was significant ( p = 0.142). Fig. 1 Probability of termite infestation and 95 % confidence intervals after 15 and 30 days. Termites significantly preferred ivermectin treated pats compared to control pats on day 15, but not on day 30. ( n = 220). Fig. 1 Section title: Wet weight decreased more slowly in ivermectin treated pats, irrespective of termite presence Educational score: 4.165825843811035 Domain: biomedical Document type: Study Language: en Both treatment and control wet weights decreased from Day 0 to Day 30. By Day 15, there was a significant effect of treatment, as control pats were 13.4 % lighter than treatment pats (control pats = 291.9 g (275.7 to 308.1) versus treatment pats = 337.2 g (321.0 to 353.3), mean difference: −45.3 g (68.2 to −22.4); df = 435.1; t = −3.89; p < 0.001, Fig. 2 ). The difference was 12.4 % by Day 30 (control pats = 277.8 g (261.6 to 294.1) versus treatment pats = 317.0 g (300.7 to 333.2), mean difference: −39.2 g (−62.1 to −16.2); df = 437.8; t = −3.35; p < 0.001). The difference was also maintained to a smaller, non-statistically significant, extent (9.3 %) on Day 45 (control pats = 238.5 g (222.2 to 254.7) versus treatment pats = 262.9 g (246.7 to 279.2), mean difference: −24.5 g (−47.4 to −1.5); df = 437.8; t = −2.09; p = 0.037); see Fig. 2 ). Fig. 2 Mean wet weight and 95 % confidence intervals of whole pats over time. Overall, ivermectin treated pats displayed significantly slower wet weight decrease in comparison to control, with the largest effect occurring from day 0 to 15. ( n = 220). Fig. 2 Section title: Wet weight decreased more slowly in ivermectin treated pats, irrespective of termite presence Educational score: 4.074062824249268 Domain: biomedical Document type: Study Language: en We tested whether termites affected the wet weight decrease, but we did not find a relationship between termite presence and wet weight decrease on Day 15 (controls: 11.1 g (−7.8 to 30.0) ivermectin: 20.3 g (2.8 to 37.9); mean difference: −9.2 g (−35.0 to 16.6); df = 214; t = −0.7; p = 0.482). Section title: Water content decreased similarly in treatment and control pats Educational score: 3.9152517318725586 Domain: biomedical Document type: Study Language: en Water content decreased over the three time points for both treatment and control dung pats. The treatments did not differ with respect to water content on day 15 (control pats = 16.4 % (14.5 to 18.2) versus treatment pats = 15.7 % (13.9 to 17.6), mean difference: 0.6 % (−2.0 to 3.3); df = 643.9; t = 0.47; p = 0.639, day 30 (control pats = 13.6 % (11.7 to 15.5) versus treatment pats = 12.4 % (10.6 to 14.3), mean difference: 1.2 % (−1.5 to 3.8); df = 644.1; t = 0.88; p = 0.381) nor day 45 (control pats = 6.5 % (4.6 to 8.3) versus treatment pats = 6.0 % (4.1 to 7.9), mean difference: 0.5 % (−2.2 to 3.1); df = 643.9; t = 0.35; p = 0.724); see Fig. 3 A). Fig. 3 Change of percent water, dry weight and organic matter content of subsamples. A Water content (%) and 95 % confidence interval over time. Ivermectin did not affect water content on day 15, 30 and 45 and the ivermectin and control pats lose water content on similar trajectories.( n = 220) B Dry matter content (%) and 95 % confidence intervals over time. Ivermectin did not affect dry weight content on day 15, 30 and 45. These results show that ivermectin and control pats dry weight content increase similarly.(n = 220) C Organic matter content and 95 % confidence intervals over time. Ivermectin did not affect organic matter content, on day 15, 30 and 45. These results show that organic matter in sub-samples decreased similarly in ivermectin and control pats.(n = 220). Fig. 3 Section title: Dry weight content changes were similar for treatment and control pats Educational score: 4.106236457824707 Domain: biomedical Document type: Study Language: en Dry weight content of sub-samples increased over the three time points for both treatment and control dung pats. The control and ivermectin treated pats did not differ on day 15 control 83.6 % (81.8 to 85.5); ivermectin: 84.3 % (82.4 to 86.1); mean difference: −0.6 % (−3.3 to 2.0); df = 643.9; t = −0.47; p = 0.639), day 30 control: 86.4 % (84.5 to 88.3); ivermectin: 87.6 % (85.7 to 89.4); mean difference: −1.2 % (−3.8 to 1.5); df = 644.1; t = −0.88; p = 0.381), or day 45 control: 93.5 % (91.7 to 95.4); ivermectin: 94.0 % (92.1 to 95.9); mean difference: −0.5 % (−3.1 to 2.2); df = 643.9; t = −0.35; p = 0.724; see Fig. 3 B). Section title: Organic matter content decreased similarly between ivermectin-treated and control pats Educational score: 4.100374698638916 Domain: biomedical Document type: Study Language: en There was no effect of treatment on organic matter content. Ivermectin treated pats and control pats did not differ at day 15 (control: 59.9 % (57.2 to 62.6); ivermectin: 59.8 % (57.1 to 62.5); mean difference: 0.0 % (−3.8 to 3.8); df = 631.8; t = 0.02; p = 0.983), day 30 (control: 46.5 % (43.7 to 49.2); ivermectin: 47.1 % (44.4 to 49.8); mean difference: −0.6 (−4.5 to 3.2); df = 632.8; t = −0.31; p = 0.758), or day 45 (control: 45.6 % (42.9 to 48.3); ivermectin: 44.8 % (42.1 to 47.5); mean difference: 0.8 % (−3.0 to 4.6); df = 632.1; t = 0.41; p = 0.682; see Fig. 3 C). Section title: Total organic matter/pat decreased slower in ivermectin pats Educational score: 4.132226943969727 Domain: biomedical Document type: Study Language: en Both treatment and control total organic matter/pat, as interpolated from subsamples to whole pats, decreased from Day 0 to Day 30. There was a significant effect of treatment on total organic matter remaining in the pats, with control pats having approx. 13.2 % lower mass, or an estimated mass at 144.1 g (135.5 to 152.7) versus treatment pats at 166.1 g (157.5 to 174.7). This is a mean absolute difference of −22.0 g (−34.1 to −9.8; df = 563.5; t = −3.55; p < 0.001). By day 30 control pats had approx. 14.7 % less mass, or 106.8 g (98.1 to 115.5) remaining of controls, compared to 125.2 g (116.5 to 133.8) of ivermectin treated pats for a mean absolute difference of −18.4 g (−30.6 to −6.1; df = 568.1; t = −2.93; p = 0.003). On day 45, this was no longer significant (control pats approx. 6.2 % lower mass; control pats: 100.5 g (91.9 to 109.2); ivermectin pats: 107.1 g (98.5 to 115.7); mean difference: −6.6 g (−18.8 to 5.6); df = 564.9; t = −1.06; p = 0.291, Fig. 4 A). Fig. 4 A Mean mass of total organic matter/pat and 95 % confidence interval over time. Ivermectin affects the average mass of total organic matter on day 15, 30 and 45 in the dung pats.( n = 220) B Mean larvae count and 95 % confidence interval over time. There were fewer larvae in ivermectin treated pats in comparison to control. These results are significant and show that ivermectin negatively impacts larvae abundance. ( n = 40). Fig. 4 Section title: Ivermectin treated pats had fewer larvae compared to control pats Educational score: 4.126142978668213 Domain: biomedical Document type: Study Language: en We found a significant effect of ivermectin treatment on larvae counts, with the difference between treatment and control increasing over time . There was a significant effect of treatment on larvae population from day 1 to day 124, (mean counts on day 124 (95 %): control pats = 82.1 (78.1 to 86.2), treatment pats = 11.9 (11.3 to12.6), corresponding to an increase by 689 % (666 to 712); df = ∞; z = 112.92; p < 0.001). Section title: Discussion and conclusion Educational score: 4.0764641761779785 Domain: biomedical Document type: Study Language: en We determined the impact of ivermectin MDA in cattle on dung degradation and dung fauna in a tropical dry savanna setting in Tanzania, focusing on dung degradation in terms of wet weight, dry weight, loss of organic matter and larval abundance. In our study, ivermectin treated pats degraded more slowly in terms of wet weight and the total organic matter/pat . Additionally, we found that more beetle larvae lived in control than ivermectin treated pats. Originally, we aimed to count adults that would have emerged from dung in the two groups. Intense hot weather in the field led to a modification of the study to a semi-field setting for further observation. During the single day in the field, adult insects visited the pats. By the end of the day, the pats had started drying out, particularly on the surface, which had become crusty. Once moved to the semi-field setting, we periodically counted larvae in the pats. Interestingly, we observed only beetle larvae and no fly larvae. The intense weather conditions and the drying dung could likely be unsuitable for fly larvae survival. We continued counting larvae until the pats completely dried out. However, no adult insects emerged during the experiment. Section title: Discussion and conclusion Educational score: 2.421816825866699 Domain: biomedical Document type: Study Language: en Together, our results suggest that ivermectin hinders colonization of dung by some arthropods mainly Coleoptera, affecting their abundance, and ultimately slowing dung degradation in tropical savanna settings. Section title: Discussion and conclusion Educational score: 4.1353654861450195 Domain: biomedical Document type: Study Language: en Wet weight of whole pats decreased in the treatments by a difference of 14 % in 15 days, 12 % by day 30 and 9 % by day 45. Dry weight of sub-samples increased and water content decreased, presumably because of moisture loss associated with dry weather for our study area . There was no statistically significant treatment difference in the organic matter content among the sub-samples. However, extrapolating the organic matter to whole pats, we found significant differences in the first month of degradation, but not the period after that. This extrapolation has uncertainties as insects moved the pats and some left hollow spaces in the dung pats. In these cases, less dung or soil remained in the samples. Still, a sensitivity analysis restricted to pats without termite infestation upheld the main result. In summary, we find these data to be compelling evidence that ivermectin given to cattle decreases dung degradation in the field of a tropical environment . A number of studies have found similar results in the temperate settings . The similarity is somewhat surprising, as ivermectin degrades quickly in sunlight and metabolites like 3′′- O -demethylivermectin (3DI) go below the threshold for detection on the upper dung layer in a week's time post dung placement . Despite the higher solar irradiation at the lower latitudes, ivermectin and its metabolites must have had an effect on dung before their degradation in our study. Section title: Discussion and conclusion Educational score: 4.08332633972168 Domain: biomedical Document type: Study Language: en We observed termites throughout the dry season, corroborating previous observations that point to their ability to tolerate harsh dry conditions . Termites specifically add soil contents in dung during mound making facilitating microbial activity. Termites also help organic matter break into nutrients taken up by plants . Despite this important role of termites in dung degradation, there was no association between termite infestation and dung degradation, measured in terms of wet weight, in this study. Maybe such an effect would become evident after the 45 days that we allotted to our experiment. Surprisingly, termites preferentially associated with ivermectin-treated pats. This was a novel observation that echoes a similar report on dung beetles [ , , ]. Wardhough and Manon suggested that some volatile metabolites of ivermectin in the dung as well as changes in the gut flora of cattle caused by ivermectin may be involved in the bias to ivermectin-treated dung . However, a recent study found no evidence that dung undergoes direct changes in the volatile compounds releases or if there is a change in gut microbiota in cattle . In our study, given the overall lower rates of colonization by insects, perhaps the termites face less competition in the ivermectin- treated pats. Further research on the phenomenon is needed. We did not observe adult helminths in any dung pats. Section title: Discussion and conclusion Educational score: 4.149043560028076 Domain: biomedical Document type: Study Language: en While less obvious in our study, colonization of pats with other dung organisms may also be crucial. For example, beetles remove dung , and adult beetles consume the liquid portion of dung, but their survival seems to be unaffected by ivermectin with further evidence of past exposures not impacting the future ecosystem at the area of exposure . Although as dung organisms make their movements, they facilitate drying and decomposition . Less larval activity in ivermectin pats is problematic since larvae contribute to dung degradation by direct decomposition of organic matter, mixing, aerating, and breaking dung into smaller particles enabling microbial decomposition . The number of larvae in dung increases with time; however, in the field, their numbers quickly decreased as dung lost its moisture in the tropical dry season tropical setting , explaining their inability to survive . In our study, adult Diptera were only observed on the surface of dung pats for one day, when they were still moist on the top layer. Afterwards, conditions for Diptera development were not favorable . Adult beetles, on the other hand, seemed less affected, but the lower larvae counts in ivermectin treated pats in the semi-field experiment points to a reduction in the ability to produce viable offspring under these conditions [ , , ]. Section title: Discussion and conclusion Educational score: 4.101031303405762 Domain: biomedical Document type: Study Language: en We conducted our study during the dry season and loss of dung diversity due to dry conditions may exacerbate the effects of ivermectin during that time . On the other hand, increased ivermectin photodegradation may have lessened the impact of ivermectin treatments on dung degradation . There is a critical need to conduct similar studies during wet season in the tropics to rule out the dry condition effect on microbial and insect activity and investigate further the ivermectin effect during this period crucial to malaria spread. Furthermore, it is unknown what level of difference in dung degradation rate is environmentally relevant and may lead to downstream effects like pasture fouling due to prolonged presence of dung pats, particularly in the extensive livestock management systems found in tropical climates. Despite these limitations, our study represents an important first step if we want to evaluate the environmental impact of mass drug administration in cattle to curb the spread of malaria in humans. Section title: Discussion and conclusion Educational score: 4.01359224319458 Domain: biomedical Document type: Study Language: en In summary, results from tropical Tanzania add to the previous findings from the temperate region studies which demonstrate that ivermectin negatively affects dung degradation and larval development. Therefore, if ivermectin MDA in cattle proves to be an effective method for malaria control, its non-target environmental consequences should be further quantified and evaluated using a One Health approach to better account for the full costs and benefits of such a control strategy. Section title: Author contribution Educational score: 0.9184009432792664 Domain: other Document type: Other Language: en N. Regina Rabinovich, Carlos Chaccour, Kang Xia, Cassidy Rist, Roger Schürch, Carlyle Brewster, Issa N. Lyimo: Conceptualisation, Funding acquisition. Kang Xia, Cassidy Rist, Issa N. Lyimo, Roger Schürch: Supervision and oversight. Miriam Ruhinda, Felician Meza and Gerald Shija: Data collection. Miriam Ruhinda, Roger Schürch: Methodology, Data curation, Data analysis, Writing of original draft. All authors: Review & editing, approval of the final version. Section title: CRediT authorship contribution statement Educational score: 1.0361393690109253 Domain: other Document type: Other Language: en Miriam Ruhinda: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Formal analysis, Data curation. Kang Xia: Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Cassidy Rist: Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Gerald Shija: Writing – review & editing, Methodology, Investigation. Issa N. Lyimo: Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization. Felician Meza: Writing – review & editing, Investigation. Carlyle Brewster: Writing – review & editing, Methodology, Funding acquisition, Conceptualization. Carlos Chaccour: Writing – review & editing, Project administration, Funding acquisition, Conceptualization. N. Regina Rabinovich: Writing – review & editing, Project administration, Funding acquisition, Conceptualization. Roger Schürch: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Section title: Declaration of competing interest Educational score: 0.8710333704948425 Domain: other Document type: Other Language: en The authors declare no conflict of interest. | Review | biomedical | en | 0.999997 |
PMC11699457 | Section title: Introduction Educational score: 4.170616149902344 Domain: biomedical Document type: Clinical case Language: en The eyebrows are critical facial structures serving a multitude of functions. They play a significant role in facial recognition, emotional expression (sadness, surprise, anger), gender identification, and aesthetics. Additionally, they serve a protective role, shielding the eyes from sweat and minor injuries. 1 Eyebrow abnormalities can significantly impact a patient's confidence and quality of life. 2 Reconstructing an eyebrow defect presents a unique challenge in plastic surgery due to the location and characteristics of eyebrow hair. Existing techniques for eyebrow reconstruction range from simple closure to more complex procedures, including local flaps, scalp flaps, hair grafts, and composite grafts. 3 While pedicle superficial temporal artery flaps have been used successfully for large eyebrow defects, there is a lack of documented cases utilizing free superficial temporal artery flaps for this purpose. This report presents the first documented case of total eyebrow reconstruction using a free superficial temporal artery flap. This novel approach offers a potential solution for patients with large eyebrow defects, potentially surpassing the limitations of existing techniques. Section title: Case report Educational score: 3.4205150604248047 Domain: clinical Document type: Clinical case Language: en An 18-year-old female patient presented with a giant congenital melanocytic naevus involving her left eyebrow and temporal region . The naevus was excised in 2015 and reconstructed with a supercharged superficial cervical artery perforator flap harvested from the scapular region. The flap's blood supply was enhanced by anastomosing the circumflex scapular artery to the superficial temporal artery . Six months later, she desired eyebrow reconstruction. Figure 1 Pre-operative views: giant congenital melanocytic naevus. Figure 1 Figure 2 Post-tumour removal view: absence of left eyebrow. Figure 2 Section title: Case report Educational score: 3.271124839782715 Domain: biomedical Document type: Other Language: en For eyebrow reconstruction, a free flap based on the right superficial temporal artery was chosen. Doppler ultrasound was used to preoperatively map the right and left superficial temporal arteries for the flap pedicle and recipient vessels. The flap design considered the size and hair direction of the contralateral eyebrow. Section title: Case report Educational score: 3.9029784202575684 Domain: clinical Document type: Clinical case Language: en The surgery was performed under local anaesthesia with 3.5 times magnification. Lidocaine 2 % was injected subcutaneously and intradermally to both the donor and recipient sites prior to incision and was repeated every 45–60 min as needed for patient comfort. No general anaesthesia was required. The patient was placed in a supine position. The recipient site was prepared by exposing the left superficial temporal vessels. The planned anastomosis site and the required vascular pedicle length were determined. A skin and subcutaneous tissue flap was harvested from the right temporal region based on the frontal branch of the superficial temporal artery. The dissection aimed to preserve the underlying hair follicles. The lateral branches of the artery were ligated and divided. The vascular pedicle was approximately 10 cm in length. The flap was detached and transferred to the recipient site. The superficial temporal vessels of the flap were anastomosed in an end-to-end fashion to the left superficial temporal vessels using 9–0 Prolene sutures. The flap margins were meticulously sutured to the recipient eyebrow defect using 6–0 Prolene sutures . Haemostasis was confirmed, and the donor and recipient sites were closed in layers. A drainage tube was placed at the reconstruction site. The total surgical time was 2 h, and a total of 400 mg of lidocaine (20 ml) was used. Figure 3 Intraoperative views. Figure 3 Section title: Case report Educational score: 2.0779972076416016 Domain: clinical Document type: Clinical case Language: en The post-operative course was without any complications. The drainage tube was removed 16 h post-operatively, and the patient was discharged home. Complete flap survival with hair growth was observed. The reconstructed eyebrow showed good stability and the patient reported high satisfaction. The reconstructed eyebrow remained stable with hair growth at over five years post-operatively . Figure 4 Postoperative views. Figure 4 Section title: Discussion Educational score: 3.6963837146759033 Domain: clinical Document type: Clinical case Language: en This case report describes the successful reconstruction of a total eyebrow defect in an 18-year-old female patient following the removal of a giant congenital melanocytic naevus. While naevi are typically managed with excision, significant post-operative defects can negatively impact a patient's appearance and quality of life, particularly when facial structures like the eyebrow are involved. Section title: Discussion Educational score: 3.5106141567230225 Domain: biomedical Document type: Other Language: en Traditionally, eyebrow reconstruction employs a variety of techniques depending on the size and location of the defect. These techniques range from simple closure to more complex procedures like local flaps, scalp grafts, and follicular unit transplantation. 3 Selecting the most suitable approach involves careful consideration of factors like the defect size and colour, eyebrow orientation, and the patient's natural features. Balancing functional and aesthetic outcomes while minimizing donor site morbidity is also crucial. 3 Section title: Discussion Educational score: 3.8868510723114014 Domain: biomedical Document type: Review Language: en For partial eyebrow defects, local flaps harvested from the uninjured contralateral eyebrow remain the gold standard due to their colour and hair growth characteristics matching the recipient site. 4 However, recent decades have seen the rise of superficial temporal artery island flaps for eyebrow reconstruction. These flaps offer advantages like hair-bearing skin with appropriate texture and thickness. 5 , 6 Other authors recommended using the advancement V-Y flap with an orbicularis oculi-based pedicle for defects less than half of the eyebrow length and the superficial temporal artery island flap for defects exceeding half of the eyebrow length. 7 Section title: Discussion Educational score: 4.025519371032715 Domain: biomedical Document type: Review Language: en While effective, island flaps become unsuitable when the ipsilateral temporal region or blood vessels are compromised. 8 Free flaps offer a solution in such cases. While they can be technically demanding and require longer operative times compared to island flaps, they provide unmatched versatility and can be used even when the donor site has been damaged. 9 Notably, free superficial temporal artery flaps share the same structural and aesthetic properties as the recipient eyebrow, including hair growth characteristics. Additionally, the rich vascular supply of the superficial temporal artery allows for successful microsurgical anastomosis, enabling reconstruction under local anaesthesia with shorter hospitalization and recovery times compared to traditional free flaps. 9 While local anaesthesia is typically sufficient, the type of anaesthesia should be discussed with patients preoperatively. The amount of lidocaine administered should be carefully calculated to prevent potential toxicities. For patients with significant surgical anxiety, sedation or general anaesthesia may be considered. Section title: Discussion Educational score: 3.9067306518554688 Domain: clinical Document type: Clinical case Language: en This case report presents the first documented instance of total eyebrow reconstruction using a free superficial temporal artery flap. The successful outcome, with complete flap survival and hair growth, highlights the potential of this technique as a viable option for reconstructing large or complex eyebrow defects, especially when traditional methods are not feasible. Further studies with larger patient populations are warranted to validate the long-term efficacy and refine the surgical approach. Section title: Funding Educational score: 0.9338935613632202 Domain: other Document type: Other Language: it None. Section title: Patient consent Educational score: 1.121711015701294 Domain: clinical Document type: Clinical case Language: en The patient provided written consent for the use of her images in the paper. Section title: Ethical approval Educational score: 0.9488582015037537 Domain: other Document type: Other Language: en Not required. Section title: Declaration of competing interest Educational score: 0.8285962343215942 Domain: other Document type: Other Language: it None declared. | Clinical case | biomedical | en | 0.999997 |
PMC11699482 | Section title: Introduction Educational score: 4.1358747482299805 Domain: biomedical Document type: Review Language: en Chronic orofacial neuropathic pain can arise from damage to peripheral branches of the trigeminal nerve. Such injuries often result in striking changes in sensitivity to both tactile and noxious stimuli applied to the head and orofacial regions, as well as intense episodic or persistent pain . Trigeminal neuropathic pain is described as particularly distressing and intensely disabling, and most current treatments are largely ineffective over the long term . The complexity of neural changes triggered by damage to peripheral nerves underlies the significant failures in the long-term management of most neuropathic pain conditions. Preclinical models of neuropathic pain play an important role in simplifying this complexity and are contributing significantly to defining the neural and non-neural changes triggered by nerve damage. Section title: Introduction Educational score: 4.50435733795166 Domain: biomedical Document type: Study Language: en Transection, compression, or constriction of either maxillary ( infraorbital n. ) or mandibular ( inferior alveolar n. or mental n. ) branches of the trigeminal nerve or damage to the trigeminal ganglion in the rat have each provided clinically relevant models of trigeminal neuropathy . Injury of a peripheral nerve triggers immediate changes at the site of damage, an immediate inflammatory response is mounted. The damaged axons and their associated Schwann cells release inflammatory mediators such as NGF and TNF-⍺, and immune cells, usually circulating macrophages migrate to the site, attracted by monocyte chemoattractant protein-1 (MCP-1), followed by invasion of the disrupted structures by these cells. These macrophages are usually a mixture of both pro- and anti-inflammatory phenotypes . This process often results in an increased firing of the nerve both spontaneously and in response to activation of its sensory terminals . Once established, these processes trigger changes along both damaged and undamaged nerve fibres as they project towards their central nervous system targets, i.e., the chief sensory nucleus of the trigeminal, as well as along the trigeminal tract to the spinal nucleus (SpV), and the dorsal horn of the upper cervical spinal cord (UCC). Section title: Introduction Educational score: 4.4589691162109375 Domain: biomedical Document type: Study Language: en In response to injury to a branch of the trigeminal nerve, the ganglion also shows injury related changes, both resident and infiltrating macrophages are reported to accumulate within the division of the ganglion through which the damaged nerves pass, and where their damaged cell bodies reside . This macrophage invasion and proliferation is driven both by increased cytokines, for example chemokine C-C motif ligand 2 (CCL2) released from damaged ganglion cells, as well as by the barrages of spontaneous activity triggered by the damaged fibres . Satellite glial cells are reported to show activated states in some reports and begin to express gap junctions, specifically connexin-43, which facilitates glial-glial communication, the result of which drives increased activity in ganglionic cells . This increase in neural activity is also enhanced by the release of TNF-⍺ from the infiltrating macrophages . Section title: Introduction Educational score: 4.608028411865234 Domain: biomedical Document type: Study Language: en The ganglionic changes then drive further change in brainstem recipient regions specifically in the SpV and the UCC, a large portion of the SpV shares a similar laminar architecture to the UCC and is the region that receives most of the trigeminal nociceptive input. This region sends projections to higher brainstem and thalamic pain processing regions, including the parabrachial region, the midbrain periaqueductal gray matter (PAG), hypothalamus, amygdala, medial thalamus as well as the medial part of the ventroposterior thalamus . Neuronal activity in these regions has been shown to change in preclinical models of trigeminal neuropathic pain . Microglia are the “resident macrophages” of the central nervous system, although occasionally, circulating macrophages can enter the parenchyma, including the brainstem. Trigeminal nerve injuries are reported to trigger the activation of microglia within the SpV, and they may also contribute significantly to the pain sensations in the immediate injury phase . Microglia share similar roles to macrophages, one of which is the production of cytokines that have pro-inflammatory effects (for example TNF-⍺, BDNF, IL-1b, IL-6). These cytokines act via several different mechanisms to increase neuronal excitation . This sustained neural activity is believed to contribute significantly to the pain of trigeminal nerve injury. Microglial activity can be sustained over time and in principle could contribute to the chronicity of pain, however inhibition of microglial activity has not shown consistent or substantial successes in attenuating ongoing pain in preclinical models . Section title: Introduction Educational score: 4.256635665893555 Domain: biomedical Document type: Study Language: en Microglial activation drives activity in co-located astrocytes, thus trigeminal nerve injury is also associated with significant changes in astrocyte activity in the same anatomical regions; importantly modulation of astrocyte activity appears to have a stronger attenuation on injury evoked pain . It is clear that recruitment and activation of macrophages, microglia, and astrocytes, at the site of injury, in the sensory ganglion and the brainstem nuclei, plays a critical role in the establishment of neuropathic pain by increasing neural excitability . These findings of non-neuronal contributions to the development of neuropathic pain are derived almost exclusively from post-mortem histological/immunohistochemical analyses. In this study we used translocator protein 18 kDa (TSPO) PET imaging with [ 18 F]PBR06 to visualise in-vivo , the activity of non-neuronal cells (macrophages and glia) following trigeminal nerve injury in the rat. TSPO ligands have been used successfully for brain imaging in several human pain conditions, despite suggestions of low specificity . This success highlights the great potential for use in preclinical models, thus enhancing translational opportunities, which includes identifying and understanding specific cellular contributions to the PET signal . Using chronic constriction injury of the infraorbital nerve (ION-CCI) our aim was to describe temporal changes in TSPO binding in male rats, prior to, and up to 28 days after ION-CCI compared to sham-injured and naïve counterparts. Sites of significant TSPO signal change were evaluated qualitatively for evidence of macrophage accumulation and glial reactivity in the infraorbital nerve, trigeminal ganglion, and the brainstem trigeminal complex. Section title: Materials and Methods Educational score: 0.8513433933258057 Domain: biomedical Document type: Other Language: en All experimental procedures were carried out with the approval of Alfred Medical Research and Education Precinct Animal Ethics Committee at the Precinct Animal Centre and in accordance with the guidelines of the Code for the Care and Use of Animals in Research Australia, and Ethical Guidelines for Investigation Association for the Study of Pain . Section title: Experimental Design Educational score: 3.5248706340789795 Domain: biomedical Document type: Study Language: en Male Sprague-Dawley rats (n = 79) were sourced from the Monash Animal Research Platform and kept for the duration of the experiment on a 12:12hr light:dark cycle in a temperature controlled environment. Rats at the transition from adolescence (6 weeks) to adulthood were used in these studies to ensure the optimal range of head size for the scanners over the duration of the study. Rats arrive at the facility at 6 weeks of age, and habituate for at least 1 week. Animals were housed 2–3 per cage with ad libitum access to standard laboratory chow and water. Section title: Experimental Design Educational score: 4.098471164703369 Domain: biomedical Document type: Study Language: en At seven weeks of age, rats were arbitrarily allocated to one of three groups: (i) infraorbital nerve chronic constriction injury (ION-CCI) (n = 35), (ii) sham surgical procedures (sham) (n = 23), or (iii) uninjured controls (naïve) (n = 21). The ION-CCI and sham surgical rats were then assigned to one of four imaging time points post ION-CCI or sham surgery: 2 days ( D2 ) (sham n = 6; ION-CCI n = 8), 7 days ( D7 ) (sham n = 5; ION-CCI n = 9), 14 days ( D14 ) (sham n = 7, ION-CCI n = 10), or 28 days ( D28 ) (sham, n = 5; ION-CCI n = 8). The naïve rats (n = 21) were placed into a single group for comparison with the ION-CCI and sham groups at each timepoint. On their allocated imaging day, each rat was scanned once for approximately 60 min in a 9.4 Tesla Bruker MRI scanner and then transferred to a PET/CT scanner for further imaging. On completion of the PET/CT scan, rats were permitted to recover from anaesthesia. Two days later, the naïve rats and the ION-CCI and sham rats at each time point ( D2-D28 ) were culled and their tissues were collected. Section title: Surgical Procedures: Infraorbital nerve constriction injury (ION-CCI) Educational score: 4.219339370727539 Domain: biomedical Document type: Study Language: en Rats were anaesthetised by induction with isoflurane (4 % in 100 % oxygen), delivered via an airtight induction chamber. Surgical level anaesthesia was maintained with 1.5–2 % isoflurane in 100 % oxygen administered via a custom-made facemask. Body temperature was maintained by a thermal blanket. ION-CCI was performed as described by Vos and colleagues . The following procedures were performed under x3.5 magnification using Galilean Loupes . The midline above the nasal bones and between the eyes was shaved and sterilised with povidone-iodine. Once the blink reflex and pedal reflex were abolished, an incision was made approximately 12 mm long, and 2 mm medial to the supraorbital ridge of the right orbit, following the curvature of the frontal bone. The fascia and muscle were gently teased from the bone using blunt dissection until the contents of the orbit could be gently retracted laterally. Following retraction of the orbital contents, the ION was visualised approximately 8 mm deep within the orbit, lying within the infraorbital canal of the maxillary bone. Once visualised, 5 mm of the ION was gently freed from the surrounding connective tissue via blunt dissection. Two chromic gut ligatures (Chromic Gut 5–0 sutures, #687G, Ethicon, Inc.) were loosely tied around the ION using a square knot, tight enough to slightly depress the nerve, but not enough to occlude epineural circulation. The ligatures were spaced approximately 2 mm apart, and the ION was repositioned in the infraorbital canal of the maxillary bone. The incision was sutured closed with nylon sutures . The incision site was cleaned, and antibiotic powder was dusted over the suture site. The rat was moved to a heated recovery cage and monitored closely until ambulatory and eating and drinking. Sham surgical procedures were conducted identically, the nerve was isolated and exposed for the same duration as the application of the ligatures in the nerve-injured group, but no ligatures were applied to the ION. Section title: Imaging procedures Educational score: 4.231060981750488 Domain: biomedical Document type: Study Language: en All procedures were performed at the Alfred Research Alliance-Monash Biomedical Imaging (ARA-MBI) facility (Alfred Centre, Melbourne, Australia). PET/CT imaging was performed using a Mediso NanoScan PET/CT (Mediso Ltd., Hungary). The [ 18 F]PBR06 was synthesized at the Peter MacCallum Cancer Centre with the radiochemical purity of [ 18 F]PBR06 always exceeding 95 %, and the molar activity ∼ 18,500 MBq/µmol. A maximum of six animals were imaged per imaging session at the different endpoints, D2 to D28 as described above. Rats were anaesthetised with isoflurane for the duration of all scanning, they were induced in an air-tight box (3–4 %), then transferred to a custom-built facemask, where isoflurane level was maintained at 1–3 %. Each rat then underwent a series of MRI scanning sequences for 1 h and 15 min prior to be transferred to the PET/CT (animal kept under anaesthesia for the whole process). Animals were placed into the PET/CT in a supine position, headfirst, with the brain and heart in the field of view (FoV). At the start of the dynamic PET acquisition, animals were injected via the dorsal penile vein with a bolus of 0.1987 mCi to 0.6343 mCi of [ 18 F]PBR06 in volumes ranging from 0.04 to 0.45mls. PET scans were acquired continuously for a total of 60 min with 1–3 coincidence mode, a coincidence time-window of 5 ns and an energy window of 400–600 keV. Following the PET scan, two CT scans were acquired over a period of 10 min. The first CT image was acquired using the same field of view as the PET and was used for attenuation correction (70 kVp at 700 µA, 480 projections per bed position, helical mode, scan length of 98.2 mm, 300 ms exposure time and 1:4 binning). The second CT image covered the head only and was used for co-registration (70 kVp at 700 µA, 720 projections per bed position, semi-circular single FoV mode, with medium zoom, scan length of 37.1 mm, 300 ms exposure time and 1:1 binning). Section title: PET image processing and analysis Educational score: 4.2215471267700195 Domain: biomedical Document type: Study Language: en The raw PET list-mode data was reconstructed using the Mediso software, Tera-Tomo 3D reconstruction method, 2 iterations of a 3-dimensional ordered subsets expectation maximization (3D OSEM) algorithm (6 subsets) and with a matrix size of 142x142x159, an isotropic voxel size of 0.6 mm and with the following corrections, attenuation, scatter, random and 18 F decay. The length of the dynamic scan frames were 5x1min, 5x2min, 3x5min, 3x10min. Time-activity-curves were obtained to assess the kinetics of the [ 18 F]PBR06 radiotracer in the brain. The last 30 min, corresponding to the relatively stable transient equilibrium, were used to calculate the Standard Uptake Values (SUVs 30-60min ), which is SUV 30-60min body weight [g/ml] = (Tissue activity/Decay corrected injected dose)*Weight*1000. To account for the amount of radiotracer that crossed the blood–brain barrier, these SUV values were divided by the whole brain SUV for each rat to obtain the SUV ratio (SUVr 30-60min ) . PET images were then pre-processed using Amide’s A Medical Image Data Examiner , Statistical Parametric Mapping 12 (SPM12) and in-house Matlab scripts . CT and SUV and SUVr images were co-registered to the Waxholm Space atlas of the Sprague Dawley rat brain . These co-registered brain maps were then resliced at 0.3x0.3x0.3 mm voxel size and smoothed using a 0.9 mm full-width-at-half-maximum (FWHM) Gaussian kernel. Section title: PET image processing and analysis Educational score: 4.0814971923828125 Domain: biomedical Document type: Study Language: en To determine significant differences between groups, using SPM12, voxel-by-voxel comparisons were made using the co-registered, resliced, and smoothed SUV and SUVr brain images. Our primary goal was to determine the effects of ION-CCI over time on TSPO binding, we compared ION-CCI groups at each timepoint (D2, D7, D14, D28) with naïve and sham-injured rats. There were no significant differences between ION-CCI rats and sham-injured rats, therefore we analysed each group compared to naïves. Significant differences in TSPO binding (SUVr brain maps) between groups were determined using second level, two sample, random effects analyses (p < 0.05, family wise error [FWE] corrected for multiple comparisons). Because there were no differences between ION-CCI and sham groups, for each significant cluster derived from the ION-CCI versus naïve analysis at a particular time point, we extracted the SUVr values from the naïve group and the ION-CCI and sham groups at each timepoint and the mean (±SEM) was calculated and plotted. Significant differences between groups then were determined between groups using two-sample t-tests (p < 0.05, Bonferroni corrected for multiple comparisons). Comparisons between ION-CCI and sham groups did not reveal significant differences between nerve-injured and sham-injured groups. Section title: PET image processing and analysis Educational score: 4.116299629211426 Domain: biomedical Document type: Study Language: en Since we hypothesised that changes in glial reactivity would occur along the ascending trigeminal pain pathway, we restricted this initial analysis to the right (ipsilateral to injury) spinal trigeminal nucleus (SpV), left ventroposterior medial thalamus (VPM) and left primary somatosensory cortex (S1). These masks were created using the volume-of-interest (VOIs) masks within the Waxholm Space atlas of the Sprague Dawley rat brain. To further explore changes in SpV, we also created effect size maps for each animal and then created a mean effect size map for each ION-CCI group and for the naïve group. We created effect size difference maps by subtracting each ION-CCI group from the naïve group, masked the right (ipsilateral to injury) SpV, and overlaid the resultant SpV maps onto a rat brain template for each of the four timepoints. In addition, we conducted a non-masked voxel-by-voxel analysis of the brain to determine significant increases or decreases in TSPO binding between ION-CCI groups at each time point and the naïve group. Since we found no significant differences at FEW corrected levels, we used a more liberal threshold of p < 0.001, uncorrected to assess more subtle group differences. To limit the potential for Type 1 errors we used a minimum cluster size of 20 contiguous voxels. Section title: PET image processing and analysis Educational score: 4.085886001586914 Domain: biomedical Document type: Study Language: en In addition to exploring TSPO binding changes in the brain, we hypothesized that in the ION-CCI groups, the injured trigeminal nerve/ganglion would also display significantly increased TSPO binding. For each timepoint, significant differences in TSPO binding (SUV maps) between the ION-CCI groups and naïve group were determined using second level, two sample, random effects analyses. We restricted this initial analysis to the right trigeminal nerve/ganglion using a custom mask. Since we found no significant differences at FWE corrected levels, we used a liberal threshold of p < 0.05, uncorrected to assess more subtle group differences. To limit the potential for Type 1 errors we used a minimum cluster size of 20 contiguous voxels. For each significant cluster at a particular time point, SUV values were extracted for all ION-CCI, sham and naïve groups and the mean (±SEM) calculated and plotted. SUV was used for analysis of peripheral structures because of the relatively small volumes of the structures analysed, whereas for the much larger brain structures SUVr was used for analyses. Significant differences were determined between groups using two-sample t -test (p < 0.05, Bonferroni corrected for multiple comparisons). Comparison between ION-CCI and sham groups did not reveal significant differences between nerve-injured and sham-injured groups. Section title: Euthanasia and Perfusion Educational score: 4.183634281158447 Domain: biomedical Document type: Study Language: en Forty-eight hours following the imaging session, animals were briefly anaesthetised with 5 % isoflurane mixed in oxygen 1.5 L/minute and a lethal dose of pentobarbitone administered (130 mg/kg, intraperitoneally). Each rat was then perfused trans -cardially with 400 ml of ice cold heparinised 0.9 % (w/v) saline followed by 400 ml of 4 % paraformaldehyde in sodium acetate-borate buffer (pH 9.6, 4 °C) (PFA). The left and right infraorbital nerves (ION), their corresponding trigeminal ganglion (TG) and the brain and cervical spinal cord were dissected free and post-fixed in PFA for two hours at room temperature, then cryoprotected in 10 % (w/v) sucrose in 0.1 M phosphate buffered saline, pH 7.4 (PBS) and stored at 4 °C until processing. Section title: Qualitative Histological Verification Educational score: 4.1951117515563965 Domain: biomedical Document type: Study Language: en All tissue was sectioned using a cryostat , a 1 in 6 series of transverse sections of the infraorbital nerve (14 µm); and the trigeminal ganglia (14 µm) were cut onto 2 % (w/v) gelatinised slides and stored at -20 °C until processing. The medulla was blocked caudally with a coronal cut at approximately the C3 level of the cervical spinal cord, and rostrally at the level of the subnucleus interpolaris of the SpV (−12.00 mm bregma . Coronal sections of the medulla were cut at 40 µm, as a 1 in 10 series and stored in antifreeze at −20 °C until processed. Section title: Qualitative Histological Verification Educational score: 4.127444267272949 Domain: biomedical Document type: Study Language: en ION, TGs, and sections of medulla were immunoreacted to determine the co-localisation of CD68-immunoreactive (CD68-IR) macrophages, Peripheral-Type Benzodiazepine Receptor (PBR) immunoreactivity (PBR-IR), blood vessels (tomato lectin immunoreactivity [TL + ]), ionized calcium binding adaptor molecular 1 (IBA1) immunoreactive microglia, or Glial Fibrillary Acidic Protein (GFAP) immunoreactive (GFAP-IR) astrocytes. Section title: Infraorbital nerve and trigeminal ganglion Educational score: 4.131454944610596 Domain: biomedical Document type: Study Language: en ION and TG mounted slides were washed in 0.1 M phosphate buffered saline, pH7.4 (PBS) and blocked in PBS containing 5 % (v/v) normal horse serum (NHS) and 0.05 % (v/v) Tween-20 (Sigma-Aldrich, Australia). Sections were incubated for 16-hours at room temperature with mouse anti-CD68 [ED-1] and rabbit anti-PBR [EPR5384] , in PBS containing 5 % (v/v) NHS and 0.05 % (v/v) Tween-20. Sections were then washed in PBS and incubated for 2-hours in Alexa-488 conjugated donkey anti-mouse and Cy3 conjugated Donkey anti-rabbit . Sections were washed in PBS and then incubated for 3-hours with DyLight™ 594 conjugated Tomato [Lycopersicon Esculentum] Lectin (TL) in PBS . Slides were washed in PBS and then incubated for 20-mins with DAPI in PBS. Slides were washed in PBS before being cover slipped with Prolong Gold Antifade mounting medium and stored at 4 °C until microscopy. Section title: Medulla Educational score: 4.148443222045898 Domain: biomedical Document type: Study Language: en Medulla sections were washed in PBS and then blocked in 5 % (v/v) NHS in PBS for 30-minutes at room temperature. PBR/GFAP/TL immunofluorescence: Sections were incubated for 16-hours at 4 °C with rabbit anti-PBR [EPR5384] and mouse anti-GFAP . Sections were then washed in PBS and incubated for 2-hours in Alexa-488 conjugated donkey anti-mouse and Cy3 conjugated donkey anti-rabbit . PBR/IBA1/TL immunofluorescence: Sections were incubated for 16-hours at 4 °C with rabbit anti-PBR [EPR5384] , sections were then washed in PBS and incubated for 2-hours in Cy3 conjugated donkey anti-rabbit . Sections were washed and then incubated for 16-hours at 4 °C with rabbit anti-IBA1 , washed in PBS and incubated for 2-hours in Alexa-488 conjugated donkey anti-rabbit . Following antibody incubations all sections were then washed in PBS and incubated for 3-hours with DyLight™ 594 conjugated Tomato [Lycopersicon Esculentum] Lectin (TL) in PBS . Slides were washed in PBS and then incubated for 20-mins with DAPI in PBS. Slides were washed in PBS before being mounted and cover slipped with Prolong Gold Antifade mounting medium and stored at 4 °C until analysed under the microscope. Section title: Microscopy Educational score: 4.1023101806640625 Domain: biomedical Document type: Study Language: en Slides were imaged using a Nikon C2 confocal microscope. The large image function was used to ‘stitch’ multiple images (100x magnification) to allow the visualisation of the entire infraorbital nerve including ligated areas, trigeminal ganglia, and coronal medulla sections. Representative z-stack images were captured at 600x magnification to visualise co-localisation of CD68-IR macrophages, PBR-IR, TL + blood vessels, IBA1-IR microglia, or GFAP-IR astrocytes. Section title: Trigeminal Pain Pathway: Trigeminal Ganglion Educational score: 4.202059745788574 Domain: biomedical Document type: Study Language: en Analysis of the trigeminal ganglion and nerve revealed that relative to the naïve group, there was a significant increase in binding in the trigeminal ganglion ipsilateral to the nerve constriction in the ION-CCI rats at day 14, and no differences at days 2, 7 or 28 . A similar increase was observed also in the sham-injured rats at this time point , although the TSPO binding was somewhat higher in sham-injured rats, at days 2 and 7 post-ION-CCI, this was not significantly different. Qualitative histological observations of the ganglion of ION-CCI rats revealed PBR-IR in the maxillary division of the ganglion co-localized with CD68-IR macrophages, and TL + endothelial cells of the ganglionic vasculature . In ganglia from sham-injured rats, PBR-IR was also observed . Naïve rats showed little PBR-IR. At the site of injury, PBR-IR was frequently colocalized with CD68-IR macrophages and TL + endoneurial vasculature . Fig. 1 TSPO binding assessed in infra-orbital nerve/ganglion in chronic constriction injury (ION-CCI), sham-injured and naïve groups. Significant binding increases (hot colour scale) at day 14 (D14) in the ION-CCI compared with naive groups occurred in the region encompassing the right (ipsilateral to nerve injury) trigeminal (V) ganglion. Clusters are overlaid onto a T2-weighted rat brain template anatomical image set. To the top right of each image is the approximate location relative to bregma derived from Paxinos and Watson . To the right are plots of mean ± SEM standard uptake value (SUV) for all three groups at day 2 ( D2 ), day 7 ( D7 ), day 14 ( D14 ) and day 28 ( D28 ). The horizontal grey line and grey shading indicates the mean and SEM of the naïve group. *p < 0.05 derived from voxel-by-voxel analysis, # p < 0.05 post hoc two-sample t-tests sham versus naïve (values extracted from significant clusters derived from ION-CCI vs naïve voxel-by-voxel analysis). Table 1 Significant cluster properties including SUVr values in the ascending trigeminal pathway and SUV values for the trigeminal ganglion. * p < 0.05 voxel-by-voxel analysis. # p < 0.05, two sample t -test. Day 2: day 2; D7: day 7; D14: day 14; D28: day 28; ION-CCI: infra-orbital nerve chronic constriction injury; SEM: standard error mean; SpV: spinal trigeminal nucleus; SUV: standard uptake values; SUVr: standard uptake values ratio. Ascending trigeminal pathway: ION-CCI > naive cluster size t- value Day SUVr mean ± SEM naive sham-injured ION-CCI D2: SpV cluster 8 3.27 D2 1.77 ± 0.03 # 1.80 ± 0.08* D7 1.59 ± 0.03 1.65 ± 0.07 1.80 ± 0.07 # D14 1.54 ± 0.09 1.70 ± 0.05 # D28 1.47 ± 0.08 1.53 ± 0.03 D7: SpV cluster 53 3.91 D2 1.79 ± 0.03 # 1.79 ± 0.07 # D7 1.61 ± 0.03 1.69 ± 0.06 1.84 ± 0.06* D14 1.57 ± 0.09 1.72 ± 0.05 # D28 1.48 ± 0.07 1.52 ± 0.04 Trigeminal ganglion/nerve: ION-CCI > naive SUV mean ± SEM D14: trigeminal ganglion cluster 116 2.31 D2 4.38 ± 0.37 3.90 ± 0.30 D7 3.96 ± 0.19 4.25 ± 0.35 3.81 ± 0.19 D14 4.75 ± 0.17 # 4.64 ± 0.23* D28 3.90 ± 0.34 3.84 ± 0.27 Fig. 2 Representative photomicrographs of PBR-IR co-localization. A: trigeminal (V) ganglion TSPO binding cluster overlaid on a T2-weighted rat brain template anatomical image. B: stitched 40X magnification photomicrograph of the trigeminal ganglion and corresponding ophthalmic (V1), maxillary (V2) and mandibular (V3) divisions with PBR-IR (orange), CD68-IR (green) and TL (red) co-localization with immunofluorescence. The PBR-IR in the V2 of (C) naïve, (D) sham-injured and (E) ION-CCI rats are shown at 600X magnification. Example of co-localization of (F) CD68-IR and PBR-IR, (G) TL + and PBR-IR and (H) CD68-IR, PBR-IR and TL + . White arrows show co-localized immunofluorescent signals. Scale bars represent 20 µm or 500 µm in panel B. Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.2162184715271 Domain: biomedical Document type: Study Language: en Analysis of TSPO binding in the brainstem trigeminal complex, revealed significant increases in the SpV/paratrigeminal region (Pa5) of the ION-CCI group compared to the naïve group at day 2 and day 7 . Increased binding was restricted to the right Pa5. Extraction of SUVr values from the significant Pa5 cluster at day 2, revealed that compared with the naïve group, ION-CCI groups displayed greater TSPO binding at days 2, 7 and 14, but not at day 28 ( Table 1 ). Sham-injured rats showed a significant increase in TSPO binding compared to the naïve group at day 2, but not at days 7, 14 and 28. Once again, extraction of SUVr values from the significant Pa5 cluster at day 7, revealed that compared with the naïve group, ION-CCI rats had greater TSPO binding at days 2, 7 and 14, but not day 28. Sham-injured rats displayed a significant increase in TSPO binding compared with naïve group at day 2, but not at days 7, 14 and 28. Qualitative observations of the Pa5 region confirmed PBR-IR localised to this region which was usually co-localized with IBA1-IR microglia, and TL + endothelial cells in the vasculature, but never in GFAP-IR astrocytes in the sections observed (not illustrated). Fig. 3 TSPO binding assessed in the ascending trigeminal pathway in infra-orbital nerve chronic constriction injury (ION-CCI), sham-injured and naïve groups. A : Significant binding increases (hot colour scale) at day 2 ( D2 ) and day 7 ( D7 ) in the ION-CCI compared with naive groups. Binding increases occurred in the right (ipsilateral to nerve injury) brainstem trigeminal complex. Clusters are overlaid onto a T2-weighted rat brain template anatomical image set. To the top right of each image is the approximate location relative to bregma derived from a rat atlas Paxinos and Watson . Below the overlays are plots of mean ± SEM standard uptake value ratios (SUVr) for all three groups. The horizontal grey line and grey shading indicates the mean and SEM of the naïve group. *p < 0.05 derived from voxel-by-voxel analysis, # p < 0.05 post hoc two-sample t-tests sham versus naïve (values extracted from significant clusters derived from ION-CCI vs naïve voxel-by-voxel analysis). Inset to the right shows the volumes of interest (light-blue) used for analysis. B: Representative photomicrographs of PBR-IR co-localization within the Brainstem Trigeminal Complex. (i) TSPO binding cluster overlaid on a T2-weighted rat brain template anatomical image of the brainstem trigeminal complex at approximately −13.44 mm bregma (ii) stitched 40x magnification photomicrograph of the brainstem trigeminal complex, with the paratrigeminal nucleus (Pa5) outlined in white and PBR-IR (orange), Iba1-IR (green) and TL (red) co-localization with immunofluorescence. Example of co-localization of (iii) PBR-IR, Iba1-IR and TL+; (iv) PBR-IR and TL+; (v) Iba1-IR and TL+; and (vi) PBR-IR and Iba1-IR. White arrows depict co-localized immunofluorescent signals. Scalebars represent 20 µm or 500 µm in panels ii-vi. Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.247824668884277 Domain: biomedical Document type: Study Language: en Fig. 4 shows overlays of the mean effect size differences within the right SpV and shows increased TSPO binding across the rostro-caudal extent of the SpV at days 2–14 and by day 28, these increases remain only in the Pa5 region, at approximately −12.8 mm to 13.5 mm from bregma. Histological analysis of the SpV region confirmed PBR-IR specific to this region and was co-localized with IBA1-IR microglia, and TL + endothelial cells in the vasculature, and GFAP-IR astrocytes . Fig. 4 Effect size increases (hot colour scale) of TSPO binding in infra-orbital nerve chronic constriction injury (ION-CCI) compared with naïve groups within the right brainstem trigeminal complex volumes of interest at day 2, day 7, day 14 and day 28 overlaid onto coronal slices of a T2-weighted rat brain anatomical image set. On the left are shown the terminal projections of the infraorbital nerve traced using WGA- conjugated horse-radish peroxidase, the data are adapted from Fig. 4 of . To the top right of each image is the approximate location relative to bregma derived from a rat atlas. Fig. 5 A: Top row shows significantly greater TSPO binding in the infra-orbital nerve chronic constriction injury (ION-CCI) compared with naïve groups at day 7 overlaid onto a series of coronal T2-weighted anatomical images between −13.68 mm and −14.76 mm relative to bregma. Below are photomicrographs of coronal sections of the brainstem trigeminal complex at equivalent levels stained for PBR-IR (orange), GFAP-IR (green) and TL (red) co-localization with immunofluorescence. The upper row of photomicrographs shows sections from naïve control rats and the lower row of rats following ION-CCI. All data are from rats at day 7 post-injury. Scale bar 500 µm. B: Photomicrographs of coronal sections of the brainstem trigeminal complex at day 7 post-injury. Top (i) large photomicrograph, stitched 40x magnification of the brainstem trigeminal complex, with high magnification insets (to right) taken from the region of the SpV outlined in white. PBR-IR (orange), GFAP-IR (green) and TL+ (red) co-localization with immunofluorescence. To the right, insets show examples of co-localization of: (ii) PBR-IR, & GFAP; (iii) PBR-IR & TL+; (iv) GFAP-IR & TL+; and (v) PBR-IR, GFAP-IR & TL + . White arrows depict co-localized immunofluorescent signals. Scalebars represent 20 µm or 500 µm. Bottom Panel: Left: large photomicrograph, stitched 40x magnification of the brainstem trigeminal complex, with high magnification insets (to right) taken from the region of the SpV outlined in white. PBR-IR (orange), Iba1-IR (green) and TL+ (red) co-localization with immunofluorescence. To the right, insets show examples of co-localization of: (a) PBR-IR, & Iba1-IR; (b) PBR-IR & TL+; (c) Iba1-IR −IR & TL+; and (d) PBR-IR, Iba1-IR −IR & TL + . White arrows depict co-localized immunofluorescent signals. Scalebars represent 20 µm or 500 µm. Section title: Trigeminal Pain Pathway: Forebrain Structures: Educational score: 4.231260299682617 Domain: biomedical Document type: Study Language: en Analysis of supra-medullary brain regions revealed a number, of TSPO binding differences between ION-CCI and naïve rats within the 28-day period. On day 2, TSPO binding was significantly increased in the area encompassing both the ipsilateral primary motor cortex (M1) and primary somatosensory cortex (S1) . Extraction of SUVr values from the cluster located in this combined M1/S1 cortical region, revealed that compared with the naïve group, the ION-CCI rats also displayed greater TSPO binding at day 28 whereas the sham-injury group did not display a significant TSPO binding change at any time point. On day 7, there was a significant decrease in TSPO binding in the ION-CCI group relative to the naïve group within the midline cerebellar cortex. No change occurred in this region at any other timepoint in either the ION-CCI or sham-injury groups. On day 14, there were multiple regions where TSPO binding was significantly reduced in the ION-CCI compared with naïve groups. Binding decreases occurred in the region of the right septal nucleus, right hippocampus and in two clusters in the right caudate/putamen. These decreases did not occur at any other timepoint and in the sham-injury group only occurred at day 14 in the septal nucleus and one of the caudate/putamen clusters. Finally, on day 28, there were two regions that displayed increased binding in the ION-CCI compared with naïve groups. One cluster in the region of the right M1 that also increased at day 2, and another at day 28 only in the region of the right S1. The sham-injury group also displayed increased binding in these two regions only at day 28. Fig. 6 TSPO binding assessed over the entire brain in infra-orbital nerve chronic constriction injury (ION-CCI), sham-injury and naïve groups. Significant binding increases (hot colour scale) or decreases in ION-CCI compared with naïve groups at day 2 ( D2 ), day 7 ( D7 ), day 14 ( D14 ) and day 28 ( D28 ) are displayed. Clusters are overlaid onto a T2-weighted rat brain template anatomical image set. To the top right of each image is the approximate location relative to bregma derived from Paxinos and Watson . To the right are plots of mean ± SEM standard uptake value ratios (SUVr) for all three groups. The horizontal grey line and grey shading indicates the mean and SEM of the naïve group. *p < 0.05 derived from voxel-by-voxel analysis, # p < 0.05 post hoc two-sample t-tests sham-injury versus naïve (values extracted from significant clusters derived from ION-CCI vs naïve voxel-by-voxel analysis). CuP: caudate/putamen; M1: primary motor cortex; S1: primary somatosensory cortex; SpV: spinal trigeminal nucleus; SUVr: standard uptake values ratio. Table 2 Significant cluster properties including SUVr values in the whole brain analysis. * p < 0.05 voxel-by-voxel analysis. # p < 0.05, two sample t -test. Day 2: day 2; D7: day 7; D14: day 14; D28: day 28; ION-CCI: infra-orbital nerve chronic constriction injury; M1: primary motor cortex; S1: primary somatosensory cortex; SEM: standard error mean; SpV: spinal trigeminal nucleus; SUVr: standard uptake values ratio. cluster size t-value day SUVr mean ± SEM naive sham ION-CCI D2: ION-CCI > naive right M1/SI 273 6.00 D2 1.30 ± 0.05 1.42 ± 0.03* D7 1.16 ± 0.03 1.33 ± 0.06 1.23 ± 0.03 D14 1.14 ± 0.04 1.16 ± 0.04 D28 1.37 ± 0.09 1.36 ± 0.05 # D7: ION-CCI < naive Right SpV 50 4.04 D2 1.63 ± 0.02 # 1.64 ± 0.06 # D7 1.49 ± 0.03 1.53 ± 0.05 1.68 ± 0.05* D14 1.42 ± 0.08 1.55 ± 0.04 D28 1.40 ± 0.08 1.42 ± 0.02 cerebellar cortex 51 3.85 D2 1.60 ± 0.03 1.71 ± 0.09 D7 1.63 ± 0.04 1.50 ± 0.04 1.41 ± 0.04* D14 1.56 ± 0.06 1.66 ± 0.07 D28 1.75 ± 0.10 1.76 ± 0.05 D14: ION-CCI < naive right septal nucleus 38 3.59 D2 0.79 ± 0.01 0.78 ± 0.01 D7 0.83 ± 0.02 0.77 ± 0.03 0.82 ± 0.02 D14 0.75 ± 0.03 # 0.73 ± 0.02* D28 0.93 ± 0.08 0.86 ± 0.04 right hippocampus 27 3.69 D2 1.07 ± 0.02 0.97 ± 0.02 D7 1.03 ± 0.01 0.96 ± 0.02 # 0.99 ± 0.02 D14 0.99 ± 0.03 0.94 ± 0.01* D28 0.97 ± 0.03 1.00 ± 0.02 right caudate/putamen 1 26 3.71 D2 0.90 ± 0.02 0.85 ± 0.02 D7 0.88 ± 0.01 0.85 ± 0.02 0.86 ± 0.02 D14 0.79 ± 0.02 # 0.78 ± 0.01* D28 0.93 ± 0.05 0.90 ± 0.02 right caudate/putamen 2 22 3.50 D2 0.75 ± 0.03 0.74 ± 0.02 D7 0.77 ± 0.02 0.73 ± 0.02 0.75 ± 0.02 D14 0.69 ± 0.03 0.66 ± 0.02* D28 0.84 ± 0.05 0.80 ± 0.02 D28: ION-CCI > naive right M1 55 4.12 D2 1.11 ± 0.06 1.24 ± 0.03 # D7 1.02 ± 0.03 1.09 ± 0.03 1.06 ± 0.03 D14 0.97 ± 0.03 0.99 ± 0.03 D28 1.25 ± 0.11 # 1.23 ± 0.04* right S1 373 4.12 D2 1.16 ± 0.02 1.18 ± 0.04 D7 1.07 ± 0.03 1.15 ± 0.05 1.09 ± 0.03 D14 1.06 ± 0.02 1.03 ± 0.03 D28 1.21 ± 0.04 # 1.27 ± 0.03* Section title: Discussion Educational score: 4.377962589263916 Domain: biomedical Document type: Study Language: en In this study our specific aim was to use PET imaging to visualise in-vivo, the activity of macrophages and microglia during the development of trigeminal neuropathic pain in male rats. Using chronic constriction injury of the infraorbital nerve (ION-CCI) we aimed to describe temporal changes in TSPO binding 2, 7, 14, and 28 days after ION-CCI, and to compared this to the binding in both sham-injured and naïve control rats. Unexpectedly, we identified almost identical changes in TSPO binding in rats that had undergone the sham-injury procedures, ie., the surgical isolation of the ION without ligation, to those seen in nerve injured rats. It is clear from these data that within this 28 day window, the surgical approach taken for nerve isolation, which involves skin incision and retraction of orbital contents is sufficient to evoke TSPO binding changes akin to those that are suggested to underly the development of the neuropathic pain state. The sham-injury effect that we have detected may share similarities to the model of persistent post-surgical pain triggered by skin-muscle incision and retraction (SMIR model) described in the hindlimb . This procedure tiggers microglial activity in lumbar spinal segments attributed to the duration of exposure of the surgical site . We note however, that similar levels of microglial activity are not reported for sham-injury conditions in hindlimb neuropathic models, suggesting that sham-injury procedures in these instances are brief and perhaps more discrete, and likely that in preclinical trigeminal neuropathy models, the surgery for sham-injury is potentially more disruptive. We reflect that the sham condition is often considered a condition closer to the uninjured state, which is not always the case as surgical procedures still trigger significant tissue disruption with associated sensory hypersensitivity, as capitalised on in models of post-surgical pain. Further, we have recently described significant neuroimmune changes following identical sham-injury procedures on the infraorbital nerve and so the fact that we detected TSPO binding changes in the sham-injured rats is perhaps not quite so surprising. We evaluated the main PET findings with immunohistochemistry using a combination of antibodies to identify TSPO binding sites (anti-PBR), combined with markers for glial cells and macrophages. Our immunofluorescence results indicated that in both infra-orbital nerve injured, and sham-injured rats, TSPO was likely binding to microglia, macrophages, astrocytes, vascular endothelial cells, as well as other non-identified classes of cell. Therefore, changes in TSPO signal are unlikely to reflect only macrophage accumulation or changes in glial cells. Secondly, we found that TSPO binds to specific sites along the trigeminal pain pathway at distinct timepoints. In addition, we found changes in TSPO binding in additional central sites including the cerebellum, the motor and somatosensory cortices, septum, hippocampus, and the striatum. To reduce the testing burden for both ethical and practical reasons, in this study we did not evaluate the rat’s behavioural responses to facial stimulation. We are however confident of the overall trajectories of both nerve injury-evoked, and sham-injury evoked changes in behaviour, based on data from a large-scale study that was conducted concurrently, and recently published . We consider the timing and location of the TSPO binding changes with regards to the changes described in that study and current literature. There is broad agreement that there are three distinct phases in the response to injury of the infraorbital nerve, these are characterized by both behavioural and neuroimmune markers. The first of the phases, described as the “early” period, lasts for up to one week, it correlates with the period of Wallerian degeneration and is characterized by hypo-responsiveness . The second phase appears to be an “intermediate”, or transition period (12–21 days post-injury) where behavioural reactivity increases and animals show aversive or defensive responses to tactile stimulation . The final or “late” phase appears after 28 days and was described by Vos and colleagues , to last at least 130 days post ION-CCI and is characterised by well-established hyper-responsiveness and clear reductions in facial grooming. We will consider our PET findings using this temporal framework of ION-CCI evoked changes, from the site of injury and then at each level of the trigeminal pain pathway. As highlighted above, TSPO was selected so that we could identify changes in microglia and macrophages, however it is important to note however that we need to broaden this consideration to include reactive astrocytes, vascular endothelial cells, and possibly smooth muscle cells and neurons . Section title: Trigeminal Pain Pathway: Trigeminal Ganglion: Educational score: 4.368085861206055 Domain: biomedical Document type: Study Language: en At the site of nerve injury, our histological analysis showed a significant accumulation of macrophages with “frothy” and vacuolated appearances, some of these cells colocalized with PBR-IR, indicating a likely site for TSPO binding. Following sham-surgery, there were small numbers of macrophages at the site of nerve exposure on each of the days of testing. PBR-IR was also colocalized with the endoneurial vasculature, we expect that this would be largely attributed to staining of the vascular endothelial cells. PET scans did not detect significant TSPO binding at the nerve-injury site at any timepoint. TSPO binding in the trigeminal ganglion was increased at day 14 in both the ION-CCI and sham-injury groups when compared to naïve rats. We also noted an increase in PBR-IR at this time point, however the numbers of CD68-IR macrophages in the ganglion were quite low and few of these contained PBR-IR. This raises the likelihood that the TSPO binding is not an indicator of macrophage accumulation. We noted that PBR-IR was present in the lumen of TL + vessels raising the possibility that TSPO was binding to endothelial cells, as well as other non-identified classes of cells. Binding of TSPO to endothelial cells has previously been reported in a mouse model of CRPS triggered by tibial fractures . It has been reported that nerve injury can lead to increased vascularization of ganglia associated with the damaged nerve . These vessels can play a critical role in the development of neuropathic pain, through local release of inflammatory mediators such as cytokines and chemokines that directly, and indirectly alter the function of the resident satellite glial cells and sensory neurons . The day 14 timepoint at which these observations were made is during the transitional phase, during which we have previously observed decreased numbers of macrophages relative to the acute injury phase, i.e., 2–7 days post ION-CCI, and prior to an apparent second wave of macrophage accumulation seen during the late phase, which includes 28 days post-injury and beyond . This transitional phase may well reflect the change from a phase of clearance of injury related cell damage, to a later phase of nerve repair and attempted functional recovery. Our observations in sham-injured rats suggest that the surgical procedures for nerve injury might also contribute significantly to these changes. Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.186338424682617 Domain: biomedical Document type: Study Language: en Similar to the trigeminal ganglia, in both nerve-injured and sham-injured rats, PBR-IR was co-localized with the neurovasculature along the rostro-caudal extent of the brainstem trigeminal complex ( pars interpolaris , pars caudalis , and Pa5) and the NTS, as well as in astrocytes and microglia. As such, the changes in TSPO binding that we observed along the brainstem do not appear to correspond to a single cell type, rather it emphasizes the close relationship between glial activity and the neurovasculature. Astrocytes are well known to regulate neuronal function and directly interact with the vasculature as well as other glial cells . Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.472445011138916 Domain: biomedical Document type: Study Language: en Increased TSPO binding was observed in a discrete region of the brainstem trigeminal complex ipsilateral to the injury, and surgical site, this region included the Pa5, the dorsal portion of SpV ( pars interpolaris and pars caudalis ), and the trigeminal portion of the NTS. These areas have all been described to receive direct inputs from fibres of the infraorbital nerve specifically and more generally from the maxiliary division of the trigeminal nerve. The paratrigeminal nucleus is a small interstitial system of the spinal trigeminal tract, which consists of small diffuse nuclei located in the dorsal lateral medulla . It receives sensory inputs not only from the trigeminal nerve, but also from the glossopharyngeal and vagal nerves and is involved in the integration of both cardiovascular, and respiratory responses, in addition to pain mechanisms . Its cells send efferent projections to both the parabrachial nucleus, the nucleus of the solitary tract, lamina 1 of the pars caudalis of the SpV, and the ventroposterior medial nucleus of the thalamus (VPM) . Similarly, the NTS receives direct sensory input from the trigeminal nerve, the glossopharyngeal and vagal nerves as well as inputs from the superficial laminae of the SpV, pars caudalis . The pars interpolaris and pars caudalis of SpV also receive direct inputs from the infraorbital nerve, via both the maxillary and ophthalmic divisions of the trigeminal nerve . These anatomical distributions of sensory inputs correspond well with the effect size analysis that we report here, which revealed increased TSPO binding ipsilateral to the nerve-injury, and the associated surgical site in the dorsolateral aspect of the laminar part of the SpV, as well as the Pa5 region and the dorsal NTS (trigeminal portion). Our data suggest that the surgical procedures for ION-CCI contributes significantly to these changes based on our observations of significant binding in sham-injured rats. Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.439244270324707 Domain: biomedical Document type: Study Language: en The dynamic changes observed in TSPO binding in the brain over the experimental period were similar to those seen at the site of nerve injury, and the trigeminal ganglion and suggest a common phasic response profile to both nerve injury and sham surgery. The largest increases in TSPO binding were noted 2 days after nerve injury and sham surgery, within the early response phase. In rats, an initial and rapid microglial response in SpV pars caudalis has been observed after inferior alveolar nerve transection and peaks between 1–3 days this response profile is also seen following ION-CCI . We note, as discussed above, that such a microglial response is not reported in sham-injured counterparts in other studies of ION-CCI, although it is reported for the SMIR model of post-surgical pain, which our sham-injury procedure may more closely resemble. This microglial response is not related to an immediate (1–3 days) increase in neuronal activity, however 7 days after ION-CCI, phosphorylated ERK (pERK) expression, a marker of neuronal excitation, provides evidence of increased neuronal excitation in SpV pars caudalis . Similarly, cFos expression increases at 9–12 days after ION-CCI, again indicating an injury evoked increase in neuronal activity sometime after the microglial peak response . Furthermore, at 7 days post injury the mean background discharge and after-discharge rates of wide dynamic neurons were significantly larger in ION-CCI rats in the superficial laminae of the pars caudalis . The delay between the peak microglial response and the increased neuronal activation may be a result of microglial modulation of the extracellular matrix, whereby the microglia degrade the perineuronal nets forming the matrix, which facilitates neuronal excitation in a similar fashion to that described following sciatic nerve injury and dorsal horn neurons . Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.353111743927002 Domain: biomedical Document type: Study Language: en Glia-glial interactions can also result in the initiation and maintenance of chronic neuropathic pain. At 7 days post injury, although microglia are still present, their numbers are decreased, and the presence of GFAP-IR hypertrophic astrocytes are increased following trigeminal nerve transection . Microglial derived factors such as C1q are known to increase astrocyte reactivity and subsequently nociceptive signaling 7 days after ION-CCI . TSPO binding at 14 days post injury appears to show a more elongated distribution along the rostro-caudal axis of the SpV, and extends into its deeper laminae, the binding in the dorsal NTS and Pa5 is maintained at this timepoint also. This timepoint corresponds to the transitional phase where it is suggested that the primary glial activity is that of astrocytes. GFAP-IR is sustained in astrocytes 14- and 21-days post trigeminal transection injury as well as ION-CCI . It is important to note that GFAP-IR astrocytes during this transitional phase are present across both superficial and deep laminae of pars caudalis . We note that in the literature, there are fewer studies of glial expression following trigeminal nerve injuries at these extended timepoints, with most studies focusing on 7–14 days post injury. Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.223631858825684 Domain: biomedical Document type: Study Language: en In our rats at 28 days post injury or sham-injury, the TSPO binding contracts along the rostrocaudal axis, and becomes most concentrated in the dorsolateral aspect of the SpV/Pa5 region and the dorsal NTS. At this late phase of injury, microglia are reported to be still present following trigeminal nerve transection, albeit in much smaller numbers, and GFAP-IR astrocytes are sustained but also in smaller numbers following trigeminal nerve transection and ION-CCI . The contraction of TSPO signal around the Pa5 region is intriguing as this subregion has been shown to be more sensitive to nociceptive-specific C-fibre inputs than the SpV, and is thought to drive the affective-emotional elements of pain via its outputs to midbrain structures including the parabrachial nucleus and the PAG . Section title: Trigeminal Pain Pathway: Brainstem Trigeminal Complex: Educational score: 4.130361080169678 Domain: biomedical Document type: Study Language: en Our analyses of TSPO using binding density versus effect size analysis revealed differences in peak signals within the trigeminal brainstem complex. While these signals all fall well within the boundaries of termination of the infraorbital nerve derived from anatomical tracing studies as illustrated in Fig. 4 , the shift deserves comment. These differences may reflect limitations of the resolution of the PET technique, or the TSPO ligand in particular, or they may in fact reflect salient neurobiological features of ION-CCI and sham surgical procedures. Whether the locations with the greatest effect sizes contribute to the sensory and behavioural consequences of ION-CCI and sham-injury , when compared with the sites showing the statistically significant binding increases, warrants future investigation. Section title: Trigeminal Pain Pathway: Cortical Structures: Educational score: 4.249355792999268 Domain: biomedical Document type: Study Language: en Primary motor cortex (M1) and S1): In the current study, we also observed increased TSPO binding in a variety of forebrain structures including S1 and M1, ipsilateral to the nerve injury and surgical site, in both ION-CCI and sham-injured rats. This was evident during both the early phase ( D2 ) and late phase ( D28 ) after both ION-CCI and sham-injury. While contrary to the classical view that ascending nociceptive pathways terminate exclusively contralaterally, there is substantial evidence for an ipsilateral nociceptive pathway transmitting trigeminal pain . Our observations could suggest surgery and nerve injury-evoked cortical plasticity; this proposal is based on observations that sciatic nerve ligations trigger astrocyte activation in the S1 within the first week of injury, and that this correlates with changes in extracellular glutamate concentrations and dendritic spine turnover indicative of neuroplasticity . Of note are data from Loggia and colleagues, who have shown in humans with chronic back pain, that significant increases in TSPO binding are present in both the S1 and M1 which provides strong evidence for prolonged glial activity that persists long after the initial injury . Section title: Trigeminal Pain Pathway: Cortical Structures: Educational score: 4.089430809020996 Domain: biomedical Document type: Study Language: en TSPO binding was also noted in a region of the midline cerebellar cortex, at approximately lobules 4 and 5 of the anterior lobe. This region receives direct sensory projections from the trigeminal nerve via the superior cerebellar peduncles , providing a route for injury and surgery mediated, direct modulation of glial-neurovascular contributions to cerebellar function. Section title: Trigeminal Pain Pathway: Cortical Structures: Educational score: 4.3806352615356445 Domain: biomedical Document type: Study Language: en Sub-cortical Forebrain: During the transitional phase (D14) the septal nuclei, hippocampus, and dorsal striatum showed decreased TSPO binding on the side ipsilateral to the nerve injury and sham-injury , consistent with the cortical changes described above. TSPO binding decreases are difficult to interpret, they may indicate a transient decrease in glial-neurovascular interactions. Altered metabolism, measured by [ 18 F]fluorodeoxyglucose in awake rats has been shown at this timepoint in a range of subcortical forebrain structures following the spinal nerve ligation model of neuropathic pain and striatal monoamine levels are significantly disrupted 13 days after a peripheral sciatic nerve CCI . A second possibility is nerve injury and surgery triggered mitochondrial dysfunction at specific central sites. TSPO is mitochondrial protein, to which [ 18 F]PBR06 binds, decreased mitochondrial function/expression may lead to decreased [ 18 F]PBR06 binding identified by regional reductions of the PET signal. It is of note that mitochondrial dysfunction is often associated with neuropathic pain-like symptoms . Both of these suggested changes likely correlate with disruptions to both cognitive and affective behaviours, resulting for example, in impaired decision making. Section title: Conclusion Educational score: 4.187074661254883 Domain: biomedical Document type: Study Language: en We provide in vivo evidence of transient increases in TSPO binding in the trigeminal ganglion and trigeminal brainstem complex following both infra-orbital nerve constriction injury and sham-injury procedures that isolate the ION in identical fashion, but do not ligate it. The use of in vivo imaging of glial cells in combination with other imaging techniques such as structural and functional magnetic resonance imaging, can provide a novel view of the complex interactions between neural and non-neural cells in preclinical models of neuropathic pain, and as suggested by observations in sham-injured rats in this study, in models of persistent post-operative pain. Given that these in vivo techniques can be used to acquire similar information in humans, our results provide the platform to begin to translate findings from preclinical models into human patients with chronic pain or the risk of persistent post-surgical pain and offer an opportunity to begin to explore the effects of treatments on neural and non-neural cells in both preclinical models and humans. Section title: Ethics approval and consent to participate Educational score: 0.8513433933258057 Domain: biomedical Document type: Other Language: en All experimental procedures were carried out with the approval of Alfred Medical Research and Education Precinct Animal Ethics Committee at the Precinct Animal Centre and in accordance with the guidelines of the Code for the Care and Use of Animals in Research Australia, and Ethical Guidelines for Investigation Association for the Study of Pain . Section title: Consent for publication Educational score: 1.006712794303894 Domain: other Document type: Other Language: en this manuscript does not contain any individual person’s data in any form. Section title: CRediT authorship contribution statement Educational score: 1.0033072233200073 Domain: other Document type: Other Language: en Gaelle M. Emvalomenos: Writing – review & editing, Visualization, Investigation, Formal analysis, Data curation. James W.M. Kang: Writing – review & editing, Writing – original draft, Visualization, Investigation, Formal analysis, Data curation. Sabrina Salberg: Writing – review & editing, Investigation, Data curation. Crystal Li: Writing – review & editing, Investigation, Data curation. Bianca Jupp: Writing – review & editing, Resources, Methodology, Conceptualization. Matthew Long: Resources, Methodology. Mohammad B. Haskali: Resources, Methodology. Sunil Kellapatha: Resources, Methodology. OIivia I. Davanzo: Visualization, Investigation. Hyunsol Lim: Visualization, Investigation. Richelle Mychasiuk: Writing – review & editing, Supervision, Resources, Conceptualization. Kevin A. Keay: . Luke A. Henderson: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. Section title: Funding Educational score: 1.0118926763534546 Domain: other Document type: Other Language: en This work was supported by funding from the Australian National Health and Medical Research Council . Section title: Declaration of competing interest Educational score: 0.9818206429481506 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Review | biomedical | en | 0.999998 |
PMC11699484 | Section title: Introduction: the Yamnaya phenomenon and its origins Educational score: 2.593174934387207 Domain: other Document type: Study Language: en Recent large-scale studies of ancient DNA (aDNA) show that the Bronze Age populations in the steppes of present-day Russia and Ukraine migrated across both Europe and Asia during the Early Bronze Age (EBA) and the Middle Bronze Age (MBA) in the chronology of the western steppes . Between 3100–2500 BCE the Yamnaya culture expanded out of its steppe homeland westward into central Europe, where people with ca. 70% Yamnaya ancestry created the Corded Ware horizon ; and eastward to the Altai Mountains, where people almost identical to the Yamnaya population in genetic ancestry appeared as the Afanasievo culture . Today the origin and nature of the Yamnaya archaeological culture is a question with new relevance across northern Eurasia. Yet none of the key archaeological sites most important for understanding the evolution of Yamnaya customs and economy is published in a western language. These key sites would include Khvalynsk on the Volga (Eneolithic), Repin on the Don (EBA), and Mikhailovka (EBA) on the Dnieper rivers. 2 This essay is about Khvalynsk, an Eneolithic cemetery on the Volga River dated ca. 4500–4300 BCE. Section title: Introduction: the Yamnaya phenomenon and its origins Educational score: 4.143272399902344 Domain: biomedical Document type: Study Language: en In this report we present new aDNA-based studies of the Khvalynsk population in combination with traditional archaeological and anthropological studies. The formal presentation of the ancient DNA data along with a comprehensive set of genetic analyses will be made in a separate publication; here, we summarize results where they are relevant to understanding the Khvalynsk cemetery population. In addition, we present specific analytical summaries of radiocarbon dates and stable isotopes, copper artifacts, animal sacrifices, and polished stone maces. We argue that Khvalynsk exhibits remarkable diversity in its population and equally remarkable segmentation between groups of individuals in its grave offerings . We discuss how the Khvalynsk cemetery was related to other sites and archaeological cultures during the steppe Eneolithic. We also discuss cranio-facial types, skeletal pathologies, and new data from aDNA on family relationships within the Khvalynsk cemetery, with broader comments on the evolution of “steppe ancestry” that later characterized the Yamnaya populations. Section title: Introduction: the Yamnaya phenomenon and its origins Educational score: 2.021413564682007 Domain: other Document type: Study Language: en Western archaeologists have asked if, by applying a cultural label such as ‘Yamnaya’ to a biological unit of analysis, as in ‘the typical Yamnaya pattern of genetic ancestry’, we imply that all Yamnaya-culture individuals had not only the same ancestry, but the same pottery, ornaments, and economy . However, to speak of a typical measurement or pottery type is never to imply that no outliers or variation existed; indeed, the concept of the average or typical observation implies the opposite. The Eneolithic populations of the Pontic-Caspian steppes (north of the Black and Caspian Seas) can be divided into regional groups defined by both material/cultural customs and genetic/morphological traits. Funeral customs (body pose, grave shape) varied regionally, as did pottery styles and economies. Ceramic types were more varied than cranio-facial or genetically defined groups. Figure 2 shows that Khvalynsk-style ceramics were the predominant ceramic type only in the middle and lower Volga steppes, while people with genetic ancestry like Khvalynsk can be found 1000 km to the south in the North Caucasus steppes. The Khvalynsk mating network extended far beyond the Khvalynsk pottery style. Section title: Introduction: the Yamnaya phenomenon and its origins Educational score: 3.950871229171753 Domain: biomedical Document type: Study Language: en Anthony refers to groups defined by similarities in their aDNA as mating networks . A mating network is a population that shared a distinctive cluster of autosomal genetic traits such that individuals from that chronological period and region can be assigned to a space in a principal components analysis (PCA) plot that does not overlap significantly with the spaces occupied by other contemporary mating networks. Mating networks were maintained by the long-term, multi-generational exchange of daughters and/or sons as mates, creating significant gene flow between groups within the network. It cannot be assumed that mating networks were culturally relevant or even known to ancient populations, although borders between mating networks probably were recognized. Section title: Introduction: the Yamnaya phenomenon and its origins Educational score: 2.940364122390747 Domain: other Document type: Study Language: en One interesting aspect of the Yamnaya mating network was its narrowness, more homogeneous (a more restricted space in a PCA) than the earlier Eneolithic populations. This reduction in genetic diversity between Eneolithic and EBA populations is interesting partly because EBA material culture (archaeology) exhibited contradictory trends at this transition—Yamnaya funeral rituals became more standardized and homogeneous than in the Eneolithic, like Yamnaya aDNA; but many regional Eneolithic artifact types, including regional ceramic types, continued into the EBA and appeared as regional variants that contrasted with the genetic and ritual homogeneity of Yamnaya ancestry and grave types. In the Pontic-Caspian steppes, mating networks and cultural traditions had a dynamic, changing relationship. Khvalynsk provides a data-rich window through which to examine the material and genetic variability of the pre-Yamnaya population at one of the largest and most important Eneolithic cemeteries. Section title: The importance of Khvalynsk Educational score: 4.011945724487305 Domain: biomedical Document type: Study Language: en Khvalynsk is the largest excavated Eneolithic cemetery in the Don-Volga-Ural steppes (201 recorded graves). It has the largest copper assemblage of the late fifth millennium BC in the steppes (373 objects) and the largest assemblage of sacrificed domesticated animals (at least 106 sheep-goat, 29 cattle, and 16 horses). The human skeletons have been sampled extensively for ancient DNA, but genome-wide data from only three individuals has been published to date . Here we discuss relevant results from 32 analyzed individuals. The whole genomes of three additional Eneolithic individuals from graves in the North Caucasus steppes dated 4400–4100 BC at the Progress-2 and Vonyuchka-1 3 cemeteries, broadly contemporary with Khvalynsk, were previously recognized as similar in ancestry and in PCA space to the published three from Khvalynsk . This discovery expanded the range of the Khvalynsk mating network 1000 km to the south, from the Middle Volga steppes to the North Caucasus steppes. Unpublished samples from Volga Eneolithic cemeteries do not significantly alter the relationships or PCA space observed in the initial published samples, but rather form a cline between Khvalynsk and Progress-2. The Khvalynsk/Progress-2 ancestry cline represents a distant genetic ancestor, although not the exclusive or proximate ancestor, for the typical pattern of genetic ancestry exhibited in Yamnaya individuals. Yamnaya individuals cluster near Khvalynsk/Progress-2 in PCA space, but their distributions overlap only marginally . Yamnaya genomes had additional Anatolian Farmer ancestry (typical for agricultural populations in southeastern Europe and the North Caucasus) not present in Khvalynsk/Progress-2 . Khvalynsk is important because of its size, its unique concentration of copper artifacts and domesticated animal sacrifices, and its genetic and ritual connections with the Yamnaya culture. Section title: The importance of Khvalynsk Educational score: 2.1344850063323975 Domain: other Document type: Study Language: en When Khvalynsk was discovered in 1977 it was recognized immediately, and with some excitement, as a good candidate for the elusive pre-Yamnaya archaeological phase in the Volga steppes . The shell-tempered, round-bottomed pottery of Khvalynsk, decorated with small comb stamps and shell-edge impressions, was like one of the earliest Yamnaya pottery types, found in Yamnaya kurgans in the southern Volga steppes . Also, the position of the body in most graves, on the back with tightly raised knees, was identical to a distinctive Yamnaya body pose often called ‘the Yamnaya position’ . The abundant red ochre on the grave floor also was like Yamnaya. The differences between them (ornament and weapon types, absence of kurgans at Khvalynsk) were ascribed to the earlier chronological position of Khvalynsk . It was anticipated that Khvalynsk would have radiocarbon dates in the early to middle fourth millennium BCE , not long before the oldest cluster of Yamnaya radiocarbon dates, 3300–3000 BCE. But radiocarbon dates calculated on human bone instead suggested that Khvalynsk dated ca 5200–4500 BC, almost 2000 years older than Yamnaya . Archaeologists did not yet know that radiocarbon dates on Eneolithic human bones were skewed older by the absorption of old carbon in the bones of populations that regularly ate riverine fish, a phenomenon now known as a freshwater reservoir effect (FRE). The long chronological gap between Khvalynsk and Yamnaya was regarded uneasily as a ‘hiatus’ . Here we show that Khvalynsk was in use about 4500–4300 BCE, about 1000 years before the Yamnaya culture appeared, contemporary with Skelya and early Sredni Stog 4 in the Pontic steppes and Varna I in the Danube valley. Section title: The importance of Khvalynsk Educational score: 1.0620571374893188 Domain: other Document type: Other Language: en A significant chronological gap still exists between Khvalynsk and Yamnaya. Subsequent discoveries of a few graves similar in ritual details to Khvalysnk and dated to the fourth millennium BCE have filled the gap to some extent, but the early fourth millennium BCE remains surprisingly poorly documented in the Volga-Ural steppes . This intermediate period is better documented in the North Caucasus steppes and the Black Sea steppes west of the Don River, where the Sredni Stog culture introduced Khvalynsk-like grave rituals with Khvalynsk-like DNA traits 4500–3500 BCE . Sredni Stog is often seen as an ancestor of Yamnaya in Ukraine . Section title: The importance of Khvalynsk Educational score: 0.9888182282447815 Domain: other Document type: Other Language: en Other Eneolithic cemeteries were smaller than Khvalynsk, usually less than 30 graves. Khlopkov Bugor, 130 km south of Khvalynsk on the west Volga bank, had 24 excavated Eneolithic graves, including one person who was a 2 nd -degree relative of a person buried at Khvalynsk (see below). A cemetery on the Volga 160 km north of Khvalynsk, at Ekaterinovka Mys, dated 150–200 years earlier, had more than 100 excavated graves . Nalchik in the North Caucasus, approximately contemporary with Khvalynsk, had 121 graves . A few other large Eneolithic cemeteries are known in the Volga-Caucasus steppes, but Khvalynsk was the largest. Section title: The importance of Khvalynsk Educational score: 2.825833559036255 Domain: other Document type: Study Language: en The exceptional size of the Khvalynsk cemetery, as well the morphological (according to cranio-facial measurements) and genetic heterogeneity of those interred there, were linked to its integrative ritual position during an era of population movements and economic change in the Volga steppes. The Khvalynsk people showed ancestry from a southern population that can be derived, using both cranio-facial measurements and aDNA, from regions including the Caucasus and the lower Don steppes . These southern-derived people mingled in the Volga steppes, around Khvalynsk and south of Khvalynsk, with a population that can be derived, using both cranio-facial and aDNA data, from the northern forest zone . The admixed population that resulted from this north-south combination gathered at Khvalynsk to conduct funeral activities, feast on the meat of newly acquired domesticated animals, and celebrate alliance-making symbols (polished stone maces, see below). The earliest domesticated animals appeared in the middle Volga steppes around 4800–4600 BCE (using dates on animal bones), just 100–300 years before Khvalynsk . Khvalynsk appears to have been a central place for the performance of these relatively new sacrificial rituals, a gathering place for genetically diverse populations participating in a new funeral cult focused on the ritual power and value of domesticated animals. Section title: The importance of Khvalynsk Educational score: 1.2333275079727173 Domain: other Document type: Other Language: en Copper ornaments were another introduced innovation used in a new way. Khvalynsk was part of a network of cultures that participated between 4500–4200 BC in the exchange of copper, exotic shells, domesticated animals, and emerging symbols of hierarchical leadership (polished stone maces) between the Volga steppes, the North Caucasus steppes, the Dnieper steppes, and the tell towns of the lower Danube valley and the Varna region in Bulgaria . The agricultural communities of the Karanovo VI/Gumelniţa/Tripol’ye B1 period were the copper-producing centers of the network, which Chernykh named the ‘Carpatho-Balkan Metallurgical Province’. Khvalynsk is the easternmost site included in this ‘province’ . Khvalynsk is thus an essential site for understanding the introduction of domesticated animals and copper metallurgy to the steppes, and the genetic, morphological, and cultural origins of the population that would later become Yamnaya. Up to now, no English-language summary of the site exists beyond short descriptions contained in longer works . Section title: History and ecological setting of Khvalynsk I and II Educational score: 0.9781113862991333 Domain: other Document type: Other Language: en Before 1971, when the hydroelectric dam was completed at Balakovo 40km downstream, the Khvalynsk cemetery was located on the west bank of the Volga River 16 km south of the town of Khvalynsk at 52°21’14.58”N, 48° 4’44.80”E . The site is now under the Saratov Reservoir created by the dam. Here dry limestone hills overlooked the Volga on its west side, and a flat steppe plain rolled away to its east. The Eneolithic cemetery was at the foot of the western hills at a place where their white peaks stood 3–5 km away from the river. The Volga River, more than a kilometer wide, flowed through a wetland of large forested islands and marshes 5km wide at this location, now a 5km-wide reservoir . Section title: History and ecological setting of Khvalynsk I and II Educational score: 2.0490949153900146 Domain: other Document type: Study Language: en Vegetation varied significantly between wooded ravines on the high west bank, a flat steppe on the east bank, and forests and marshes on the Volga bottomlands. At the Eneolithic settlement of Lebyazhinka VI north of Samara, with radiocarbon dates contemporary with Khvalynsk , the most frequent fish bones were those of northern pike ( Esox lucius ), which in this region can weigh up to 25 kg; followed by catfish ( Silurus glanis ), up to 300 kg; and zander ( Sander lucioperca ), up to 20 kg . Large birds whose tubular bones were used for flutes or whistles in the Khvalynsk graves included white-tailed eagles ( Haliaeetus albicilla ), bustards ( Otis tarda ), cranes ( Grus grus ), and swans ( Cygnus sp .) . Broad-leaf forests on the 5-km-wide floodplain hosted moose ( Alces alces ), red deer ( Cervus elaphus ), roe deer ( Capreolus capreolus ), wild boar ( Sus scrofa ) and aurochs ( Bos primigenius ), as well as smaller game . East of the gallery forest a dry steppe plain rolled unbroken to the Altai Mountains in Siberia. In the Volga-Ural region, the steppes contained wild horses ( Equus caballus ) in the northern steppes near Khvalynsk and saiga antelope ( Saiga tartarica ) and onagers ( Equus hemionus ) in the drier southern Volga steppes bordering the Caspian Sea . Section title: History and ecological setting of Khvalynsk I and II Educational score: 0.938602864742279 Domain: other Document type: Other Language: en Erosion of the western bank after the reservoir was filled led to the discovery and salvage excavation in 1977–79 of the northern cemetery of 158 individuals, later designated Khvalynsk I . An unknown number of graves was lost before the archaeologists arrived. The director was I.B. Vasiliev, the energetic leader of the archaeology faculty at the Samara (then Kuibyshev) Pedagogical Institute, now the Samara State Social-Pedagogical University. The Volga continued to erode the cemetery during the excavation, swallowing 4m in the two years between 1977 and 1979, indicated in Figures 4 and 15 by the two parallel lines on the right side of Khvalynsk I. The graves were isolated in the center of the explored area, with no additional graves found to the north, south, or west, leading the archaeologists to believe that they had established the limits of the cemetery on all sides except the rapidly eroding east . A report was published in the Russian language in 1990 describing each grave and surface sacrificial deposit. Section title: History and ecological setting of Khvalynsk I and II Educational score: 0.9165042638778687 Domain: other Document type: Other Language: en Khvalynsk II was discovered 10 years after Khvalynsk I when continuing erosion exposed additional human skeletons 120m southwest of the first cemetery. Khvalynsk II was excavated in 1987–1988 by many of the same archaeologists from Samara (Kuibyshev) who had worked on Khvalynsk I, including A. Khokhlov and S. Agapov, co-authors of this report. I.B. Vasiliev again directed the first part of the excavation, and V.I. Pestrikova directed the second part. The Khvalynsk II excavation recovered 43 individuals , again after an unknown number were lost to the Volga. A monograph describing both Khvalynsk I and II was published 22 years after the excavation ended . It contains many specialist studies (lithics, ceramics, metallurgy, fauna, shells, skeletal measurements, etc) in the Russian language. Pestrikova’s unpublished dissertation on Khvalynsk I was revised by D. Agapov for the opening essay of the 2010 monograph . The 1990 and 2010 monographs are the most important sources of information for this report. Section title: History and ecological setting of Khvalynsk I and II Educational score: 1.1037300825119019 Domain: other Document type: Other Language: en The funeral rituals (body pose, grave form, use of ochre, etc), ceramic types, lithics, ornaments, and radiocarbon dates from the two cemeteries are alike. Khvalynsk I and II were used during the same era by people from the same archaeological culture. But the demographic traits of I and II were sharply different. At Khvalynsk I males and females were buried in nearly equal numbers. Among 83 adults or adolescents to whom a sex could be assigned based on skeletal features, 45 (54%) were males and 38 (46%) were females . Copper ornaments accompanied six adult males and five adult females at Khvalynsk I, again about equal, and were found with one infant and two adolescents. Section title: History and ecological setting of Khvalynsk I and II Educational score: 2.2317140102386475 Domain: other Document type: Study Language: en The Khvalynsk II burial plot, in contrast, was heavily weighted toward adult males: more than three adult males for each adult female. Of 26 skeletons assignable to a sex, 20 (77%) were males and only six (23%) were females . This was much like the proportion of males and females buried later in Yamnaya graves in the Volga steppes (80% males). This raises the question if Yamnaya gender practices evolved not from the preceding ‘culture’ but specifically from an Eneolithic sodality or other male-focused sub-group like the one buried in the Khvalynsk II cemetery. The absolute number of copper ornaments buried with 158 individuals at Khvalynsk I (35) was one tenth of the number found with 43 individuals at Khvalynsk II (338) . The abundant copper at Khvalynsk II accompanied about one third of both males and females: nine adult males and two adult females. Copper ornaments also were found with an infant and a child. Section title: History and ecological setting of Khvalynsk I and II Educational score: 1.0184884071350098 Domain: other Document type: Other Language: en Khvalynsk I looks like a ‘family’ cemetery where all ages and sexes were buried, while Khvalynsk II was a more specialized cemetery for a copper-rich group of males (possibly warriors or traders), most of whom were related by family descent (see genetics section below), with a few unrelated males, females and immatures. Section title: History and ecological setting of Khvalynsk I and II Educational score: 1.7182769775390625 Domain: other Document type: Study Language: en Unfortunately, another difference between I and II is that most of the skeletons from I were lost in a Volga River flood that destroyed the storage area where they were curated in the river-port city of Samara. One might say that the Volga reached for these graves twice. Contemporary studies, including ancient DNA studies, can be conducted only on male-dominated II and the small number of individuals from I that survived the flood (9 of 158—see Table 1 radiocarbon dates). Section title: Radiocarbon chronology and stable isotopes Educational score: 1.105140209197998 Domain: other Document type: Other Language: en Twenty radiocarbon dates were previously published from Khvalynsk: ten from Khvalynsk I and ten from Khvalynsk II ( Table 1 ). Most were in Russian-language publications . Some dates were reported from the Soviet-era laboratory UPI, which operated briefly in Ekaterinburg in the 1980s, but never was included in the journal Radiocarbon ’s global list of current and former radiocarbon laboratories. The 20 published dates are compiled here for the first time. Also, 28 new dates are presented here for the first time. Section title: Radiocarbon chronology and stable isotopes Educational score: 3.11505389213562 Domain: biomedical Document type: Study Language: en For Khvalynsk I, the ten dates previously published were from the Kiiv, UPI, and Groningen laboratories ( Table 1 ). Three dates on shell beads are clearly subject to a variable freshwater reservoir effect; they are usually ignored in discussions of Khvalynsk chronology. Four new dates from the Pennsylvania State University Accelerator Mass Spectrometry laboratory (PSUAMS) are presented here, making 14 dates from Khvalynsk I. For Khvalynsk II, eight dates were published previously by the Oxford, Groningen, and Arizona laboratories, and two dates by PSUAMS . To these ten previously published dates we now add 24 new PSUAMS dates from Khvalynsk II, making 34 dates from Khvalynsk II ( Table 1 ). Table 1 presents 48 dates for the Khvalynsk cemetery, 34 from Khvalynsk II and 14 from Khvalynsk I, including 28 new dates. In addition, Table 1 presents data on dietary stable isotopes (δ 13 C and δ 15 N) from 30 individuals, not all dated by radiocarbon. Section title: Radiocarbon chronology and stable isotopes Educational score: 1.6641744375228882 Domain: other Document type: Study Language: en Most of the dates are on human bones or teeth. This is a problem, because studies by Shishlina and van der Plicht have shown that radiocarbon dates from Eneolithic human bones can be more than 1000 years too old in this region, a result of freshwater reservoir effects (FRE) . Therefore, most of the radiocarbon dates in Table 1 are skewed too old. Section title: Radiocarbon chronology and stable isotopes Educational score: 3.9824748039245605 Domain: biomedical Document type: Study Language: en We have direct evidence of such skewing from two graves published by Shishlina et al. , one at Khvalynsk I and the other at Khvalynsk II, with radiocarbon dates from domesticated cattle and sheep bones, not subject to reservoir effects . Calibrated, the two samples produced statistically the same age: 4450–4350 BCE. A date of 4450–4355 BCE was obtained on a ring made of sheep bone from grave 147 at Khvalynsk I . The human female buried with this bone ring was dated 4789–4618 BCE , about 300 years older ( Table 1 ). The second date, 4448–4362 BCE , was obtained on a cow bone from grave 10 at Khvalynsk II. The human female in this grave was dated by Oxford to 4730–4530 BC , about 300 years older; but recently has been re-dated to 5210–5017 BC , about 600 years older than the cow bone in the same grave ( Table 1 ). It seems possible that a mistake was made in labeling one of these two human samples, but in any case, we can be confident that both came from Khvalynsk II. 5 Section title: Radiocarbon chronology and stable isotopes Educational score: 3.840442419052124 Domain: biomedical Document type: Study Language: en The offsets between faunal and human dates from the same grave indicate the presence of an FRE, in which consumption of aquatic resources (fish, shellfish, aquatic birds) leads to the incorporation of ‘old carbon’ into human tissues . Therefore, the faunal dates of 4450–4355 calBCE from Khvalynsk I and 4448–4362 calBCE from Khvalynsk II provide the best estimate currently available for the true age of the two cemeteries. If we compare the midpoint of these dates, ca. 4400 calBCE, to the midpoints of the calibrated age ranges for the humans, the resulting offsets range between 43 yr (essentially no offset given the inbuilt uncertainty in radiocarbon dating) and 860 yr, with a mean FRE offset of 401 ± 288 yr ( Table 1 ). Section title: Radiocarbon chronology and stable isotopes Educational score: 3.7481586933135986 Domain: biomedical Document type: Study Language: en The details of the genetically-determined family trees at Khvalynsk are examined below . Here our narrow purpose is to use family relationships as a chronological check on the FRE connected with radiocarbon dates. In three of the five families at Khvalynsk II (Grey, Purple, and Orange), individuals who were nearly contemporary (1st to 3rd degree relatives) have 14 C dates more than 100 years apart, and the older 14 C dates are associated with lower δ 13 C values. In the extreme case (Orange), a father and son are dated minimally 247 years apart (between the 95% confidence intervals of the two dates), and the older date is linked to a lower δ 13 C value. The dates for related individuals confirm that the 14 C dates at Khvalynsk II do not identify contemporary graves, so they are not reliable relative to each other. However, in some related pairs of individuals the older 14 C date (indicating depleted 14 C) is from the individual with lower δ 13 C values (indicating depleted δ 13 C). Section title: Radiocarbon chronology and stable isotopes Educational score: 4.193973064422607 Domain: biomedical Document type: Study Language: en This is reflected in a moderate negative correlation between δ 13 C values and calibrated age, accounting for nearly half the variation in the latter ( r 2 = 0.474, p < 0.001, n = 29) . There is a clear outlier (K-I, grave 17) with a predicted offset removed by nearly three standardized residuals from the assumed date of 4400 calBCE. Its removal improves the regression considerably ( r 2 = 0.663, p < 0.001, n = 28; the slope of the regression line remains similar). Extending the slope to the y-intercept suggests that a diet with no 14 C offset would result in a δ 13 C value of ca. −20.3‰ (or ca. −20.0‰ if the outlier is excluded). In contrast, there is no relationship between δ 15 N values and calibrated age ( r 2 < 0.001, p = 0.995, n = 29) . This is unexpected, since aquatic foods are typically significantly 15 N-enriched compared to terrestrial flora and fauna , and therefore a positive relationship with radiocarbon offsets is often observed . Since most of the analyses were made on the petrous bone, the core of which forms in infancy and does not remodel , it is possible that some samples retain a partial nursing signal , which could obscure the relationship between δ 13 C and δ 15 N values. Section title: Radiocarbon chronology and stable isotopes Educational score: 3.8914060592651367 Domain: biomedical Document type: Study Language: en However, the relationship between radiocarbon offsets and both δ 13 C and δ 15 N values is complex . Aquatic systems are often 13 C-depleted, as seems to be the case on the Volga, but they may also be elevated relative to C 3 terrestrial ecosystems . And fish from adjacent watersheds, or even different parts of the same river, can exhibit variable 14 C offsets, leading to different relationships with both stable isotopes . In the Upper Lena river system north of Lake Baikal, Siberia, a program of paired human–fauna dating from the same graves identified a comparable relationship in which 14 C offsets were better predicted by δ 13 C values than by δ 15 N . This differed from Lake Baikal itself, where both isotopes were significant predictors, but δ 15 N accounted for the larger amount of the variability in 14 C offsets. Section title: Radiocarbon chronology and stable isotopes Educational score: 2.61639666557312 Domain: biomedical Document type: Study Language: en The implication of the variability in the FRE at Khvalynsk is that individuals were acquiring aquatic resources from different catchments, subject to different 14 C reservoir offsets. This is consistent with the cranio-facial metric and genetic data indicating that the cemetery served for communities of different origins to the south and to the north, though it places this within a context of the immediate lifetimes of individuals rather than their more distant ancestry. One possibility is that such access was held within families or clans, as was the case with the best fishing places on the salmon rivers of the Interior Plateau culture area of northwestern North America . If so, we might expect to see a link between the genetic ancestry evidence and the FRE offsets. Section title: Radiocarbon chronology and stable isotopes Educational score: 4.1258864402771 Domain: biomedical Document type: Study Language: en While this is not the case for Y-chromosome haplogroups, there is some indication of such a relationship between the estimated FRE offset and mitochondrial haplogroups. Limiting the comparison to haplogroups with more than five samples, the estimated mean FRE offsets relative to the faunal date of 4400 cal BC differ significantly for mt-haplogroups U2, U4 and U5 (ANOVA, F = 4.268, p = 0.031, n = 20). Bonferroni post-hoc tests show that the significant difference is between U2 and U4 ( p = 0.029), with mean 14 C offsets of 295 ± 256 yr and 624 ± 114 yr, respectively . Note that the same result would obtain if the means of the calibrated dates were used directly, since the same offset is applied to all the individuals. Section title: Radiocarbon chronology and stable isotopes Educational score: 3.837775945663452 Domain: biomedical Document type: Study Language: en The Volga River appears to have been depleted in both δ 13 C and 14 C in some of its catchments, creating a mild correlation between older ages and lower δ 13 C in the bones of people who regularly ate Volga fish from those parts of the river. The maternal mtDNA haplogroup U2 (represented in two lineages, U2e1b and U2e2a) differed significantly from U4 in its smaller average FRE offsets (with U5 being intermediate), perhaps suggesting that U2 females came from a riverine catchment with less depleted δ 13 C and 14 C. Females with U2 maternal ancestry occur in both Khvalynsk I and II ( Table 6 ). The richest grave at Khvalynsk II contained an older brother (Khvalynsk II:24) and a younger sister (II:25) who carried U2 mtDNA ancestry. Section title: Radiocarbon chronology and stable isotopes Educational score: 3.8484671115875244 Domain: biomedical Document type: Study Language: en The mean faunal date of 4400 calBC probably is the most accurate estimate of the midpoint date for the Khvalynsk cemetery. A relatively short span of time is suggested by the fact that 70% of the individuals analyzed from Khvalynsk II were related to other individuals in ways that could fit within a 5-or-6 generation span, or about 140–170 years . Stable isotopes indicate a diet in which riverine fish played a large role, causing a strong FRE in radiocarbon dates on human bones and teeth. Variation in δ 13 C seems to identify Volga riverine catchments that were depleted in carbon. δ 13 C also correlated with mtDNA haplogroups, suggesting that females at Khvalynsk came from different riverine catchments, while the men’s Y-haplogroups did not display such patterning. Section title: Copper artifacts and trade Educational score: 1.8752251863479614 Domain: other Document type: Study Language: en The two Khvalynsk cemeteries together yielded 373 copper objects, the largest assemblage of copper items from any Eneolithic cemetery in the steppes. Almost all were ornaments (beads, rings, or bracelets) made of hammered sheet copper or wire, bent into tubes and rings. Four melted lumps of copper in two graves at Khvalynsk II were possibly unshaped, primitive trade ingots or possibly were evidence of local production (but Khvalynsk pyrotechnology probably was not sufficient for production, see below). Two similar lumps, interpreted as ‘ingots’, were found at Khvalynsk I in the ‘cultural stratum’, but were not associated with a specific grave . As noted above, the number of copper objects at Khvalynsk I (35) was one tenth of the number at Khvalynsk II (338). The count of 338 objects from Khvalynsk II includes 332 preserved objects and an additional six copper stains/traces that were recorded during the excavation but could not be catalogued . Similarly, the count of 35 from Khvalynsk I includes one grave distinguished only by a copper stain. It is necessary to include the stains to identify the individuals who had copper objects. At Khvalynsk I, 9% of the individuals (15/158) had copper objects on or near their bodies, about one in ten; and at Khvalynsk II 30% of individuals (13/43), about one in three. Adding the two cemeteries together, 28 individuals (14% of 201) had at least one copper object. Section title: Copper artifacts and trade Educational score: 1.473376989364624 Domain: other Document type: Study Language: en Within the 14% minority that had access to copper ornaments, most had one to four pieces . A single bead of copper was an important find at Khvalynsk; presumably, it was just as important to the person who wore it. Most of the pieces were combined into sets such as beads strung together or connected rings made into a hanging ornament. One male aged 20–30 in Khvalynsk II: grave 12 was buried with 297 copper objects, most of them (293) simple copper beads strung on at least two necklaces also adorned with small sheet-copper oval pendants . This single individual had 80% of the copper objects found in both cemeteries combined. If we exclude grave II:12 to see if it alone was responsible for the difference between Khvalynsk I and II, Khvalynsk II still would have 41 copper objects, more than Khvalynsk I (35) in one third the number of graves. A higher proportion of graves at Khvalynsk II (1/3 compared to 1/10) contained copper objects, so even without II:12 the two cemeteries differed significantly in their access to copper. Section title: Copper artifacts and trade Educational score: 1.6762810945510864 Domain: other Document type: Other Language: en The copper-rich male in grave II:12 was the brother of the male in II:13, and the uncle of the male in grave II:22, who was the son of II:13 ( family relationships below). The brothers in II:12&13 were the center of a cluster of seven related males that included II:4, II:7, II:22, II:27, and II:31 as second or third-degree relatives . This patriline accounted for one of seven individuals at Khvalynsk II, the largest single family identified, and modeling described below suggests that the relationships within it should be distributed over four or five generations, so it was a persistent presence over more than 100 years. Its wealth in copper could have been related to its central position in the male-dominated group buried at Khvalynsk II. No female relatives—no mothers, daughters, sisters, or female cousins of the seven related men were buried with them. Khvalynsk II could have been a burial place for a multi-generational male sodality or society engaged in long-distance expeditions that brought Balkan copper to the Volga. The paternally central man in II:12 had much more copper than anyone else at Khvalynsk. Section title: Copper artifacts and trade Educational score: 2.929882287979126 Domain: other Document type: Study Language: en Family relationships also might suggest that the beginning of the Balkan copper trade occurred suddenly on the Volga, with copper changing from absent to abundant over the span of two generations, between grandparent and grandchild. The male in grave II:4 was a second-degree relative of a female in grave 7 at Khlopkov Bugor (KB7), a Khvalynsk-culture cemetery 130 km south near Saratov. No copper was found at Khlopkov Bugor, so it is generally thought to be older than Khvalynsk, although the artifact and ceramic types are quite similar. (We established above that their radiocarbon dates are variably affected by FRE and cannot be relied on to indicate their relative age.) If Khlopkov Bugor was older, then the Yellow-family female KB7 was a paternal grandmother or paternal aunt (given their different mtDNA haplogroups) of the male at Khvalynsk II:4. The chronological difference between them was no more than two generations, perhaps 50–60 years. If the absence of copper at Khlopkov Bugor is explained by its earlier position, then the copper trade began suddenly and abundantly when the Khvalynsk cemetery began to be used, about 4500 BCE. Section title: Copper artifacts and trade Educational score: 2.127347946166992 Domain: other Document type: Study Language: en An artifact linked to the copper-using minority was the bird-bone tube, possibly used as a flute or whistle . With one exception (II:4, the Yellow-family male related to KB7) bird-bone tubes appeared only in graves with copper ornaments, and only with adult males, or in one case, an adolescent buried with an adult male . They were not modified to create musical notes—they had no holes—so their function is uncertain. At Khvalynsk I, graves 19, 30, 57 , and 90 had bird-bone tubes , and Table 3 shows that all these graves contained copper ornaments with a male (or an adolescent). At Khvalynsk II, only grave 24 was described by the zoologist Bogatkina as containing a bird-bone tube, and this was the richest grave at Khvalynsk, discussed below, belonging to a male equipped with many copper items. The Moscow zoologist Kirillova found five more bird-bone tubes in collections that had moved to Moscow, from four graves at Khvalynsk II:4, 13 (two tubes), 18, and 35. All nine individuals in both cemeteries with bird-bone tubes were adult males (or an adolescent buried with an adult male), and all but one (II:4) were buried with copper artifacts ( Table 3 ). It interesting that II:4 is modeled in the Yellow family relationships as the oldest Yellow family grave at Khvalynsk II, so perhaps the copper trade had not yet started when II:4 died. Kirillova specified that the bones were ulnas from large birds, which she tentatively identified as a swan, a white-tailed eagle, and three bones that were in the size class of swan-crane-bustard, among locally available large birds. Two of the three mace graves at Khvalynsk (see below) contained bird-bone tubes, which seem to have symbolized an office or status among the copper-using men (and one boy) at both Khvalynsk I and II. This restriction in the use of bird-bone tubes was one of many shared customs that connected I and II in the same ‘culture’. Section title: Copper artifacts and trade Educational score: 3.3264787197113037 Domain: other Document type: Study Language: en Balkan ores probably were the source of the copper imported to Khvalynsk, although most of the imported metal was worked into rings and beads by local artisans. A Balkan source is surprising given the distance between Khvalynsk and the lower Danube valley. But ‘clean’ Balkan ores, specifically copper ores of groups B1-B2 and B3-B6 from Ai Bunar in Bulgaria, match the trace elements in Khvalynsk copper better than Caucasus ores do . Courcier argued that relatively ‘clean’ copper ores also were found in the Caucasus in some Chalcolithic artifacts, as at Menteshtepe . But the Menteshtepe ‘clean’ copper had trace amounts of arsenic measured in the high tenths of one percent (range 0.6–0.9% arsenic). E.N. Chernykh analyzed 41 copper objects from Khvalynsk with methods capable of detecting arsenic, and only ten (12.2%) had any arsenic trace elements; more than 80% had no detectable arsenic. Of the ten exhibiting some arsenic, seven were in the range 0.0034–.1% , like the copper from Cucuteni-Tripolye sites, which ranged 0.007–0.1% . The trace elements in 70% of the tested Khvalynsk copper objects with arsenic fell into the range of the trace elements in Balkan copper rather than Caucasian copper. Three of the ten tested objects had arsenic outside the range of the tested Balkan copper objects, but not by very much: 0.2, 0.3, and 0.42. These three rings all were worn by adult females. Their slightly elevated arsenic might have resulted from a mixture with copper from Caucasian ores, so might indicate trade with the south. Section title: Copper artifacts and trade Educational score: 1.1907325983047485 Domain: other Document type: Other Language: en ‘Clean’ oxide copper ores are abundant locally in the Volga-Ural steppes , not far from Khvalynsk, but ore mining and smelting probably was not yet possible locally during the Eneolithic. To smelt copper from a multi-mineral sandstone ore usually requires charcoal heated to 1200–1300 °C, much higher than the maximum temperature (700–800 °C) attained in making Khvalynsk ceramics . Khvalynsk pyrotechnology probably was not sufficient to smelt local copper oxide ores, which began to be mined in the Yamnaya period, by present evidence . Eneolithic experimentation with metallurgy ultimately led to the beginning of extractive copper ore mining and productive metallurgy in the steppes during the fourth millennium BCE. Section title: Copper artifacts and trade Educational score: 1.3726173639297485 Domain: other Document type: Other Language: en At least five copper ornaments examined by Ryndina were made at temperatures of 900–1000° C and must have been imported as finished objects; three of these were spiral rings like ornaments at Varna . But most of the other copper beads and rings were shaped at temperatures between 300–800° C, were rather crudely finished, and seem to have been bent and welded into shape locally (but using imported metal). Ryndina noted that the methods used for wire-making and welding on the Khvalynsk copper artifacts seem to have been copied after the methods used by Tripol’ye A and B1 metalsmiths, including the same welding method (adding a small strip of heated copper), but the Khvalynsk artisans used lower working temperatures, their work was cruder, and their welds often failed to join completely. One individual at Khvalynsk II: 21 was named ‘the smith’ by the excavators because his grave contained an unworked lump of copper , an antler hammer and a grooved stone hammer that might have been used to make sheet copper, and a beaver incisor that could have been used as an edge tool to cut sheet copper. He was not related to any of the known families at Khvalynsk II but had similar genetic ancestry. Section title: Copper artifacts and trade Educational score: 1.7354178428649902 Domain: other Document type: Study Language: en Rassamakin proposed that the Dnieper Rapids region emerged in this era as a secondary center of ‘Skelya-culture’ metalworking between Varna and the North Caucasus steppes. Most of the Khvalynsk copper is consistent with this kind of secondary source, among local steppe artisans. This could also be the source of a copper bead found at Svobodnoe, made of Balkan copper . Svobodnoe was one of a series of agricultural settlements established in the Kuban River drainage after 4700 BCE by immigrant farmers who crossed the North Caucasus peaks from Georgia . They participated in the trading network that brought Balkan copper into the steppes. Svobodnoe also produced many polished greenstone axes with faceted butts, like the axe found at Khvalynsk in grave I:105, probably made in the North Caucasus. A polished serpentine bracelet at Khvalynsk found in grave I: 8 probably was made in the North Caucasus ; it was like bracelets at Nalchik. The Khvalynsk population was active in inter-regional exchange systems (Danube-Dnieper-Caucasus-Volga) that were stimulated by the heightened production of Balkan copper after 4500 BCE. Section title: Animal sacrifices: a new funeral cult Educational score: 1.8528121709823608 Domain: other Document type: Study Language: en A complete zoological report on the Khvalynsk fauna has not been published, but partial descriptions are contained in four sources . These sources occasionally contradict each other. We arrived at the numbers presented in this text and in Tables 3 and 4 by following this rule: where one source contradicted another, Bogatkina was authoritative for the Khvalynsk II fauna, and Agapov et al. for the Khvalynsk I fauna. Bogatkina and Morgunova attempted to re-count the Khvalynsk I fauna, but both gave numbers much smaller than Agapov et al. . They apparently described only the Khvalynsk I bones that survived in the Samara laboratory in the early 1990s. The faunal data in the original 1990 report must be presumed to be accurate. That report had no separate chapter by the site zoologist, A.B. Petrenko, but she is credited on the first page where fauna is described , and the animal bones are identified to taxa and briefly described within the text by grave number or sacrificial deposit (bones found in ochre-stained deposits above the graves at both I and II). Summary tables of the fauna from the graves and above-grave sacrificial deposits provide only the number of individuals, not the number of bones, which was not reported for Khvalynsk I. Therefore, to compare I and II, we can use only the number of individuals, as in Table 2 . Section title: Animal sacrifices: a new funeral cult Educational score: 3.7615511417388916 Domain: biomedical Document type: Study Language: en According to our interpretation of these sources, the animal bones recovered from Khvalynsk I and II represented the funeral sacrifices of at least 151 mammals. Three mammalian taxa were sacrificed: at least 106 domesticated sheep-goat (70%), 29 domesticated cattle (19%), and 16 horses (11%) whose domesticated status is debated. No obviously wild mammals were included in the funeral sacrifices, although wild species were represented in bone tools, ornaments, and flutes or whistles; and moose ( Alces alces ), red deer, horses, beavers, and fish were important in the diet at regional Eneolithic settlements . At the Eneolithic Ivanovska settlement on the upper Samara River, dated 4360–4220 BCE (68%) , with pottery of the ‘Samara’ type, distinct from Khvalynsk pottery, horses contributed 40.2% of the 6068 animal bones, domesticated cattle 11.4%, domesticated sheep-goat 7%, moose 17%, and beaver 22.5% , not counting fish or birds. Sheep-goat were ten times more frequent in the funeral deposits at Khvalynsk than at the Ivanovska settlement. However, in seasonal (winter?) camps containing Khvalynsk pottery on the lower Volga, as at Kair-Shak VI, dated 4400 BCE, sheep-goat were 60–70% of bones, and wild saiga antelope and onagers were 15% . The sacrifices at Khvalynsk did not include the wild game animals that were prominent in the diet at both settlements. Instead, domesticated mammals were exclusively used to communicate with the spirit world. Section title: Animal sacrifices: a new funeral cult Educational score: 1.5663394927978516 Domain: other Document type: Other Language: en What segments of domesticated mammals carried the prayers of the mourners? At Khvalynsk, horses were represented by one or two bones of the lower leg, usually a single phalange, in contrast to cattle and sheep-goats, which were represented by head and lower leg bones. Most of the described elements for cattle and sheep-goat were distal leg bones (principally metapodials and phalanges) and skulls, mandibles, or teeth . Head and leg bones might be the result of ‘head-and-hoof’ deposits, in which the skin or hide of the animal with head and hooves attached is left at a ritual site as the symbol of the gods’ portion, while the meat is consumed by the human participants. Head and hoof deposits occurred throughout Eurasian steppe prehistory and into the modern era . In the Eneolithic they are indicated at Khvalynsk and at another late 5 th millennium BCE cemetery on the Samara River, a tributary of the Volga, at a site known as S’yezzh’e, containing ‘Samara’ style pottery, like Ivanovska. At S’yezzh’e parts of two horse heads and distal legs were found in an ochre-stained sacrificial deposit above nine Eneolithic graves, arranged in head-and-hoof offerings like the cattle and sheep-goats at Khvalynsk . Section title: Animal sacrifices: a new funeral cult Educational score: 1.7089587450027466 Domain: other Document type: Other Language: en Where were the sacrificed animals deposited? About two thirds of the animal sacrifices at Khvalynsk were found in graves, associated with individual humans. These animal bones were connected to individual human deaths. One third of the sacrifices were in red-ochre-stained sacrificial deposits above the graves, possibly not connected with individual deaths but rather conducted for the public . The sacrifices at S’yezzh’e were like these, in a red-ochre-stained deposit above the graves. Eleven of the 13 sacrificial deposits at Khvalynsk I (85%) contained the bones of domesticated sheep-goats, domesticated cattle, and/or horses, the same three taxa found in the graves. The two sacrificial deposits that did not contain these taxa (SD 9 & 13) contained a greenstone adze in a red ochre deposit in SD 13, and a bird (not identified) skeleton decorated with two shell beads and one copper bead lying on a red-ochre-stained bark plate in SD 9. Domesticated animals and horses were the exclusive mammalian sacrificial offerings in the sacrificial deposits as well as in the graves at both I and II. Section title: Animal sacrifices: a new funeral cult Educational score: 3.127983570098877 Domain: other Document type: Study Language: en The inclusion of horses in graves with humans and domesticated animals, and the equally interesting exclusion of obviously wild animals such as moose, suggests that at Khvalynsk the symbolic status of horses had started to move toward the domesticated pole on the wild-domesticated continuum by 4500 BC. Horses were treated like domesticated animals in three ways: they were buried with humans and domesticated animals in graves that excluded obviously wild animals; at S’yezzhe they were arranged in head-and-hoof deposits like the cattle and sheep-goats at Khvalynsk; and horse images were new symbolic artifacts. Decorative bone plaques shaped like horses were found at S’yezzhe and zoomorphic mace-heads that might represent horse heads were found at Khlopkov Bugor, 130 km south of Khvalynsk; and at Lebyazhinka IV, an Eneolithic settlement near Samara . The evidence for a significant change in the human treatment of horses during the fifth millennium BC is symbolic rather than zoological, but it should not be ignored. In addition, recent studies of ancient horse DNA indicate that the horses in the Don-Volga steppes in this era were the genetic ancestors of the modern domesticated horses that first appeared in fully modern form about 2200–2100 BCE in the Don-Volga region. The symbolic changes in the human treatment of horses seen at Khvalynsk, S’yezzhe, and other Volga sites signal the earliest phase in an experimental selection process between humans and horses in this region that produced a gradually improving partnership over the next two millennia, culminating in horses genetically and behaviorally suited for warfare, like modern horses. Perhaps the Khvalynsk horses could be trained to ride in quiet settings such as herding. Section title: Animal sacrifices: a new funeral cult Educational score: 2.6305572986602783 Domain: biomedical Document type: Study Language: en The proportion of individuals buried with domesticated mammal sacrifices was 14% at Khvalynsk I (23 of 158 individuals) and 14% at Khvalynsk II (6 of 43 individuals). Counting only adults preserved well enough to be assigned a sex, the percent receiving sacrifices was higher: at Khvalynsk I, seven females had an animal sacrifice, or 18% of adult females; and 12 males, 27% of adult males; four immature individuals also received animal sacrifices. At Khvalynsk II, animal bones occurred with two adult men (II:38 and the mace chief II:24, together 10% of adult males), three females (50% of females), and two immatures . Section title: Animal sacrifices: a new funeral cult Educational score: 1.658315896987915 Domain: other Document type: Other Language: en The largest single sacrifice associated with a specific grave was the complex grave in Khvalynsk I:142–144, where two adult men aged 45–60 and 30–40 and an adult woman aged 40–50 were buried together on their backs with tightly raised knees . With them were a first phalange of a horse and the skulls of eight cattle . We can estimate edible meat weight as about 40% of adult body weight—for example, a 500 kg steer yields about 200 kg of ‘retail’ meat. Neolithic domesticated cattle in eastern Europe weighed between 350–500 kg ; let us use 400kg. If the cattle at Khvalynsk weighed 400 kg, eight cattle would produce 1280 kg of meat, and the horse another 120 kg (assuming a pony-sized body weight of 300 kg), equaling a total meat weight of 1400 kg. for the mammals in I:142–144. While no precise estimate is possible, this quantity of meat implies that the guests numbered in the hundreds. At Khvalynsk II, a similar large sacrifice was found in a sacrificial deposit above the graves in Quadrat I/8 . This deposit contained heads and hoofs of at least two cattle and two sheep-goats, and the lower limbs of two horses. These animals again would have yielded around 1400 kg of meat, like the large sacrifice at Khvalynsk I, and again imply hundreds of guests. Large-scale feasts are implied by the large mortuary animal sacrifices at Khvalynsk. Section title: Animal sacrifices: a new funeral cult Educational score: 0.9922341704368591 Domain: other Document type: Other Language: en Five hallmarks of competitive feasts conducted to create and maintain socio-political power, according to a recent analysis of feasting by Kassabaum , are large quantities of special foods shared between large groups at special places in the presence of special markers of elite status (maces and copper, here). In kin-based societies with competitive sections, feasts are an important arena for competition between lineages and clans , while at the same time they channel that competition into non-violent rituals that often play an integrative, peace-making role . Section title: Animal sacrifices: a new funeral cult Educational score: 1.291735291481018 Domain: other Document type: Other Language: en The feasts associated with funerals were sponsored or channeled through 14% of the population, and the animals sacrificed were not representative of the complex diet of fish, wild game (moose and deer), horses, and domesticated mammals that characterized everyday food consumption in Eneolithic settlement faunas in the middle Volga region . Domesticated mammals, segmented and represented in funeral rituals by their parts, were used at Khvalynsk as a ritual currency to mark and symbolize social segments among the funeral guests and their families. Males, females, children, and even infants were among the designated minority to receive sacrifices. The status connected with mortuary mammal sacrifice seems to have resided in multi-generational families or in the role played by the sacrifice receiver in the funeral ritual rather than in the lifetime accomplishments of the deceased. Section title: Animal sacrifices: a new funeral cult Educational score: 1.0903834104537964 Domain: other Document type: Other Language: en Domesticated animals, first adopted in the Volga-Ural steppes about 4800–4600 BCE, had triumphed by 4500 BC as the principal means of communication with the gods and ancestors, who apparently desired only sheep and goats, cattle, and an occasional horse. The horse was the only acceptable mammal that was indigenous. This new system of belief about the desires of the spirit world necessarily post-dated the arrival of domesticated animals, so it was a recently established ritual in 4500 BC. Yet this was the exclusive sacrificial ritual in the funerals at Khvalynsk. Khvalynsk was a central cemetery (because of its size) for a new funeral cult in which domesticated animals were the preferred channel of communication with the spirit world. If the Volga steppes were part of the Proto-Indo-European homeland, as many have argued then from the point of view of Indo-European religion, this was the moment when the world, made from the pieces of a cosmic cow , began. Section title: Four depositional groups: social segments at Khvalynsk Educational score: 0.9769650101661682 Domain: other Document type: Other Language: en At least four depositional groups can be identified archaeologically at Khvalynsk. Three were defined by the presence of copper, animal sacrifices, and polished stone maces in graves; and the fourth, the majority, by their absence. The ca. 70% of graves that contained neither copper nor animal sacrifices nor maces did contain some notable bone, stone, and antler artifacts and many beads made of exotic imported shells. Section title: Four depositional groups: social segments at Khvalynsk Educational score: 1.114510178565979 Domain: other Document type: Other Language: en About 14% of the population was buried wearing copper ornaments, and a different 14% with sacrificed domesticated animals. It is remarkable that these two minority groups were so similar in size and that they did not overlap more than expected, if the deposition of grave goods had occurred at random. An excessive overlap might be expected if people of higher social status had an elevated probability of receiving both types of grave goods, but with a few important exceptions noted below, the two groups were separate. Section title: Four depositional groups: social segments at Khvalynsk Educational score: 1.8178505897521973 Domain: other Document type: Study Language: en At Khvalynsk I, among 158 excavated individuals, 34 (17%) were buried with copper and/or mammal bones. In 32 of these 34 cases (94%), copper and sacrificed animal parts occurred separately, with different individuals . Copper ornaments occurred without animal bones with 13 individuals (8% of 158), and animal bones without copper with 20 different individuals (13%). Both exceptions at Khvalynsk I, two adult males with both copper and animal sacrifices (I:57 and I:108–110), also had polished stone maces, and in fact were the only individuals at Khvalynsk I with stone maces, suggesting that apart from the stone mace holders (see below), there was a disassociation between copper users and sacrifice receivers. Section title: Four depositional groups: social segments at Khvalynsk Educational score: 2.1247007846832275 Domain: other Document type: Study Language: en Khvalynsk II exhibited a similar separation between an animal-receiving minority and a copper-receiving minority, but with much more copper in the graves. Here out of 43 excavated individuals, 19 (44%, more than 2x the percentage at Khvalynsk I) had copper artifacts and/or animal sacrifices. Copper ornaments occurred without animal bones in the graves of 13 individuals, the same absolute number found at Khvalynsk I. These included nine males, two females, and two immatures. Animal sacrifices occurred without copper with three different individuals, one male, one female and one immature. In 16 of the 19 graves that had copper and/or animal sacrifices—84% of cases—they again occurred separately. The remaining three cases at Khvalynsk II where copper and animal sacrifices occurred in the same grave were divided between a richly equipped adult female (II:6), the isolated skull of an infant (II:14) buried with a string of copper beads and two horse phalanges, the only horse bones at Khvalynsk II; and an adult male with a stone mace (II: 24), the richest grave at Khvalynsk, discussed below. Section title: Four depositional groups: social segments at Khvalynsk Educational score: 1.2218326330184937 Domain: other Document type: Other Language: en At both Khvalynsk I and II, people buried with animal sacrifices did not in general have copper ornaments, and people who wore copper ornaments into the grave did not in general receive animal sacrifices. (Bird bone tubes are counted as an artifact, not a sacrifice.) The segregation between these groups is surprising. Under a model in which higher social status confers an elevated chance of receiving both offerings, they should have overlapped more. Instead, their segregation suggests that they represented distinct statuses. The copper ornaments were worked and welded locally, but the metal probably was obtained from Balkan cultures where smelting was practiced. Copper represented contacts with distant others. It symbolized foreign adventures and long-distance travels . The mammals for sacrifices were, in contrast, herded and produced nearer to Khvalynsk, so came from and symbolized a different set of locations and behaviors. Animal sacrifices were sacral (connected with funerals and spirits of dead ancestors) and local. A sacrifice was shared during integrative feasts attended by hundreds. Copper ornaments, in contrast, were deployed on the bodies of specific individuals, a minority, presumably with pride and its companion envy. A feast animal was partible and belonged at least temporarily to everyone, while a shining metal ornament decorated the individual who wore it. Section title: Four depositional groups: social segments at Khvalynsk Educational score: 1.1045626401901245 Domain: other Document type: Other Language: en It is tempting to interpret the sacrifice-receivers as members of a local sacral group such as shamans or priests (and their families) who were buried primarily at Khvalynsk I; and the metal-users (with their bird-bone whistles) as members of a male-biased, far-ranging group, such as traders or warriors, defined by their visits to different cultural worlds and/or access to metals obtained abroad, buried primarily at Khvalynsk II. Horseback riding perhaps already facilitated long-distance travel. Sacrifice-receivers were buried at Khvalynsk II, but it is interesting that none of them except the mace chief was related genetically to any other person at Khvalynsk II, while many of the copper-receiving males were related to other males in that cemetery. As a cemetery, Khvalynsk II was organized around related copper-receiving males, while the sacrifice-receivers were perhaps wives or sacrifices themselves (the infant in II:14). Polished stone maces identified a special class of leaders who united these two groups, and occurred in both cemeteries, implying that leadership was not limited to one cemetery or to one of the minority groups. Section title: The mace-holders: Eneolithic chiefs Educational score: 1.298304557800293 Domain: other Document type: Study Language: en Only three individuals among the 201 excavated at both cemeteries had a polished stone mace (I:57; I:108; and II:24). One was the adult male in Khvalynsk I:57, the only adult male with copper objects and an animal sacrifice among 158 individuals at Khvalynsk I. This rare combination was recognized by a polished stone mace. The second mace holder was another adult male buried in grave 24 at Khvalynsk II with the most diverse and numerous assortment of artifacts at Khvalynsk, including an animal sacrifice (a sheep and a goat) and a large number of copper objects . Like I:57, he was the only adult male at Khvalynsk II who had both copper objects and an animal sacrifice; and he was distinguished by the only polished stone mace. The third mace holder deviated from this pattern. Grave 108 at Khvalynsk I contained an adult male buried with two polished stone maces, one broken and one whole. Of the three mace graves, his was the only one without copper ornaments, animal sacrifices, or a bird-bone tube. He was buried in a cluster of four individuals (I:108–110), the other three all immature. One had an animal sacrifice and another had a copper item, so together they had the animal sacrifice+copper+ adult male combination that distinguished the other two mace graves. The difference is that in I:108 the mace holder did not unite these categories within himself by combining them. Let us look more closely at these three graves. Section title: The mace-holders: Eneolithic chiefs Educational score: 1.3918451070785522 Domain: other Document type: Other Language: en The mace in I:57 , was deposited on the skull of a male aged 40–50, whose red-ochre-painted skull and long bones were buried in a complex grave. His was a secondary burial, including only his skull and some long bones. With these were strings of bone rings, bone beads, and Unio shell beads; three copper rings (copper-user); two unifacial flint blades; a miniature ceramic cup (several other miniature cups were included in graves at Khvalynsk); a large bone fish-hook; an abraded stone polisher; and a bird-bone tube. On top of his red-ochre-painted skull was the polished stone mace-head. The burned skulls and lower limb bones of one Bos and one Ovis were found near his skull (sacrifice-receiver). These animal bones were burned outside the grave—the grave itself showed no signs of fire—then were placed in the grave. The meat from these animals, around 170 kg, was sufficient for more than 100 mourners. Section title: The mace-holders: Eneolithic chiefs Educational score: 1.1250073909759521 Domain: other Document type: Other Language: en The remains of four other people were placed above individual 57, under a stone pavement: a male aged 40–50 intensely colored with red ochre, and an adolescent aged 13–14 without red ochre, on their backs with tightly raised knees (I:55 and I:56); and mingled with 55 and 56, the isolated bones of a woman aged 40–50 (not assigned a number) and a child aged 6–10 (I:36), curated and re-buried, like the mace-chief. Because these bones do not survive, we do not know if the five individuals in grave 55–57 were related genetically. The bones of the mace chief, a woman about his age, and a child seem to have been curated until the deaths of 55 and 56, when these two were buried ‘in the flesh’ with the curated bones of the other three. The remains of the mace chief were interred with his mace, ornaments, and the head-and-hoof remains of a funeral feast; then 55 and 56 were posed above him, 55 being placed on soil stained with red ochre; then the curated bones of the woman and child were scattered over the bodies of 55 and 56; and finally a pavement of flat stones was placed above the grave. Section title: The mace-holders: Eneolithic chiefs Educational score: 1.452832579612732 Domain: other Document type: Other Language: en The other mace grave at Khvalynsk I was I:108 . This grave held two maces, one whole and one broken . They were on the upper chest of a male described as ‘young adult’, age not given . His skeleton is now unfortunately lost, like I: 57. Male I:108 was on his back with tightly raised knees, fallen to one side, with red ochre around his pelvis. The partial bones of three individuals were at his feet—a ‘young’ female contracted on her right side (I:110), the skull of a child aged 3–5 (I:107), and the skull and long bones of another child (I:109), age not recorded. Near the skull of this child (I:109) were Unio shell beads stained with red ochre, and under the skull was a fragment of a copper ring. The partial skull of a sheep-goat was found under the skull of female 110, and a miniature ceramic cup 11 cm high was placed with her . Male I:108 had a polished stone ring and wore strands of shell beads . His was the only mace-grave that did not contain a bird-bone tube, a copper object, or an animal sacrifice, although an animal sacrifice (110) and a copper item (109) were found in the same burial cluster. The broken mace buried with him was missing its narrow end, the end that was mounted in a haft . The matching broken end piece was found 2m to the north in grave I:104 , reportedly in a rodent burrow in the floor of the grave . Grave I:104 contained an adult female aged 25–35, wearing copper and shell ornaments, lying on top of the mace fragment, with the curated skull fragments and long bones of a child 3–7 years old (I:106) beside her. Section title: The mace-holders: Eneolithic chiefs Educational score: 0.9850019216537476 Domain: other Document type: Other Language: en The male in I:108 curated the broken mace made of yellow-brown stone but also acquired a new, unbroken mace of a different, cruciform type, made of dark grey stone . The curation of the broken mace, its replacement by a new whole mace, and the possibility that the broken piece was intentionally placed in the adult woman’s grave vividly attest to the power infused into these objects by the people at Khvalynsk. Discussing religion at Çatalhöyük, Hodder observed that the Neolithic population there seems to have regarded some material objects, including houses, as imbued with a vital, living force that empowered them with spiritual agency. The context of the two maces in Khvalynsk grave I: 108 suggests that these iconic symbols were regarded as possessing vitality and agency in a similar way, perhaps related to the vital power of their owners. Section title: The mace-holders: Eneolithic chiefs Educational score: 1.0914205312728882 Domain: other Document type: Other Language: en The third mace grave was the only grave at Khvalynsk II that contained copper and animal sacrifices and an adult male, like I:57 at Khvalynsk I. Although grave II:12 contained more pieces of copper, grave II:24 was the richest grave at Khvalynsk, defined by the most diverse collection of grave gifts, including an eared stone mace . This is the only Khvalynsk mace-chief that is preserved, so can be analyzed using modern methods. Section title: The mace-holders: Eneolithic chiefs Educational score: 3.383514642715454 Domain: biomedical Document type: Study Language: en The male aged 20–25 (II:24) was buried with a female aged 8–9 (II: 25), his sister, with the partial remains of three other individuals at their feet: a male 16–19 years old (a 3 rd -degree relative), and two infants (not analyzed for aDNA). Whole genome analysis revealed that 24 & 25 were brother and sister, although born at least 10 years apart. She wore many decorative belts of riverine Unio shell beads. Copper rings and beads were found only on the male, and beside him was an ‘eared’ mace like the broken one in Grave 108, and 14 bones from a sheep and a goat . He was buried wearing multiple mid-body belts of Unio shell beads, multiple mid-body belts made of 194 beaver incisors (or a shirt covered with beaver incisors?); a boar’s tusk chest pendant; fossil Glycemeris shell pendants (a marine shell also used at Varna for ornaments); a tubular bird bone; and 14 copper ornaments consisting of beads, rings, a spiral ornament or coil of wire , and bands that might have been wrapped around wooden shafts . He also had two lumps of melted copper, perhaps signs of metal craft working, or perhaps copper trade ingots. The male’s Y-chromosome haplogroup was Q1a1b, a Siberian, northern haplogroup; for example, almost all the males at Murzikha, a contemporary cemetery in the forest zone, were Q1a . His and his sister’s MtDNA haplogroup was U2e1b, also found in Mesolithic individuals in Latvia and Siberia, so again a northern lineage. His paternal ancestry contrasted with the paternal ancestry of most of the males at Khvalynsk II. Section title: The mace-holders: Eneolithic chiefs Educational score: 1.10019052028656 Domain: other Document type: Other Language: en Maces at Khvalynsk represented a specific and unique status reserved for the two adult males who belonged to both the copper and animal-sacrifice depositional groups simultaneously, and for one other adult male who was buried in a complex group grave that contained copper and an animal sacrifice deposited with other individuals in the group. The intersection between adult male + copper user + sacrifice receiver was marked using polished stone maces by the ancient population itself. The repetition of this prehistoric act twice, in the only two graves at Khvalynsk where copper + animal sacrifice + adult male clearly coincided, and perhaps a third time if we relax the rules a little, suggests that the copper-receivers and the sacrifice-receivers had real social salience. We do not know what these groups represented, whether they had broad Dumézilian social functions as we have suggested (priest vs. warrior/trader), or if they symbolized something more specific. But whatever they meant, they were recognized by the Khvalynsk people—and their intersection was marked with maces. The maces therefore perhaps symbolized generally the integration of socially separate groups through the medium of a single adult male who symbolically balanced their interests and facilitated their union—a chief. Section title: Maces and head trauma at other sites Educational score: 0.9188359379768372 Domain: other Document type: Other Language: en Polished stone mace-heads were buried in Eneolithic graves in the Dnieper steppes (Mariupol) and the North Caucasus steppes (Merekli-Tekeb), and they later appeared as imports in Varna-era agricultural towns in the Danube valley and in Cucuteni A/Tripol’ye B1 towns in the eastern Carpathian piedmont. It was once argued that they were products of the Varna-era agricultural towns that diffused eastward into the steppes , but later studies claimed that they are more numerous in the steppes, with older dates . In relation to these debates, the four maces at Khvalynsk are important because of their well-dated archaeological contexts. But they were not the earliest dated maces in the region. That distinction belongs to the polished stone mace-heads from the Ekaterinovka Mys cemetery . Section title: Maces and head trauma at other sites Educational score: 0.9280099272727966 Domain: other Document type: Other Language: en Ekaterinovka Mys (‘Mys’ means ‘peninsula’) is a Volga riverside Eneolithic cemetery of 100+ graves 150 km north of Khvalynsk, below the big Volga loop known as the ‘Samarskaya Luka’, or the Samara bow, near the city of Samara. Here the Volga makes a 160-km loop around a limestone mountain that rises more than 300m above the river, a prominent feature that coincides broadly with the ecological border between the steppe and forest-steppe vegetation zones. Ekaterinovka Mys was situated at the southern or steppe end of the Luka, at the transition to the steppes. Section title: Maces and head trauma at other sites Educational score: 3.54490327835083 Domain: biomedical Document type: Study Language: en The radiocarbon dates on human bone are skewed too old by FRE, but we also have dates on animal bones or teeth unaffected by reservoir effects. In Table 4 we present five dates from Ekaterinovka Mys from terrestrial animals, including a goat, a sheep, and a beaver. Like beavers at other sites , the beaver incisor dated here has stable isotopes indicating a terrestrial diet (δ13C −20.7, δ15N 6.7). The human from grave 45, buried with the dated goat kid, yielded a FRE-skewed radiocarbon date 800 years older . An organic residue from a potsherd gave a date like three animal bone/tooth dates; one date on a sheep tooth was somewhat older. The average midline for the five dates was 4618 BCE, about 200 years older than the average midline from terrestrial animals at Khvalynsk, 4400 BCE. The sheep and goat dates are among the oldest dates for domesticated sheep and goats in the Volga steppes (excluding anomalous dates on organic residues) . But the male in grave 45 was buried with much more than a domesticated goat. He had three maces. Section title: Maces and head trauma at other sites Educational score: 1.6099660396575928 Domain: other Document type: Other Language: en Figure 14:C illustrates the three drilled polished stone mace-heads placed on the right arm of the man in grave 45, reproduced from Korolev et al. . They are surprisingly diverse in shapes and colors, made of different stones: one ovoid, one four-lobed, and one zoomorphic, probably meant to resemble a fish head (more like a catfish than any other Volga fish). No copper was found in this or any other grave at Ekaterinovka Mys, a strong contrast to Khvalynsk ; and very few domesticated animals were sacrificed during the funerals at Ekaterinovka Mys, another contrast to Khvalynsk. But the young male (aged 20–25) in grave 45 was provided with a domesticated goat kid deposited on his left arm . Also, in addition to the three maces deposited on his right arm, the young male had two severed hands from two different people placed on his left hip; and two severed lower legs (from the knee down, including tibias and some foot bones) of two people, probably the same two victims. One severed lower leg was on the young male’s right side with the three mace-heads; the other was placed between his lower legs . These severed body parts from two people appear to be war trophies. It is possible that two of the three mace-heads in grave 45 also were war trophies and were associated with the two victims. Section title: Maces and head trauma at other sites Educational score: 1.2646163702011108 Domain: other Document type: Other Language: en A piece of elk antler carved in the shape of a long-beaked bird lay across the head and face of grave 45 . It had use-worn, polished serrated notches on the bird’s ‘neck’, as if used for the attachment of decorative suspensions, like bundles of feathers. Figure 14:B shows the carved antler bird arranged over the reconstructed head and face of the young male in grave 45 as if it were the crest of a feathered hat . Figure 14:D shows an artist’s impression of how the hat might have been worn, created by free-lance artist Russell Story, who creates imagery for Industrial Light and Magic within LucasFilms. The species of long-beaked bird represented in the carving (13:B) is unknown, but here is represented as a glossy ibis ( Plegadis falcinellus ) because this is the longest-beaked Volga bird, and the carved beak looks longer than a crane or heron beak. In the artist’s rendition (12:D), the feather mantle is made of glossy ibis wing feathers, and the paint around the man’s eyes uses white and red colors from glossy ibis eye-patch feather colors. In the lower Volga marshes, near the Caspian Sea, glossy ibises gather each summer to feed and breed before migrating south for the winter. If the carved antler bird was meant to represent a glossy ibis, it is interesting that Ekaterinovka Mys is hundreds of kilometers north of the ibis breeding zone on the lower Volga, so an ibis head-dress might suggest a southern provenance, possibly a gift from the south. The annual migrations of glossy ibis could be an animal-world metaphor for the north-south movements of human groups that are reflected in the heterogeneous genes and cranio-facial types at Khvalynsk and to a much lesser extent at Ekaterinovka Mys. Section title: Maces and head trauma at other sites Educational score: 1.0813170671463013 Domain: other Document type: Other Language: en The Eneolithic maces at Khvalynsk, Ekaterinovka Mys, and other steppe sites must be understood not only as aesthetically attractive symbols, but also as status weapons that threatened violence. Unlike a knife or axe, a mace has no non-violent function; it is designed to break skulls. Those skulls might be of large fish (Volga catfish could weigh more than 300kg and probably were killed by blows to the head) or sacrificial animals. But grave 45 at Ekaterinovka Mys shows that inter-human violence also was associated with mace-holders in the centuries before Khvalynsk. Section title: Maces and head trauma at other sites Educational score: 1.4374345541000366 Domain: other Document type: Other Language: en Increased violence is not evident in the few studies of pathologies in Eneolithic skeletons in the Volga steppes. Khokhlov did not feel that violent trauma was significant in the Khvalynsk population. But this is largely because an odd and puzzling trait on many Eneolithic skulls that would normally be interpreted as indicating violent blows to the head has instead been interpreted by three different experts as ritual, not connected with violent blows but rather with intentional scraping or gouging of the skull. Section title: Maces and head trauma at other sites Educational score: 3.9412930011749268 Domain: biomedical Document type: Study Language: en These oval, saucer-shaped depressions in the parietal bone are called “ritual trepanations” by Khokhlov, who counted 10 cases at Khvalynsk II, all adults , and nine cases at Ekaterinovka Mys, including the male in grave 45. The gouged-but-not-hit skulls at Khvalynsk II were noticed also by Murphy in her internal report for the Samara Valley Project, but she did not describe them in print . In the cases at Khvalynsk and Ekaterinovka Mys, the outer layer of skull bone was scraped away, making small ovoid depressions that did not penetrate through the inner layer of bone. Gresky et al. documented a similar but more extreme skull modification ritual in the North Caucasus steppes at Progress-2 and Vonyuchka-1 (also known as Konstantinovskii-1), in which full trepanations were conducted, with penetration through both the inner and outer layers of bone, on people who showed no sign of skull trauma or injury. These features did not exhibit the radiating cracks or crushed edges that accompany a violent blow, but were created for unknown reasons, perhaps (by analogy with actual trepanations) to relieve other sources of head pain. They represent a confusing factor in attempting to evaluate the level of inter-personal violence in the Eneolithic, because they look very much like head trauma and might disguise trauma, but they are not themselves the result of violence. Gresky et al. counted these features on fully 10% of the Eneolithic skulls they examined from graves between the North Caucasus steppes and the lower Don. They document a ritual that was shared across the Volga-Don-Caucasus steppes among Eneolithic people who also exhibited similar genetic ancestries and similar styles of polished stone maces. Section title: Maces and head trauma at other sites Educational score: 1.2964372634887695 Domain: other Document type: Other Language: en The integrative, inter-group bridging function suggested here for the copper-rich mace-chiefs at Khvalynsk could have been a peace-making reaction to the violent trophy-taking of the previous century, illustrated at the Ekaterinovka Mys cemetery. An ethnohistoric analogy might be the creation of the League of the Iroquois in what is now New York state. The League was a peacemaking alliance between five powerful tribes that had previously experienced chronic inter-tribal warfare . A document-based interpretation of the League suggests that it functioned to sustain and facilitate access among the five tribes to novel and highly desirable European trade goods. These are paralleled on the Volga by copper objects introduced by the Danubian centers of what Chernykh named the ‘Carpatho-Balkan Metallurgical Province’. Iroquoian belief systems present a different picture of the founding of the League, describing it as a religious awakening inspired by a culture hero (known as the Peacemaker) who introduced new, integrative rituals. Similarly, at Khvalynsk we witness the appearance of new sacrificial rituals in which domesticated mammals were a mandatory medium for the funeral ceremonies of a minority that distributed funeral feasts to hundreds of mourners. These funeral gatherings, and the integrative symbolism of the chiefs who oversaw them, could be signals of a broader social integration or inter-regional confederation, partially inspired by the desire to reduce conflict and facilitate trade in novel copper artifacts. Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 1.0496598482131958 Domain: other Document type: Other Language: en The spatial arrangement of the graves at Khvalynsk I was described in maps, plans, and figures in Agapov et al. . Small errors on the cemetery plan were corrected by Vasiliev , the basis for Figure 15 . The only published plan of Khvalynsk II is in the article on copper metallurgy by D. Agapov in the Khvalynsk monograph , which is the basis for Figure 17 . Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 1.2936865091323853 Domain: other Document type: Other Language: en The arrangement of graves within Khvalynsk I and II did not follow a consistent rule. The cemetery plan does not reveal straight rows of graves, or clearly separated groups. The linear distance between graves was highly variable, so some graves were crowded near each other, while other graves were scattered apart. Genetic analysis of family relationships at Khvalynsk II raised the possibility that the individuals belonging to the largest family (designated the Yellow family) were buried in what appears to be an east-west row; but this pattern, if we can call it that, was not maintained. Graves of other families clustered around the ‘Yellow’ row in no apparent pattern, creating a cemetery plan in which no rows or alignments could be perceived. Rows of graves were more apparent at Ekaterinovka Mys, 150 km north, where the rows were aligned NW-SE with heads oriented NE. At Khvalynsk I, graves were aligned toward the N, NW, or NE, most to the NE. At Khvalynsk II, the same N-NE orientation was standard, although the richest grave, the mace chief (II:24), was oriented SE. Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 1.3695036172866821 Domain: other Document type: Other Language: en The standard body pose at Khvalynsk was highly distinctive—on the back with tightly raised knees—and would later be characteristic of early Yamnaya graves. About 55% of the individuals at Khvalynsk I and II were arranged in this pose, which predominated among both men and women ; another 10% were buried in a contracted position on one side; and less than 10% were buried in a ‘sitting’ position with the upper body slightly raised and the back curved, originally posed with elbows down, and the knees raised. The sitting position occurred as a minority pose also at other regional cemeteries, including Ekaterinovka Mys. Most of the other individuals were secondary or partial skeletons, often just the skull and a few other bones, in which pose could not be determined. Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 1.0193771123886108 Domain: other Document type: Other Language: en The supine-with-raised knees burial pose was a defining steppe-zone Volga funeral custom, seen also at Khlopkov Bugor and in Eneolithic graves at Engels and on the lower Volga . A different position—supine with legs extended straight—was standard in graves at Ekaterinovka Mys and S’yezzh’e at the northern edge of the steppes, and in Neolithic cemeteries on the Dnieper. Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 1.4666398763656616 Domain: other Document type: Other Language: en In the North Caucasus steppes at older Eneolithic cemeteries such as Nalchik , the flexed pose, contracted on the side, was used for most individuals, but even here a few individuals were buried on the back with raised knees. When the first small earthen mounds, or kurgans, began to appear in the North Caucasus steppes during the Eneolithic, after 4500 BCE, they were erected over graves in which the deceased was positioned supine with raised knees, usually oriented to the east . The Eneolithic individuals at Progress-2 and Vonyuchka in the North Caucasus steppes who had genetic ancestry similar to Khvalynsk were buried in the Khvalynsk position, in graves intensely colored with red ochre, beneath small (less than 1m high, ca. 15m diameter) earthen mounds . These mounds were among the oldest kurgans in the Pontic-Caspian steppes ; the other region where small kurgans appeared this early was in the steppes north of the Danube delta, as at Suvorovo , again at a cultural, economic, and genetic border . Although they were small compared to later Yamnaya kurgans, the Eneolithic kurgans in the upper Tersek steppes east of the Svobodnoe-Meshoko agriculturalists perhaps were a boundary-marking practice that emerged during the late fifth millennium BCE. This was a millennium before the Yamnaya culture made the kurgan type of funeral monument universal across the Pontic-Caspian steppes. Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 2.071126937866211 Domain: other Document type: Study Language: en The Skelya and Sredni Stog cultures in Ukraine, contemporary with Khvalynsk, also used the supine-with-raised-knee posture, unlike the supine-extended burial pose in the Dnieper Neolithic cemeteries . Sredni Stog individuals also had genetic ancestry more like Khvalynsk and Progress-2 than the Dnieper Neolithic ancestry type (see below). Sredni Stog lithics also were similar to Khvalynsk, particularly the use of large lanceolate projectile points and long unifacial lamellar flint blades. Sredni Stog pottery was tempered with crushed shell, like Khvalynsk pottery, and unlike the Neolithic pottery of the Dnieper valley, where sand or mineral temper had been used. The high percentage of horse bones, averaging more than 50% of all animal bones in Dnieper-valley Sredni Stog sites, was consistent with the high importance of horses in the diets of many fifth- and fourth-millennium BCE Eneolithic settlements in the Volga and Don valleys . Many traits indicate ‘eastern’ influences on Sredni Stog material culture, economy, and genetic ancestry, and the supine-with-raised-knee burial pose is one of these. Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 1.8074522018432617 Domain: other Document type: Study Language: en At Khvalynsk I about 30% of the graves were single, about 20% held a pair of individuals (eg, I: 68 & 69), about 20% had three individuals (I: 61–63), and the most complex graves contained individuals buried with parts (often skull parts) of multiple other individuals and with additional individuals at their feet (I: 107–110), or individuals arranged in layers with multiple adults laid out over other adults who had additional individuals at their feet (I: 126–130). All three mace graves were complex multiple interments (I;55–57, I: 107–110, and II: 23–25, 23a, 23b). At Khvalynsk I, the two mace graves (I: 57 and I: 108) were only two meters apart in the most crowded part of the cemetery. Graves with animal sacrifices seem to be arranged on the periphery of the cemetery and copper-users seem concentrated more in the center, but they overlap spatially considerably. The spatial layout of Khvalynsk II is considered below with the DNA evidence. Section title: Funeral rituals in the Volga-Caucasus Eneolithic Educational score: 1.0092538595199585 Domain: other Document type: Other Language: en Pestrikova attempted to discern sub-groups within Khvalynsk I based on the depth of the grave, the age and sex of the individual, the use of red ochre, special artifact types such as bird-bone tubes, and other traits. She suggested five principal social groups or categories, with the two highest-prestige categories being interpreted as ‘leaders’ and ‘shamans’. This is not so different from the suggestion here that animal sacrifices designated a sacral minority group and copper ornaments designated a warrior/trader minority group. Pestrikova did not consider animal sacrifices in her analysis, and her suggested five groups were not borne out at Khvalynsk II, where the traits that defined her Khvalynsk I categories were mixed. This essay presents a hypothesis about four social categories at Khvalynsk based on animal sacrifices, copper items, maces, and the residual majority without them. These groups seem to apply equally well to Khvalynsk I and II. Other ways of looking at this cemetery would be possible and should be pursued in the future. Section title: Khvalynsk genetic relationships and cranio-facial types Educational score: 1.8617299795150757 Domain: biomedical Document type: Study Language: en Genome-wide data from more than 50 individuals from three Eneolithic cemeteries between Samara and Saratov on the Volga are under study in a parallel work. The cemeteries are Ekaterinovka Mys, Khvalynsk, and Khlopkov Bugor, mentioned above and mapped in Figure 2 . Here we summarize genetic results relevant to Khvalynsk from the comprehensive report that will be published elsewhere (for questions about the genetic analysis and early access, please write to I. Olalde and D. Reich). Section title: Khvalynsk genetic relationships and cranio-facial types Educational score: 2.4180285930633545 Domain: biomedical Document type: Study Language: en Until now, three individuals from Khvalynsk II (II:1, II:12, II:17) were the only published whole genomes of Eneolithic individuals from the Volga-Ural steppes. First referenced under the generic label Steppe Eneolithic in a report that did not mention Khvalynsk , they were cited briefly three years later in Damgaard et al , and their admixture components were illustrated in Wang et al . They were first analyzed in detail in a Supplementary Information document attached to a primary report that again did not refer to Khvalynsk . For colleagues in genetics they were easily referenced in shared online databases, but for colleagues in archaeology they remain largely unknown. What follows includes these three, but also uses preliminary information from the larger set of individuals now under study by Anthony, Reich, Olalde, and others. Section title: Relatives and families from ancient human DNA Educational score: 1.9154051542282104 Domain: biomedical Document type: Study Language: en Most of the 158 individuals from Khvalynsk I were lost in a flood. Five individuals were preserved and passed aDNA screening, and none were related (see Table 6 for their sex-linked haplogroups). This discussion is about family relationships at Khvalynsk II. Section title: Relatives and families from ancient human DNA Educational score: 2.706104040145874 Domain: biomedical Document type: Study Language: en At Khvalynsk II 43 individuals were recovered, 77% of them males, a sex ratio like later Yamnaya kurgans in the middle Volga steppes, and unlike Khvalynsk I, where the sexes were equally represented. After screening, 26 individuals had whole-genome data sufficient to analyze family relationships at the level of 1 st degree (parent, sibling, child), 2 nd degree (grandparents/grandchildren, uncles/aunts, nephews/nieces, half-siblings), or 3 rd degree (first cousins, great-grandparents, great uncles/aunts). Section title: Relatives and families from ancient human DNA Educational score: 3.138213872909546 Domain: other Document type: Study Language: en Of the 26, 18 (70%) were related to at least one other individual and 17 of the 18 (95%) were males . The only female related to another individual (II: 25) was a 9-year-old girl buried with her older brother, the mace chief (II: 24). The other five females at Khvalynsk II that passed screening were unrelated to the males or to each other, within three degrees. Assuming that they were wives of the men, cross-cousin marriage, a type ascribed to Proto-Indo-European speakers by Benveniste , is disproved for the Khvalynsk II population, since no adult female was first cousin to any man. Moreover, no mother-son or father-daughter relationships were detected at Khvalynsk II; all mothers, sisters (with one exception), and daughters were buried elsewhere. We can identify one such female relative: a grandmother or great-aunt of the Yellow-family male in II: 4 was buried 130 km downstream at Khlopkov Bugor (KB7). The absence of female relatives at Khvalynsk II was unlike the older cemetery at Ekaterinovka Mys, where three mother-son relationships were recognized in the cemetery population of about 100. In contrast, Khvalynsk II contained three father-son pairs, and brother’s sons (II:22,27) were buried near their paternal uncles (II:12, 13). Patrilineal family relationships connected 70% of the individuals analyzed at Khvalynsk II, which might have been reserved for a paternally related male sodality, with some unrelated females and immatures. In this hypothetical sodality males died at a younger average age than the males in Khvalynsk I . As noted above, 70–80% of the individuals in Yamnaya kurgans were adult males, as at Khvalynsk II, suggesting that Yamnaya funeral customs could have evolved from the conventions of an Eneolithic male sodality rather than a “culture”. Section title: Relatives and families from ancient human DNA Educational score: 1.113732099533081 Domain: other Document type: Other Language: en The 18 related individuals can be grouped into six families of at least two members. The families are given colors in Table 5 . The plan of the cemetery in Figure 17 uses the Table 5 color code to identify graves belonging to each of the six families. Their spatial patterning is discussed first. Section title: Relatives and families from ancient human DNA Educational score: 1.0586581230163574 Domain: other Document type: Other Language: en The spatial arrangement of families at Khvalynsk II shows some patterns . The Yellow family graves were arranged in an east-west line, heads to the N or NE. They are the only family group that shows such spatial coherence. The Grey family graves (II:1, 17, 34, 24, & 25), were placed north, south, and east of the central Yellow-family line. The Purple family (II: 29, 30, 38) graves were arranged on the northern and western periphery of the Yellow family line. The two members of the Orange family (II: 28, 35) seemed to intrude into the space of the Yellow family, but no other family did so. This makes it seem that the Yellow family line of graves around the brothers II:12 & 13 was established first. After these graves were made, other families arranged their graves around the central Yellow cluster. The rich Grey family brother-sister pair in II:24&25 was buried quite near the Yellow brothers II:12&13, perhaps an intentional spatial expression of proximity in power and status. Similarly, the two mace-chiefs buried at Khvalynsk I were within two meters of each other, perhaps another spatial expression of proximity in status. Section title: Relatives and families from ancient human DNA Educational score: 1.3429925441741943 Domain: other Document type: Other Language: en Table 5 lists 23 binary family relationships between individuals at Khvalynsk II. The Yellow brothers in graves II:12 and II:13 lie at the center of these relationships, participating in almost half of them (11 binary relationships) as brother, uncle, cousin, or grandfather to other males. The Yellow brother II:12 possessed 80% of the copper found at Khvalynsk, so he was identifiable archaeologically as a central figure, but his genetic centrality was not previously known. Section title: Relatives and families from ancient human DNA Educational score: 2.689823865890503 Domain: biomedical Document type: Study Language: en In addition, the Yellow-family male in grave II:4, buried with a bird-bone tube (but without copper or animal sacrifices), was a 2 rd degree relative, modeled in Figure 18 as a grandson, of a Yellow family female buried 130 km to the south in grave 7 at Khlopkov Bugor (KB7), a Khvalynsk-culture cemetery of 24 graves near modern Saratov. The absence of copper at Khlopkov Bugor makes it likely that it was older than Khvalynsk, but within the chronological limits of 2 nd -degree relatives, so no more than two generations older. Since the mtDNA haplogroup of II:4 differed from KB7, the female in KB7 was either his paternal aunt or paternal grandmother. His would then be among the oldest graves at Khvalynsk II. During the brief interval between Khlopkov Bugor and Khvalynsk, perhaps around 4500 BCE, Balkan copper began to flow through exchange relationships in the Volga steppes and was concentrated at Khvalynsk. Section title: Relatives and families from ancient human DNA Educational score: 4.22441291809082 Domain: biomedical Document type: Study Language: en The colors in Figure 18 designate sex-linked haplogroups, not families. The mtDNA haplogroup is the main color and the Y haplogroup group is the corner color. The corner colors for Y-haplogroups in Figure 18 are used again in Table 6 , which shows all sex-linked haplogroups detected at Khvalynsk I and II. Nine mtDNA haplogroups and four Y haplogroups are listed. R1b-L754 was the most common Y-haplogroup and U5a was the most common mitochondrial group. Five individuals from Khvalynsk I had four mitochondrial haplogroups (T2a, U2e, U4a, U5a), all of which were shared at Khvalynsk II; and two paternal Y-haplogroups, one of which (I2a-L699) was unique, while the other (R1b-L754) was shared at Khvalynsk II. In Table 6 , Y-haplogroups are listed with both their alpha-numeric code out to three digits (as in R1b) and their Y-full tree ( https://www.yfull.com/tree/ ) designation (as in R1b-L754). Evolutionary lineages within a Y-haplogroup branch are shown as in R1b-L754 > L389 > V1636, where L754 is basal, L389 is derived from L754, V1636 is derived from L389, and all the SNPs out to V1636 are preserved. The difference between R1b-L754 and R1b-V1636 can be caused by differential preservation of SNPs, so does not indicate different paternal ancestry. Section title: Relatives and families from ancient human DNA Educational score: 4.199887752532959 Domain: biomedical Document type: Study Language: en Figure 18:A presents a best-fitting family tree for the Yellow family individuals, limited by making II:4 among the oldest Yellow individuals, and by Y-chromosome and mtDNA haplogroups ( Table 6 ) as well as permissible degrees of relationship ( Table 5 ). Six maternal mtDNA haplogroups were present in the Yellow family , and one paternal Y-haplogroup (R1b). The oldest modeled Yellow male, II:4, was a 3 rd -degree relative, probably a paternal great-uncle, of the Yellow brothers II:12&13. If this sequence is correct, then after the brothers II:12&13 were buried, three more Yellow family males (22 and 27, then finally 31) were buried on either side of II:12&13. The Yellow family is modeled as present at Khvalynsk over five generations, although we lack graves from generation two, between II:4 and the II:12&13 brothers. The R-V1636 form of R1b seen in the Yellow family occurred also at Ekaterinovka Mys, where it was abundant among males, at Progress-2, and at the Eneolithic cemetery at Berezhnovka II on the lower Volga, so seems to have been widespread in the Volga-North Caucasus steppe mating network in the final centuries of the 5 th millennium BCE. This is a separate side branch of the R1b that led to the typical Yamnaya form of R1b. Section title: Relatives and families from ancient human DNA Educational score: 4.035240173339844 Domain: biomedical Document type: Study Language: en Figure 18:B presents a similar analysis for the Grey family, including the mace chief in II:24 and his sister in II:25, in this case representing one possible family tree among several equally plausible trees. The Grey family had Y-haplogroup Q1a2b , a patriline with northern forest-zone and Siberian connections, and three maternal mtDNA haplogroups, including U2e, linked above with an isotopically distinct riverine catchment. None of their haplogroups, maternal or paternal, were shared with the Yellow family. Like the Yellows, the Grey patriline buried at Khvalynsk was divided into two primary avuncular branches. But the Greys also included a male related only through his mother. Grey males II:34 and II:1 were 3 rd -degree relatives, probably cousins whose mothers were sisters (U5a1i), sharing only maternal relatives in a cemetery dominated by paternal relations ( Table 6 ). Also, II:1’s mother had married an R1a (R-M459) husband, making II:1 the only R1a (R-M459) male at Khvalynsk II. Male II:1 was included in the Grey family through his relationship with his mother’s sister’s son, hinting at the continuing importance of maternal marriage links in this paternally dominated society. Additionally, a male unrelated to anyone was included in grave II:26, with Y-haplogroup J1 ( Table 6 ). Some males at Khvalynsk II were associated with but not genetically related to the others. Section title: Relatives and families from ancient human DNA Educational score: 1.6972002983093262 Domain: other Document type: Study Language: en As was noted above, the Grey family graves seem to have been arranged around the pre-existing east-west row of Yellow graves, with the Grey mace chief in II:24 located near the copper-rich II:12 male. The Grey family is modeled as present at Khvalynsk over three generations. If the Yellow family was present first, and some overlap is permitted between the two families, then the Yellow and Grey family graves could fit within five generations, or 140 years at 28 years/gen. With no overlap eight generations (224 years) would be required. The two radiocarbon dates on ruminant animal bone from Khvalynsk I and II overlapped in a single century 4450–4350 BCE, supporting a short span of time. Section title: Relatives and families from ancient human DNA Educational score: 3.7599616050720215 Domain: biomedical Document type: Study Language: en The two dominant patrilines at Khvalynsk II had distinct histories and fates. The Q1a Y-haplogroup is also found at the cemetery of Murzikha II, located 400km north of Khvalynsk in the forests of the Volga-Kama region, and chronologically contemporary with Khvalynsk or slightly later . Most men at Murzikha II were Q1a, but from a different lineage (Q1a1) than the Grey family at Khvalynsk (Q1a2). A migrant from the steppes buried in Hungary at Csongrad-Kettëshalom Bastanya, contemporary with Khvalynsk, also had Y-haplogroup Q1a2, like the Grey family, and autosomal DNA similar to Khvalynsk. This steppe male was part of a diaspora of steppe males into the Danube valley that occurred about 4400–4200 BCE. The Q1a patriline was then mobile and wide-ranging, and at Khvalynsk II was accorded the richest grave at the cemetery. However, most of the men at Khvalynsk II, and all the Yellow family, were R1b of the R-L754 > R-L389 > R-V1636 lineage. A millennium later, when the Yamnaya culture appeared, the Q1a Y-haplogroup would be eliminated from steppe patrilines and a different branch of the R1b family, R-Z2103, would become dominant. Section title: Relatives and families from ancient human DNA Educational score: 1.517825961112976 Domain: other Document type: Other Language: en The society that created Khvalynsk II was organized patrilineally, although at least one maternal cousin was included. The dominant male lineage seems to have shifted from R1b to Q1a. The females were from genetically distinct and unrelated families, except for one sister buried with her brother (25&24). The male-centered ancestries at Khvalynsk II suggest a virilocal kinship system, and the absence of their mothers, daughters, and sisters might indicate that this was a burial place for a multi-generational male sodality, with some unrelated females (wives?) and children. Section title: Cranio-facial groups and genetic mating networks in the steppes Educational score: 3.986717939376831 Domain: biomedical Document type: Study Language: en The cranio-facial types of Khvalynsk and neighboring Eneolithic sites were studied by A. A. Khokhlov in Samara as part of a quantitative metric analysis of 549 skulls from the Volga-Ural region . Cranio-facial metrics showed that the Khvalynsk population was an admixture of two major components, one (robust, broad-faced) derived from the northern forest zone and the other (more gracile, narrow-faced) from the southern steppes, a conclusion borne out by aDNA data that came to the same conclusion (see below). Khokhlov further divided each major regional type into two sub-types, so two northern sub-types (Lapp-like and Uralic) and two southern (perhaps lower Don and Caucasus steppe). Khokhlov was uncertain about the exact metric source of the southern component at Khvalynsk, which is also true of the geneticists’ uncertainty about the exact source of the southern genetic component (CHG). His metrics also identified the cranio-facial similarities between most of the first-order relatives discussed above: 12 & 13 brothers, noted as very similar by Khokhlov ; 29 & 30 father-son ; 24 & 25 brother-sister ; and 18 & 33 brothers, noted as similar in Khokhlov . Also Khokhlov felt that the Khvalynsk II burial plot was designated for the burial of some special group of males who died young, compared to the males in Khvalynsk I. In many ways, cranio-facial metrics, traditional demographic research, and aDNA findings confirmed each other at Khvalynsk. Section title: Cranio-facial groups and genetic mating networks in the steppes Educational score: 3.698509931564331 Domain: biomedical Document type: Study Language: en The Khvalynsk population was genetically admixed between northern and southern ancestry types, in general agreement with Khokhlov’s interpretation based on cranio-facial data. The northern type, Eastern Hunter-Gatherers (EHG), evolved in northern Eurasia; and the southern type, designated Caucasus Hunter-Gatherers (CHG), was defined initially by Mesolithic and Early Neolithic inhabitants of Georgia and western Iran . Both the EHG and CHG labels were first applied to hunter-gatherers, but afterwards were extended to genetically similar individuals regardless of economy. Wang et al. recognized that EHG & CHG ancestry like Khvalynsk was shared by Eneolithic individuals at Progress-2 and Vonyuchka-1 (also known as Konstantinovskii-1) in the North Caucasus steppes. They are dated 4336–4173 calBCE ; and 4233–4047 calBCE . Section title: Cranio-facial groups and genetic mating networks in the steppes Educational score: 2.7736313343048096 Domain: biomedical Document type: Study Language: en We do not know the proximate source of the CHG population that mixed with EHG to create the typical Khvalynsk/Progress-2 pattern of genetic ancestry. But it must have separated from other CHG populations in the Caucasus and western Iran before about 6500–6000 BCE, because after this date the CHG populations in the Caucasus and western Iran became admixed with Anatolian Farmer (AF) ancestry. By 4700 BCE, when the first farmers migrated from Georgia across the western North Caucasus Mountains and occupied sites on the north side of the North Caucasus ridge such as Meshoko and Svobodnoe, they had up to 50% AF ancestry . The Progress-2/Khvalynsk steppe people had no AF ancestry, so they did not exchange mates with Meshoko farmers, even if archaeology shows that they did exchange material valuables (see Copper section above). Section title: Cranio-facial groups and genetic mating networks in the steppes Educational score: 4.239251136779785 Domain: biomedical Document type: Study Language: en In the aDNA literature, “steppe ancestry” is a phrase used since Allentoft et al. and Haak et al. to refer to the typical Yamnaya pattern of genetic ancestry. The principal components of steppe ancestry were EHG & CHG, each in robust proportions, like Khvalynsk, although often with more CHG than in the Khvalynsk/Progress-2 population, with an added component of Anatolian Farmer (AF) ancestry (5–15%) that was absent from the Khvalynsk/Progress-2 populations . Also, the Khvalynsk/Progress-2 mating network has not yet yielded the Y-haplogroup mutations that were directly ancestral to the typical Yamnaya form of R1b . The R-V1636 form of R1b, found in males at Khvalynsk, Ekaterinovka Mys, Berezhnovka II, and Progress-2, identifies a branch that split from the Yamnaya branch defined by R-P297 > R-M269 > R-L23 > R-Z2103 ( yfull.com ). This entire branch is absent from the sampled Eneolithic males from the steppes, appearing for the first time in Yamnaya males. The evolution of Yamnaya Y-haplogroup ancestry occurred in a still-unsampled Eneolithic population. Section title: Cranio-facial groups and genetic mating networks in the steppes Educational score: 3.494760274887085 Domain: biomedical Document type: Study Language: en The Eneolithic populations around the Dnieper Rapids were even more different from Yamnaya. All those sampled were admixtures of EHG (primarily) and Western European Hunter-Gatherers (WHG) similar to the Iron Gates Mesolithic populations . Among 30 published individuals from three Neolithic and Eneolithic cemeteries (Dereivka-1, Vil’nyaka, and Vovnigi) in the Dnieper River valley dated 5200–4400 BC, assigned to the Dnieper-Donets culture, there were a few individuals with minor (<10%) CHG ancestry, but most had none . The Khvalynsk/Progress-2 populations had substantial CHG ancestry but no WHG ancestry, ubiquitous in Dnieper-valley populations. This indicates that the Dnieper-Donets mating network did not extend eastward to the Volga, nor westward to the Criş and early Tripol’ye farmers, whose ancestry was typical of European farmers (AF or EEF) . The Dnieper-Donets people seem to have been an endogamous population focused on the rich resources of the Dnieper Rapids. Their substantial WHG ancestry, nearly absent in Yamnaya individuals, rules them out from being a major source for the Yamnaya. Section title: Cranio-facial groups and genetic mating networks in the steppes Educational score: 3.714411973953247 Domain: biomedical Document type: Study Language: en The Sredni Stog culture succeeded and replaced the Dnieper-Donets culture in the strategic Dnieper Rapids and throughout the steppes of Ukraine beginning around 4500–4300 BCE and ending in the late fourth millennium BCE with the appearance of Yamnaya. Unpublished Sredni Stog male genomes exhibit admixture ‘cocktails’ with the same basic elements as Yamnaya (EHG & CHG & AF). The CHG & EHG component was like Khvalynsk/Progress-2, suggesting an eastern origin for at least part of the Sredni Stog population, and the AF component could have come from either the early Maikop or Tripol’ye populations. Sredni Stog introduced into the Ukrainian steppes new funeral customs (the Khvalynsk or ‘Yamnaya’ position), ceramic types (shell-tempered like Khvalynsk), and economies (large numbers of horse bones) that had appeared earlier on the Volga. Sredni Stog has for decades been recognized as an Eneolithic ancestor of Yamnaya influenced by late Khvalynsk, early Maikop, and the Tripol’ye and Varna cultures. But neither R1b Z-2108 nor its immediate ancestral forms are found among sampled Sredni Stog males, most of whom belonged to the R1a or I2a haplogroups, unlike Volga males. The sampled Sredni Stog populations included individuals who autosomally resembled Yamnaya a millennium before the Yamnaya culture appeared. But within that population the Yamnaya Y-haplogroup patriline evolved in a region that has not been sampled. Section title: Conclusion: Khvalynsk as a coalescent culture Educational score: 1.9250215291976929 Domain: other Document type: Study Language: en Khvalynsk was an exceptionally large cemetery of the mid-to-late-fifth millennium , used by a heterogeneous, admixed population. Cranio-facial measurements and genetic ancestry indicate mixture between northern (forest-zone, EHG) and southern (lower Don-Caucasus, CHG) population components . The admixed Khvalynsk population exhibited significant genetic and family diversity. It seems to have been a central place for the ritual integration and unification of a population that normally lived dispersed up and down the Volga in isotopically different catchments. At least one female buried at Khlopkov Bugor, 130 km south on the Volga, was a 2 nd -degree relative (grandmother or great-aunt) of a Yellow-family male buried at Khvalynsk, but the cemetery at Khvalynsk was ten times larger than at Khlopkov Bugor. Section title: Conclusion: Khvalynsk as a coalescent culture Educational score: 1.034934639930725 Domain: other Document type: Other Language: en The exclusive use of domesticated cattle, sheep-goats, and horses for funeral sacrifices at Khvalynsk indicates the acceptance of a new set of religious ideas about the desires of the gods and ancestors, who now could be satisfied only by the sacrifice of domesticated animals (horses included). An early phase in the evolution of this cult might be indicated by the few sheep, goat, and horse sacrifices at the older cemeteries at Ekaterinovka Mys and the related site of Sy’ezzhe about 4700–4500 BCE. Section title: Conclusion: Khvalynsk as a coalescent culture Educational score: 1.379756212234497 Domain: other Document type: Other Language: en About 4500 BCE new long-distance exchange systems connected Khvalynsk with the Varna-era Balkans. Imported copper metal began to flow into the social and political world of steppe societies. These two new imports, copper ornaments and domesticated animals, gave ambitious families at Khvalynsk, ‘aggrandizers’ in Hayden’s terms , two different kinds of status enhancers that could be used to build alliances in a political context that featured war and human trophy-taking. Imported copper was consumed individually, so enhanced individual status; while locally-raised feast animals were consumed communally, so enhanced group solidarity and the generosity of the hosts. At death, people who wore copper ornaments did not normally receive animal sacrifices, and people who received animal sacrifices in the grave did not normally wear copper ornaments, signaling the presence of distinct social segments marked by the two new status enhancers. Polished stone maces were used at Khvalynsk to identify the only adult males who belonged to both segments (copper-users and sacrifice-receivers) simultaneously, arguably to represent the union of both. These mace-chiefs seem to signal the emergence of hierarchy during the Eneolithic, but their maces also symbolized alliance and the agreement of at least two social segments to accept one adult male as their joint representative. Section title: Conclusion: Khvalynsk as a coalescent culture Educational score: 1.9361933469772339 Domain: other Document type: Other Language: en Khvalynsk can be regarded as ‘coalescent culture’, a culture resulting from the integration of cultural components that originally were geographically (north-south), genetically (EHG-CHG), and culturally distinct. Coalescence is different from the concept of hybridity developed in post-colonial studies , in that coalescence focuses on hybrid communities and bridge-building institutions rather than on individual agency and alterity. The coalescent community model, originally developed to characterize multi-ethnic indigenous societies in the post-Contact U.S. southeast , was revised to provide an explanatory framework for the dramatic migrations and community reorganizations of the late thirteenth and fourteenth centuries in the U.S. southwest and the League of the Iroquois . In the American examples, coalescent communities represented the initial phase of cultural integration during a period of population movement, when cultural assimilation and hybridization processes were incomplete, under political conditions where all forms of extra-familial authority were relatively weak. Pre-existing ethnic and tribal identities as well as indigenous political authority were sustained through metaphors of kinship, common origin, or co-residence that were deeply ingrained and resistant to change even after periods of conflict, chaos, and reorganization. Coalescent communities overcame these deeply ingrained identities through the creation of new meta-identities that functioned beside the older, more limited ones. These meta-identities were founded upon new religious beliefs and rituals such as the Kachina Cult that provided new institutions and sodalities that were accorded a level of prestige equal to those of family or clan . Similarly, at Khvalynsk we witness the appearance of new funeral rituals in which domesticated mammals were the mandatory medium for the ceremonies of a social minority that apparently distributed funeral feasts to hundreds of mourners. This new set of rituals and feasts could have sustained a regional meta-identity that overcame divisions based on local kinship and co-residence. Khvalynsk II might even have been a burial place for a new multi-generational male sodality, an example of a bridging institution. Section title: Conclusion: Khvalynsk as a coalescent culture Educational score: 0.9902452826499939 Domain: other Document type: Other Language: en As was mentioned above (see Mace Holders), the League of the Iroquois shared many features of a coalescent community but was understood by those inside the league as the product of a religious awakening inspired by a culture hero who introduced new, integrative, bridge-building rituals at a time of debilitating warfare and sorcery. The most important ritual, the Condolence Ceremony, was conducted when one of the five (originally) League chiefs died. This was perhaps paralleled in the funerals of the mace holders at Khvalynsk. The League also functioned partly to facilitate and sustain trade between previously hostile communities after intensely desired European trade goods were introduced . The Balkan copper trade could have stimulated integrative institutions in the same way. Section title: Conclusion: Khvalynsk as a coalescent culture Educational score: 1.061827540397644 Domain: other Document type: Other Language: en The new religious ideas and rituals at Khvalynsk can be seen as elements in the creation of a new meta-identity that unified the previously disparate populations of the Volga-Don-Caucasus steppes. Domesticated animal sacrifices continued into the Yamnaya period, again among a minority, since only about 15% of Yamnaya graves had animal sacrifices . If Yamnaya is accepted as the material correlate of late Proto-Indo-European languages, then the new religion indicated at Khvalynsk was an important ancestor of Indo-European religious ideas. The first Indo-European priest-figure archetype sacrificed a divine cow from which the world was made, and the Indo-European warrior-hero archetype defeated a serpent who had imprisoned divine cows (or flowing water, in some versions) . Domesticated animal sacrifice was at the root of Indo-European religious ideas. Other integrative institutions probably were added to these rituals as the Eneolithic steppe cultures passed through the coalescent phase to emerge as the hybrid cultural background for the Yamnaya culture. | Study | other | en | 0.999996 |
PMC11699511 | Section title: Introduction Educational score: 2.016486883163452 Domain: other Document type: Other Language: en Dramatic declines in many pollinator populations have been documented worldwide raising concerns about the maintenance of their species through time and space and the sustainability of the ecosystem services they provide. In this context, accurate data on pollinator distribution is crucial to document their current status. Amongst these, specimens preserved in natural history collections are reliable pieces of the puzzle that describes ecological communities, habitats and ecosystem health . Opening these collections and making them accessible through digitisation and publication is, therefore, of paramount importance for generating science-driven conservation strategies. Section title: Introduction Educational score: 2.196105718612671 Domain: other Document type: Study Language: en Over the past decades, researchers at the Mediterranean Institute of marine and continental Biodiversity and Ecology (IMBE) in Marseille have been investigating relationships between pollinators and plants across a variety of habitats in France, with a focus on the Bouches-du-Rhône Department . Several projects have been undertaken by the Institute, including field courses on bee identification and field campaigns in the Calanques National Park and the City of Marseille. These campaigns used standardised sampling protocols such as netting, pan-trapping and trap-nesting (insect hotels, Geslin et al. ). This work generated the collection of many specimens that have been kept in the IMBE since 2015. Additionally, some specimens were sent to enrich the collection from some of the renowned professional taxonomists of France (namely M. Aubert, E. Dufrêne and D. Genoud). Each of the specimens collected has been meticulously pinned, labelled and was frozen at least twice a year to avoid the emergence of pests. Section title: Introduction Educational score: 2.035989999771118 Domain: biomedical Document type: Study Language: en This paper aims to publish the raw data of bee specimens currently housed in the IMBE collections. These include specimens collected during studies, as well as those donated to the Institute, with the exception of specimens provided to professional taxonomists, lost, destroyed during preparation or used for DNA sequencing, representing a small fraction of the collection. This dataset serves as a valuable resource for advancing our understanding of the Mediterranean Basin's bee fauna and supports ongoing efforts to develop national checklists and Red Lists. Section title: Purpose Educational score: 1.6675578355789185 Domain: biomedical Document type: Study Language: en The dataset presented here includes information on a total of 2,181 bee specimens, collected between 2003 and 2023, all identified to the species level, along with detailed information on the date and location of each capture. This dataset includes some Mediterranean species for which few published records are available in national (inpn.mnhn.fr) and international (www.gbif.org) databases. Section title: Additional information Educational score: 1.8452301025390625 Domain: biomedical Document type: Study Language: en The project aims to compile all recorded data on Apoidea , collected during field campaigns and from donations over the past 10 years, as well as opportunistic captures sent by entomologists to the IMBE laboratory. A total of three scientific field campaigns were conducted in the protected area of the Calanques National Park , two in the urban parks of Marseille , one in humid area and three within the Alpes-de-Haute-Provence Regional Park . A few additional specimens were gathered elsewhere in France, Spain and Italy and sent to the IMBE. Section title: Sampling description Educational score: 1.8926211595535278 Domain: biomedical Document type: Study Language: en For each specimen, the following information was retrieved: species, date and location of capture, sex and collector, as well as any relevant ecological data, such as the plant on which the specimen was captured. Specimens were captured with an entomological net or with coloured pan traps. In addition to the data on the original labels, each specimen was assigned a unique identification code written on an added label. Section title: Quality control Educational score: 1.3054300546646118 Domain: other Document type: Other Language: en The specimens were identified by Mathieu Aubert, Claire Bouchot, Eric Dufrêne, Benoît Geslin, Vincent Leclercq, Lise Ropars, Lucie Schurr, Erwin Scheuchl, Benoît Martha, Emile Gigandet, Gilles Mahé, Yvan Brugerolles, Gérard Le Goff and Nicolas J. Vereecken. The taxonomy was checked to be compatible with the French TAXREF v.17,0 from the Museum national d’Histoire naturelle (Paris) data base and with the latest checklist of the bees of the French fauna ( Ropars et al. in press ). In case of name change due to taxonomic update or identification correction, the original name given to the specimen was retained in the 'previousIdentifications' column. Section title: Step description Educational score: 3.236299991607666 Domain: biomedical Document type: Study Language: en All data on the 2181 identified specimens currently in the IMBE collection were input in a table format. Latitude and longitude coordinates of each capture location were obtained either directly from the label or inferred from the location description. In the second case, coordinates were retrieved using the geoportail.gouv.fr website. Coordinates originally in Lambert93 format and in degrees minutes seconds were transformed to standard GPS format (latitude and longitude in decimal degrees). The locations were then verified using the https://www.geoportail.gouv.fr/ website. Coordinates uncertainty (‘coordinateUncertaintyInMeters’ column) was set to at least 100 m, depending on the level of detail provided on the capture location on the specimen label. All formats follow GBIF Darwin Core specification, to ensure interoperability with other international databases. Section title: Step description Educational score: 2.3657114505767822 Domain: biomedical Document type: Study Language: en For 32 specimens, only identification to the genus level was possible. This is indicated by a value of 0 in the ‘identificationVerificationStatus’ column, with relevant additional details provided in the ‘identificationRemarks’ column. Species groups were indicated with ‘gr.’ in the ‘identificationQualifier’ column, such as for the Bombus terrestris species group, which requires genetic data for accurate species-level identification . ‘cf.’ was used to indicate an approximate identification. This was the case of some Hylaeus specimens which could only be assigned to a couple of species: H. pictipes or H. taeniolatus . Section title: Step description Educational score: 1.8263944387435913 Domain: biomedical Document type: Other Language: en All data were entered in CSV format, with the fields separated by tabs and encoded in UTF-8, thus following the protocol compatible with the GBIF database (Global Biodiversity Information Facility, https://www.gbif.org/fr/) as used in previously published datasets . Section title: Description Educational score: 1.1445976495742798 Domain: biomedical Document type: Other Language: en Most of the specimens come from France , one is from Spain and two are from Italy. Section title: Description Educational score: 1.1342543363571167 Domain: other Document type: Other Language: en In France, 93% the data come from the Bouches-du-Rhône Department, with some from Var, Alpes-Maritimes and Alpes-de-Haute-Provence . Section title: Coordinates Educational score: 1.0160740613937378 Domain: other Document type: Other Language: en 39.30 and 49.33 Latitude; -1.28 and 9.20 Longitude. Section title: Description Educational score: 2.2156946659088135 Domain: biomedical Document type: Study Language: en The dataset covers 246 species of Apoidea belonging to the six bee families found in France: Andrenidae , Apidae , Colletidae , Halictidae , Megachilidae and Melittidae (Tables 1 , 2 ). Section title: Temporal coverage Educational score: 1.0419973134994507 Domain: other Document type: Other Language: id Data range: 2003-3-20 – 2023-9-01. Section title: Notes Educational score: 1.5792781114578247 Domain: biomedical Document type: Other Language: en The specimens were captured from 2003 to 2023. Twenty specimens were collected at an unknown date. Most of the specimens were captured in April or May (the time of the year in which bees are the most abundant in this region), totalling 60% of the dated captures . Section title: Collection name Educational score: 1.339406967163086 Domain: other Document type: Other Language: en IMBE Apoidea collection ( Hymenoptera ) Section title: Collection identifier Educational score: 0.9944888949394226 Domain: biomedical Document type: Other Language: en IMBE-H Section title: Specimen preservation method Educational score: 1.3876622915267944 Domain: biomedical Document type: Other Language: en Dried and pinned specimens Section title: Curatorial unit Educational score: 0.9885762929916382 Domain: other Document type: Other Language: fr IMBE, contact: Gabriel Nève (email: gabriel.neve@imbe.fr) Section title: Usage licence Educational score: 1.0548874139785767 Domain: other Document type: Other Language: en Creative Commons Public Domain Waiver (CC-Zero) Section title: IP rights notes Educational score: 1.038129448890686 Domain: other Document type: Other Language: en This work is licensed under a Creative Commons Attribution (CC-BY) 4.0 Licence. All work derived from the present study should cite it appropriately. Section title: Data package title Educational score: 1.1948972940444946 Domain: other Document type: Other Language: en IMBE Bee collection ( Hymenoptera ) Section title: Resource link Educational score: 1.4034777879714966 Domain: biomedical Document type: Other Language: unknown https://doi.org/10.5281/zenodo.13936315 Section title: Alternative identifiers Educational score: 1.4019027948379517 Domain: biomedical Document type: Other Language: unknown https://doi.org/10.5281/zenodo.14356371 Section title: Number of data sets Educational score: 1.1899832487106323 Domain: biomedical Document type: Other Language: unknown 1 Section title: Data set name Educational score: 1.339406967163086 Domain: other Document type: Other Language: en IMBE Apoidea collection ( Hymenoptera ) Section title: Data format Educational score: 1.0696437358856201 Domain: other Document type: Other Language: et CSV (tab delimited values) Section title: Character set Educational score: 1.0613566637039185 Domain: other Document type: Other Language: en IMBE_beeColl_v02.csv Section title: Data format version Educational score: 1.037717580795288 Domain: other Document type: Other Language: en Darwin core, so that it may be transferred later into GBIF. Section title: Description Educational score: 1.5926480293273926 Domain: biomedical Document type: Other Language: en The dataset includes data on 2181 specimens of Apoidea collected or received by researchers at IMBE, in GBIF compatible format. Section title: General discussion Educational score: 1.4443778991699219 Domain: other Document type: Study Language: en At a time of global pollinator decline, this study highlights once again the French Mediterranean Region as one of the main hotspots for wild bee species . The collection holds about a quarter of the 980 species that thrive in mainland France ( Ropars et al. in press ) and the data provided here will contribute to improve knowledge about French bee species. Section title: General discussion Educational score: 1.9029855728149414 Domain: biomedical Document type: Other Language: en The collected specimens listed here, beyond enhancing our understanding of wild bees as previously mentioned, are also used as a reference collection and for the training of entomologists to ensure the accurate identification of future specimens. Moreover, such a collection acts as a 'memory of nature's diversity,' an exceptional resource available to scientists worldwide. Preserving these specimens provides tangible evidence of biodiversity over time, offering a unique opportunity to monitor its evolution in the long term — a critical aspect in the face of the current widespread biodiversity decline and the current major global changes. Section title: General discussion Educational score: 1.5313166379928589 Domain: other Document type: Other Language: en One species in the IMBE collection is classified as 'endangered' by the IUCN at the European level . This species, Lasioglossum soror , was recorded at nine different localities in the Calanques National Park in 2018. Our data suggest that this species is locally widespread and may have been overlooked in other studies as all but two of the 19 specimens in the IMBE collection were collected in coloured pan traps. Section title: General discussion Educational score: 3.449810266494751 Domain: biomedical Document type: Study Language: en Six species in the IMBE collection are classified as “Near Threatened” by the IUCN at the European level : Andrena ovatula , Colletes albomaculatus , Lasioglossum prasinum , Lasioglossum pygmaeum , Lasioglossum sexnotatum and Dasypoda argentata . In addition, D. argentata is also generally recognised as a rare species. The identification of these species is crucial for conservation efforts, as it guides habitat management and protection initiatives. Understanding the status of near-threatened species helps preserve biodiversity and maintain essential ecosystem services provided by pollinators, while raising public awareness and support for bee conservation initiatives. Finally, a total of 68 species recorded in the IMBE collection are currently classified as 'Data Deficient'. This status underscores significant gaps in our understanding of these species' biology, distribution and conservation needs. It highlights the critical importance of maintaining and enhancing monitoring efforts to gather the data necessary for informed conservation decisions. Ensuring the availability of updated and comprehensive information on these species is essential to address potential threats and support their long-term survival. Section title: General discussion Educational score: 1.497004747390747 Domain: other Document type: Other Language: en This study is part of current national effort to document the distribution of pollinators in France. Many initiatives, for example, French National Pollinator Plan, Regional Initiatives ("Plan Régional d'Action") and the French Checklist ( Ropars et al. in press ) are currently assessing the status of wild bees species in France and this topical issue is part of the research programme. By contributing valuable data to the forthcoming French Red List of bees, we aim to foster a greater awareness of the ecological significance of these species. As the IMBE continues its dedicated research, we anticipate that future studies will further enrich our knowledge and help shape effective conservation strategies, ensuring that these vital pollinators thrive for generations to come. Section title: Abbreviation used throughout Educational score: 0.9353255033493042 Domain: other Document type: Other Language: fr IMBE: Institut Méditerranen de Biodiversité et d’Ecologie marine et continentale (Marseille, France) | Other | other | en | 0.999998 |
PMC11699512 | Section title: Introduction Educational score: 3.1828057765960693 Domain: biomedical Document type: Other Language: en The Diptera family Chironomidae (also known as non-biting midges) comprise the most diverse and frequently the most abundant insect group found in freshwaters. Although larvae of some species can be semi-aquatic or terrestrial, the majority are strictly aquatic . The immature stages of chironomids can inhabit a wide range of water types, from thin water film on glaciers to plant-held waters, streams, rivers, lakes, ponds, reservoirs, brackish waters and shoreline habitats . Besides their widespread distribution, the species richness of Chironomidae is usually amongst the highest of aquatic insect families, occasionally exceeding 100 species per site in many aquatic habitats . Section title: Introduction Educational score: 1.6624665260314941 Domain: biomedical Document type: Other Language: en Globally, approximately 7,300 chironomid species are currently known , but the estimated number of species may be around 15,000 . According to Fauna Europaea , nearly 1,300 species have been recorded only from Europe. The available information on chironomid fauna varies considerably amongst European countries, with more comprehensive knowledge for Western Europe compared to other regions . In Central European countries, the chironomid fauna is still limitedly known, despite recent extensive collections and an increasing amount of available data. Section title: Introduction Educational score: 2.8897202014923096 Domain: biomedical Document type: Study Language: en The present study provides a contribution to the knowledge on chironomid fauna of three Central European countries by presenting results from detailed investigations conducted within three selected river catchments representing Croatia, Hungary and Czechia. In this research, we primarily focused on larvae, but pupae and exuviae were also included occasionally and molecular techniques have been applied for certain taxa to achieve precise identification. We provide here the first records of some species in Croatia and Czechia. Notes are given for selected taxa of taxonomic interest. Section title: Study area Educational score: 1.4651554822921753 Domain: other Document type: Study Language: en Our investigations cover three river catchment areas from three Central European countries representing three European ecoregions. Bükkösdi-víz (BUK) is located in the south-western part of Hungary (Pannonian ecoregion with Continental climate) with an area of 185 km 2 and forms a sub-catchment of Fekete-víz within the drainage area of the Drava River. The area is characterised by the hilly surroundings of Mecsek Mountains (sampling sites were located between 104–245 m a.m.s.l.). The Butižnica River (BUT) catchment is situated in the Dinaric Mountains (sampling sites were located between 235–591 m a.m.s.l.), North-Central Dalmatia, Croatia (Balkanic ecoregion, Mediterranean climate). Its drainage area is around 225 km 2 and belongs to the Krka River catchment. The Velička River (VEL) is a sub-catchment of the Morava River and it is located in the South Moravian part of Czechia in the White Carpathian Mountains (Continental ecoregion, humid continental climate). Velička catchment area of 172 km 2 was included in the sampling design of this project (sampling sites were located between 174–446 m a.m.s.l.) . Section title: Sampling, sample processing and identification Educational score: 2.517627000808716 Domain: biomedical Document type: Study Language: en Chironomidae larvae were collected using multihabitat sampling methods during two campaigns (Suppl. material 1 ): one in 2018–2019 at the Bükkösdi-víz catchment and another in 2021 at all catchments. Additionally, at some sites in Bükkösdi-víz catchment, floating chironomid pupal exuviae were collected from the water surface using a hand net (frame 25 × 25 cm, mesh size 250 µm) and a plastic tray in 2021. Section title: Sampling, sample processing and identification Educational score: 3.535428047180176 Domain: biomedical Document type: Study Language: en In case of larvae and pupae, all individuals were identified from samples containing less than 300 individuals, while subsamples of ca. 300 individuals were taken from larger samples. We used multi-level identification methods that included: separating specimens based on macroscopic characteristics; conducting detailed investigations without preparation and using light microscope; and mounting specimens on microscope slides for examination at higher magnification. Exuviae were mounted on microscope slides in every instance. For morphological identification of larvae and pupae/exuviae, multiple keys were used. For further details, see Suppl. material 1 . Section title: Sampling, sample processing and identification Educational score: 2.8420989513397217 Domain: biomedical Document type: Study Language: en In addition to morphological identification, a subset of larvae underwent molecular analysis using a traditional DNA barcoding protocol (Suppl. material 1 ). All sequences were uploaded to the Barcode of Life Data Systems . Section title: Data resources Educational score: 4.132346153259277 Domain: biomedical Document type: Study Language: en Over 140,000 chironomid individuals were collected from 456 samples. A total of 207 taxa identified to at least genus level were documented, belonging to five subfamilies (32 Tanypodinae , 8 Diamesinae , 3 Prodiamesinae , 103 Orthocladiinae , 61 Chironominae ) and 74 genera, providing 5,481 occurrence records. A total of 170 taxa were identified to species level (82% of all taxa), accounting for 3,613 occurrence data (66% of all records). We recorded 117 taxa (93 species) from Croatia, 127 taxa (102 species) from Hungary and 128 taxa (104 species) from Czechia. Across the three countries, Micropsectra sp. (representing different species) provided the highest number of occurrence records amongst taxa, while Parametriocnemus stylatus accounted for the highest number of species-level occurrences, ranking first in both Croatia and Hungary. However, in Czechia, Polypedilum convictum was the most frequent species. Section title: Data resources Educational score: 1.092047095298767 Domain: other Document type: Other Language: en All our records have been uploaded to the Global Biodiversity Information Facility (GBIF) from 78 localities with georeferenced coordinates, available at https://www.gbif.org/dataset/45b4c600-6d61-4eeb-b9a6-6d07e0cdcd1a . Section title: Newly-recorded Chironomidae species Educational score: 2.20790696144104 Domain: biomedical Document type: Other Language: en The first occurrence of 14 species, Macropelopia notata , Eukiefferiella brevicalcar , Eukiefferiella claripennis , Hydrobaenus distylus , Krenosmittia camptophleps , Orthocladius calvus Pinder, 1985, Orthocladius luteipes Goetghebuer, 1938, Orthocladius saxosus , Parakiefferiella triquetra , Rheocricotopus unidentatus Saether & Schnell, 1988, Rheosmittia spinicornis , Microtendipes rydalensis , Paratendipes nudisquama and Endochironomus donatoris Shilova, 1974 were recorded in Croatia. Additionally, the first occurrence of two species, Rheocricotopus unidentatus Saether & Schnell, 1988 and Microtendipes britteni were registered in Czechia. Section title: Molecular identifications Educational score: 4.074565887451172 Domain: biomedical Document type: Study Language: en DNA was extracted from a total of 31 specimens (14 from Hungary, 10 from Croatia and 7 from Czechia) that were uploaded to BOLD. Sequences had a median length of 658 bp, ranging from 569 to 659 bp, meaning all the sequences were high-quality barcodes (≥ 500 bp) being assigned to a Barcode Index Number . The 31 COI-sequences were assigned to 23 BINs, including eight unique BINs that are new to BOLD. Half of the specimens were identified to species level; however, in the case of 16 specimens, there were no matches passing the 1.6% threshold, preventing conclusive species-level identification. Section title: Notes on selected taxa Educational score: 1.7373548746109009 Domain: biomedical Document type: Other Language: en In this section, we summarise detailed information on taxa with taxonomic issues and/or morphological peculiarities. Our aim was not to provide full taxonomic descriptions; therefore, we only highlight the relevant differential characteristics. Section title: Notes on selected taxa Educational score: 3.954878091812134 Domain: biomedical Document type: Study Language: en Clinotanypus – In most parts of Europe, C. nervosus can be found , but from Russia and Romania, larvae identified as C. pinguis have also been recorded . The difference between the two species is that C. nervosus has a 5-tooth ligula, while C. pinguis has ligula with 6–7 teeth . However, C. nervosus is regarded as the only western Palaearctic representative of the genus, while C. pinguis is widely distributed in North America and, to our best knowledge, has never been found in the Palaearctic as adult. In our study, Clinotanypus larvae were found in Hungary and all of them had 6-toothed ligula. DNA barcoding revealed that this morphotype belongs to C. nervosus , proving that two morphotypes of this species exist and the new morphotype can be dominant in some populations. Furthermore, our results raise the possibility that the occurrence of C. pinguis in Europe might be based on misidentification. Section title: Notes on selected taxa Educational score: 2.8757944107055664 Domain: biomedical Document type: Study Language: en Conchapelopia – In our study, larvae were identified to species level only when associated pupae were available. We distinguished two pupal morphotypes in our material: pupae of C. melanops are well recognisable, while there are small differences between C. pallidula and C. triannulata , species representing the other morphotype. In Hungary, the morphology of the collected specimens clearly fits the description of C. triannulata , which was also found in a previous study close to the Bükkösdi-víz catchment . However, the Croatian specimens were not unambiguously separable and were identified as a species pair. Section title: Notes on selected taxa Educational score: 2.8453667163848877 Domain: biomedical Document type: Study Language: en Thienemannimyia – The species of the genus are not or hardly distinguishable as either larvae or pupae . In our study, larvae were morphologically identified to genus level, while one pupa from Czechia was identified as T. carnea , and one pupa from Croatia as T. lentiginosa / laeta . DNA barcoding was performed for the latter pupa and two larvae, but no match was found in BOLD systems in either case. Section title: Notes on selected taxa Educational score: 4.068066596984863 Domain: biomedical Document type: Study Language: en Zavrelimyia – According to the broadened generic conception of Zavrelimyia , the genus contains more former genera as subgenera . In our study subgenera Zavrelimyia s. str. and Zavrelimyia ( Paramerina ) occurred, with larvae identifiable only to subgenera by morphological characters. Some pupae/exuviae were identified as Z. ( Z. ) barbatipes and as Z. ( P. ) cingulata . The pupal form Paramerina spec-Griechenland was found in Croatia and sequences from DNA barcoding matched (100% and 98.48% similarity) with one sequence under the name Zavrelimyia divisa in BOLD Systems. However, these sequences differed more than 9% from other sequences of Z. divisa , which questions the correctness of species identification. The clear morphological differences between Z. ( P. ) divisa and P. spec-Griechenland and the inconsistencies in molecular results suggest that the pupal form represents a different species. Section title: Notes on selected taxa Educational score: 3.199644088745117 Domain: biomedical Document type: Study Language: en Diamesa – There are many difficulties in separating Diamesa species as larvae (e.g. Janecek , Rossaro and Lencioni ) and as pupae by morphological characters. In our study, identification was possible only to the species group level in many cases ( D. cinerella -Gr., D. zernyi -Gr.). Amongst D. insignipes / cinerella / tonsa , only pupae with very typical characters (see Langton and Visser ) were identified as D. insignipes or as D. tonsa . In the case of larvae, D. tonsa was relatively easily recognisable, based on the colouration of the head , but D. insignipes was only accepted when DNA barcoding confirmed the species identity (one specimen from Croatia and two specimens from Czechia). Section title: Notes on selected taxa Educational score: 2.5441107749938965 Domain: biomedical Document type: Study Language: en Monodiamesa – Some larvae were found in Bükkösdi-víz catchment, Hungary. Only one species, M. nitida has been recorded in the country , but neither the larva nor the pupa of this species is known . The other species that potentially might occur in Hungary is M. bathyphila , a widespread species in Europe. However, DNA barcoding revealed a high distance between M. bathyphila and our studied individual (only 89.74% similarity), suggesting that the collected specimens might belong to M. nitida . Section title: Notes on selected taxa Educational score: 2.76025128364563 Domain: biomedical Document type: Study Language: en Chaetocladius – The morphological identification of the larvae of this genus is only possible at species group level , whereas species can be distinguished as pupal exuviae . For this reason, in our study, only exuviae were identified to species ( C. piger , C. laminatus ). One pupa was identified as a pupal form ( Chaetocladius pe1) because not all characters were clearly recognisable, but, most probably, it was an atypical individual of C. piger . Section title: Notes on selected taxa Educational score: 2.7998461723327637 Domain: biomedical Document type: Study Language: en Corynoneura – From one site in Hungary, we identified the larval morphotype as C. cf. antennalis , although this name is regarded as a synonym of C. celeripes by some authors , but questioned by other experts . Accordingly, we used the name C. antennalis (as cf.) in our dataset. A larval morphotype ( Corynoneura sp. BUT) was collected from many sites in Croatia that closely resembles C. lobata (three median teeth, strongly reduced first lateral teeth), but the central median tooth on the mentum is as large as the other median teeth (see Corynoneura species A in Cranston ). Section title: Notes on selected taxa Educational score: 1.5017632246017456 Domain: other Document type: Other Language: en Cricotopus – Despite the available keys for Cricotopus species (e.g. Hirvenoja , Langton and Visser , Cuppen and Tempelman ), specimens of this genus were identified to species level only occasionally due to the little morphological differences between species in many cases. Section title: Notes on selected taxa Educational score: 3.1821372509002686 Domain: biomedical Document type: Study Language: en Epoicocladius – The Palaearctic E. ephemerae and Nearctic E. flavens are currently recognised as separate species (e.g. Ashe and O’Connor , Andersen et al. ); however, some authors suggest they are synonyms (e.g. Evenhuis and Pape ). Accordingly, we used the name E. ephemerae for our records. Larvae collected in Butižnica River catchment were somewhat different from those in other populations, as the setae on body segments were remarkably less numerous and paler. DNA barcoding resulted in no match with any Epoicocladius sequences, raising the possibility that these larvae represent a different species within the genus. Section title: Notes on selected taxa Educational score: 4.1195149421691895 Domain: biomedical Document type: Study Language: en Eukiefferiella – In the case of E. devonica , larvae were identified to species only as pupae and to species group as larvae. Eukiefferiella minor and E. fittkaui were separated neither as larvae nor as pupae due to very little morphological differences . A strange larval morphotype ( Eukiefferiella sp. BUT) was found in the Butižnica River catchment: its morphology fits the generic description of Eukiefferiella , but the thorax is covered with dense, relatively long (approx. half the width of the thoracic segment) setae , which is unique in the genus. In the Velička River catchment, a peculiar pupa ( Eukiefferiella sp. VEL) was collected. In the key of Langton and Visser , the specimen could be identified as either E. claripennis or E. brevicalcar due to the swollen base of the thoracic horn, but the length of its apical filament separates this morphotype from both species (longer than in E. claripennis , but shorter than in E. brevicalcar ). Although the pattern of the hooks on abdominal segments resembles E. claripennis , it is not clear whether it is a new pupal form or a non-typical representative of one or the other species. Section title: Notes on selected taxa Educational score: 2.418738603591919 Domain: biomedical Document type: Study Language: en Krenosmittia – The two common species of the genus ( K. boreoalpina and K. camptophlebs ) can be separated by the length of the 1 st antennal segment and the head , but this separation is considered as tentative . In the Croatian material, the dimensions of the head and the antenna fell in the range of K. camptophleps and pupae found here also confirmed the species identity. However, in the Czech material, only larvae occurred and their dimensions were ambiguous; therefore, we identified these specimens only to genus level. Section title: Notes on selected taxa Educational score: 1.8861498832702637 Domain: biomedical Document type: Other Language: en Limnophyes – Larvae of the genus are morphologically indistinguishable (e.g. Janecek , Orendt and Bendt ). DNA barcoding performed on one of the many morphologically identical larvae from the Bükkösdi-víz catchment revealed that likely they belong to L. minimus . Section title: Notes on selected taxa Educational score: 1.998963475227356 Domain: biomedical Document type: Other Language: en Metriocnemus – Larvae of the genus are hardly distinguishable due to poorly-known morphological differences and many species with undescribed larvae . DNA barcoding was performed on a larva from the Bükkösdi-víz catchment, but there was no match with valid species in BOLD systems, although the generic status was confirmed. Section title: Notes on selected taxa Educational score: 4.122554302215576 Domain: biomedical Document type: Study Language: en Orthocladius – Larvae and pupae belonging to the subgenus Orthocladius s. str. were identified to species level only occasionally due to unclear taxonomy and little morphological differences between the species . Nearly all larvae and pupae belonging to the subgenus Euorthocladius were separated to species using multiple keys . One larva was morphologically different from the identifiable ones and either the DNA barcoding resulted in no match in BOLD Systems. Accordingly, this larva represents a new morphotype and a species that, at least as larvae, has not yet been described. In the subgenus Symposiocladius , larvae were identified as O. ( S. ) lignicola and as O. ( S. ) holsatus based on the L4 hair tuft and morphology of the mentum . The occurrence of O. ( S. ) lignicola in Bükkösdi-víz catchment was also confirmed by pupae . However, both larval types might represent other species as well, for example, the larva of O. ( S. ) ruffoi is not known . Larvae from the Butižnica River catchment closely resembled O. ( S. ) holsatus , but the L4 hair tuft was much shorter than it is depicted in Cuppen and Tempelman ; accordingly, they only were identified to subgenus level. Section title: Notes on selected taxa Educational score: 3.3842594623565674 Domain: biomedical Document type: Study Language: en Parametriocnemus – Larvae from all catchments were identified as P. stylatus , based on their morphology (e.g. Janecek ) and co-occurrence of conspecific pupae . However, DNA barcoding of a larva from the Bükkösdi-víz catchment resulted in no match with P. stylatus , suggesting that it is rather a species complex (see Orendt and Bendt ). Some pupae collected in the Velička River catchment proved to be Parametriocnemus Pe1; DNA barcoding also resulted in no match in BOLD systems in this case. Section title: Notes on selected taxa Educational score: 1.9838411808013916 Domain: other Document type: Other Language: en Paraphaenocladius – From the Velička River catchment, larvae were identified as P. impensus , but only pupae of P. irritus were found at the same site. The larvae of P. irritus is not known/not included in the keys available for European Paraphaenocladius species (e.g. Klink and Moller Pillot , Orendt and Bendt ). Accordingly, the possibility arises that the larvae of P. impensus and P. irritus are morphologically identical or hardly separable and only the latter species was collected in Czechia. Section title: Notes on selected taxa Educational score: 1.820501685142517 Domain: biomedical Document type: Other Language: en Rheocricotopus – At multiple sites in the Velička River catchment, a morphotype ( Rheocricotopus sp. VEL) was found , which has the general characteristics of the genus, but does not resemble any species with currently known larva or pupa . Section title: Notes on selected taxa Educational score: 2.447326421737671 Domain: biomedical Document type: Study Language: en Tvetenia – In the Butižnica River catchment, two larval morphotypes were found that differed from any species in the genus . Tvetenia sp. A is a known larval form (e.g. Cranston , Schmid , Klink and Moller Pillot ) and was suggested to be linked with various species . Tvetenia sp. BUT clearly belongs to this genus, based on the morphology of the head and the presence of long setae on body segments, but does not resemble any other species with known larva. Section title: Notes on selected taxa Educational score: 3.500878095626831 Domain: biomedical Document type: Study Language: en Genus unknown – The morphology of some larvae from the Velička River catchment clearly matches with the taxon Orthocladiinae sp. “Berka vor dem Hainich, Thüringen” in Orendt and Bendt . Our results proved that the distribution of this taxon is not limited to Germany. Orthocladiinae larvae and a pharate larvae with unique morphological characteristics were found in Bükkösdi-víz catchment ( Orthocladiinae Gen. sp. III.). Neither larval nor pupal characteristics resemble any species or genera included in the keys used in our work. Some larval characters suggest that this morphotype belongs to Parakiefferiella , but the thoracic horn is completely different from that characteristic for the genus. DNA barcoding also resulted in no match in BOLD Systems. Section title: Notes on selected taxa Educational score: 4.326294422149658 Domain: biomedical Document type: Study Language: en Chironomus – Based on DNA barcoding, a larva from the Bükkösdi-víz catchment identified earlier as C. entis by morphological characters, proved to be C. plumosus . As sequences can also be found for C. entis in the BOLD Systems, we accept that this result is correct. In our larva, the upper surface of the antennal pedestal was completely light brown, which is a distinctive character for C. entis according to Vallenduuk . Moreover, the dimensions of the parts of the head fell in the range of this species, but it must be noted that these characters can vary and overlap between the species. According to Orendt and Spies , C. plumosus can be recognised by a distinct dark streak between the antennal pedestal and the upper eyespot. Our specimen proved that this pigmentation could spread further on the upper surface of the antennal pedestal. Accordingly, all characters used to separate the two species may overlap and should be regarded with caution. DNA barcoding performed on a larva identified as C. bernensis resulted in the closest match with a sequence under the name C. annularius . However, in this BIN, there are also specimens under the name C. bernensis MOTU7, which was analysed by Pfenninger et al. , with the result that the nominal species C. bernensis might be a cryptic species complex. However, the taxonomy of C. annularius is also not clear with more biological species mentioned under this name by various authors . Nevertheless, the two larval morphotypes that represent these names can clearly be separated by the presence of lateral gills on abdominal segment VII in C. annularius , while these gills lack in C. bernensis (e.g. Orendt and Spies , Vallenduuk ). As the larvae we found had no lateral gills on segment VII, accordingly we listed them as C. bernensis . Section title: Notes on selected taxa Educational score: 3.0556774139404297 Domain: biomedical Document type: Study Language: en Phaenopsectra – Larvae from the Bükkösdi-víz and Velička River catchments were identified as P. flavipes , which was confirmed by DNA barcoding of an associated pupa. Another larval form was collected in the Butižnica River catchment, with the mentum and mandible being identical with those depicted as Phaenopsectra sp. in Andersen et al. . Section title: Notes on selected taxa Educational score: 2.0757973194122314 Domain: biomedical Document type: Other Language: en Polypedilum – Larvae identified as P. convictum from the Butižnica River catchment had elongate anal tubules which is unusual for this species (triangular tubules are characteristic, Vallenduuk ). It is not clear if this difference is a population level variability or indicates a different species. Section title: Notes on selected taxa Educational score: 3.8890674114227295 Domain: biomedical Document type: Study Language: en Micropsectra – We had difficulties with the identification of a larval Tanytarsini morphotype (cf. Micropsectra sp.) from Bükkösdi-víz catchment. The bifid premandible suggested Micropsectra , but the remarkably small body size, the lack of spur on antennal pedestal and the small number of claws on posterior parapods rather resemble Tanytarsus . The separation of the two genera is very difficult and probably the best character is the shape of the premandible . Based on the bifid premandible, our larvae most probably belong to Micropsectra . In addition, co-occurring small Micropsectra pupae were also collected. These pupae resemble those of M. notescens , but are much smaller and less pigmented. The similar size of the larvae and pupae raises the possibility that they might represent the same species. The pupal morphotype Micropsectra sp. BUK1 was collected at one site in the Bükkösdi-víz catchment. This morphotype is most similar to M. auvergnensis due to the shagreen on the pleura of segments II-V, but, in the case of the latter species, pleura of segments II-VII have shagreen. Furthermore, male hypopygium was observable in one pupa and it clearly differed from that of M. auvergnensis . Further studies are in progress to clarify the taxonomic status of this species. Section title: Chironomidae Educational score: 1.0382393598556519 Domain: biomedical Document type: Other Language: de DAB5B22E-3830-523F-B4C6-F8358BA54902 Section title: Chironomidae Educational score: 1.3207838535308838 Domain: biomedical Document type: Other Language: unknown https://www.gbif.org/dataset/45b4c600-6d61-4eeb-b9a6-6d07e0cdcd1a Section title: Discussion Educational score: 2.064913511276245 Domain: biomedical Document type: Study Language: en The first comprehensive species list of Chironomidae of Croatia was published by Čerba et al. , presenting 239 species. Since then, more papers have been published (Suppl. material 2 ) bringing the number of species to 245. Our study added 14 new species to the Croatian fauna raising the number of species to 259. The fact that the new species came from a single, relatively small area, indicates that the chironomid species list of the country is far from complete. Section title: Discussion Educational score: 1.7832179069519043 Domain: biomedical Document type: Study Language: en The latest updated checklist of Chironomidae of Czechia was published by Bitušík and Brabec and it included 271 species. Since then, multiple papers (Suppl. material 2 ) contributed with an additional 104 species to the fauna. Our study contributed to the species list with two species raising the number of species to 377. Section title: Discussion Educational score: 1.6872471570968628 Domain: other Document type: Study Language: en The first comprehensive checklist of Hungarian chironomid fauna was published by Móra and Dévai listing 228 species. Since then, more than 20 faunistic studies were published containing new species to the Hungarian fauna (Suppl. material 2 ) and the number of recorded species has expanded to 371 (own data). Although the fauna of the country is far from fully documented, we did not find new species, most probably because the Mecsek Mts. and its surroundings are amongst the better studied areas , but we provided new occurrence data for many rare species. Section title: Discussion Educational score: 1.934155821800232 Domain: biomedical Document type: Study Language: en In addition to species, morphotypes without clear taxonomic state (see Notes on selected taxa) have also been found in each country, with the possibility that some of them might be new to science. Our results are in concordance with the conclusion that undescribed species are still being discovered, even in well-documented areas like the Western Palearctic Region and, in the taxonomy of many groups remain unresolved even within Europe . Section title: Discussion Educational score: 2.2550766468048096 Domain: other Document type: Study Language: en Although our study focused on small streams representing a limited range of habitat types, a remarkably high number of taxa have been detected. It might be due to the extensive sampling and accurate sorting process that increase the chance to find rare species . Additionally, the multi-level subsampling for detailed species-level identification, which is frequently neglected in ecological, biodiversity or biomonitoring research (e.g. Raunio et al. , Dorić et al. ), increased remarkably the number of detected taxa. Section title: Discussion Educational score: 4.116321086883545 Domain: biomedical Document type: Study Language: en Using DNA barcoding is an effective method for species identification; however, its success largely depends on the availability of appropriate reference databases . With the increasing effort in barcoding campaigns, more data are becoming available for Chironomidae . However, reference databases of the Central European Chironomidae are incomplete, as the number of public barcodes from our study area demonstrates: from Czechia, 21 COI-sequences are available, while the number of public barcodes is five from both Hungary and Croatia. In addition, we found some inconsistencies in molecular results (e.g. Zavrelimyia species, see Notes on selected taxa) and these did not fit with the results based on morphological investigation in some cases, highlighting the conspicuous gaps in the databases. It is widely recognised that combining traditional taxonomy with molecular tools can contribute to the better documentation of a region’s Chironomidae fauna and, accordingly, DNA barcoding has contributed to our taxa list in a few cases. Section title: Discussion Educational score: 2.9951164722442627 Domain: biomedical Document type: Study Language: en Finally, we can conclude that extensive studies in relatively small areas with a limited range of habitats (only streams in hilly regions) can remarkably contribute to the local and global knowledge on Chironomidae fauna, especially when taxonomically difficult and often problematic larvae are investigated. However, the number of species might increase and taxonomic problems might be solved more easily, if all life stages and molecular methods are included in these studies. | Other | biomedical | en | 0.999994 |
PMC11699514 | Section title: Introduction Educational score: 3.5412163734436035 Domain: biomedical Document type: Other Language: en The class Dothideomycetes is megadiverse and the largest class of fungi in the phylum Ascomycota . Currently, the class is divided into 54 orders and 226 families and contains about 20,000 described species . The best-known and studied orders are Capnodiales s. lato, Dothideales , Botryosphaeriales , and Pleosporales . Other orders of Dothideomycetes are less known, primarily because their members are less common in the environment, although some of them are important for human life and the economy . Section title: Introduction Educational score: 4.183639049530029 Domain: biomedical Document type: Study Language: en The high species diversity observed in Dothideomycetes is reflected by the wide geographical range and diversity of lifestyles of its members. They are known from all areas of the world and habitats, even capable of colonizing extreme habitats such as cold deserts , deep-sea sediments, including methane sediments , saline waters , acidic environments , and resin exudates . Species of Dothideomycetes are plant pathogens, saprobes, rock-inhabiting fungi, lichens, endophytes, and epiphytes. Hongsanan et al. defined fungal epiphytes as species occurring on the living plant surfaces, especially leaves, which belong to the following orders of Dothideomycetes : Asterinales , Capnodiales , Microthyriales , Zeloasperisporiales , and Meliolales in Sordariomycetes . However, epiphytes are also known in other, less obvious lineages . The special kind of epiphytes are sooty moulds, which live on leaves/needles covered with exudates of phloem-feeding insects, especially honeydew secreted by aphids . Section title: Introduction Educational score: 3.7893459796905518 Domain: biomedical Document type: Study Language: en Recently, we isolated an enigmatic fungus from a sooty mould colony on Symphoricarpos albus in southern Poland, which showed affinities to members of the poorly known order Neodactylariales . In this study, a novel species accommodated in a new genus is described for this fungus. The phylogenetic placement of this new genus and its most closely related genera are analysed, and emended descriptions of the order Neodactylariales and family Neodactylariaceae are provided. Section title: Strains and morphological analyses Educational score: 4.177570819854736 Domain: biomedical Document type: Study Language: en The strain was isolated from the sooty mould community on Symphoricarpos albus leaves planted in municipal greenery in southern Poland . Macroscopic features of cultures were observed and photographed using 4-week-old colonies grown on MEA and PDA at 6 °C, 15 °C, and 25 °C, as well as 15-week-old colonies grown on MEA and PDA at 15 °C. Description of culture characteristics is based on 4-week-old colonies. The morphology of colonies observed after 15 weeks is briefly mentioned in the subsection “notes”. Growth at different temperatures was assessed by measuring the colony diameter after 4 weeks. Microscopic features were analysed using colonies older than 8 weeks grown on MEA at 15 °C. Hyphae and conidia taken from the edge of the colony were mounted in lactic acid (80%) on microscope slides and examined under a Nikon Eclipse 80i light microscope. Microscopic structures were measured and photographed using NIS‐Elements BR 3.0 imaging software. The holotype is a dried specimen obtained from culture and deposited in the fungal collection of the W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków ( KRAM F ). Culture is preserved in the culture collection of the Westerdijk Fungal Biodiversity Institute ( CBS ) and in the W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków. The morphological characteristics of Beaucarneamyces and Pseudoarthrographis used for the emendation of Neodactylariales and Neodactylariaceae are taken from the literature . Section title: DNA isolation, PCR , and sequencing Educational score: 4.166259288787842 Domain: biomedical Document type: Study Language: en After growth of cultures for about one month, genomic DNA was extracted from a portion of mycelium using the DNeasy® Plant Mini Kit (Qiagen, Germany). To conduct molecular studies, four genetic loci were amplified, namely ITS1-5.8S-ITS2 rDNA (= ITS), 28S D1–D2 rDNA (= LSU), a small subunit rDNA (= SSU ), and a protein-coding gene—a partial DNA-directed RNA polymerase II second largest subunit ( rpb2 ). To amplify the loci mentioned above, four different primer pairs were used: ITS1 and LR5 for a fragment containing ITS and LSU , NS1 and NS4 for SSU , and fRPB2-5F and fRPB2-7cR for rpb2 . Polymerase chain reactions ( PCR ) for all loci were performed in a reaction mixture prepared as described in Piątek et al. . Amplification conditions for PCR reactions of the fragment containing ITS and LSU were performed as described by Piątek et al. ; in turn, amplification conditions for SSU and rpb2 were described by Piątek et al. . The PCR products were checked on 1% agarose gels and enzymatically purified using Exo-BAP Mix (EURx, Gdańsk, Poland). DNA sequencing was carried out in both directions by Macrogen Europe B.V. (Amsterdam, the Netherlands). ITS was sequenced using primers ITS1 and ITS4; LSU was sequenced using primers LSU1Fd and LR5; and SSU and rpb2 were sequenced with the same pairs of primers that were used for amplification. Section title: Phylogenetic analysis Educational score: 4.204723834991455 Domain: biomedical Document type: Study Language: en The affinities of obtained ITS, LSU, SSU , and rpb2 sequences of the isolated fungus were performed in the NCBI’s GenBank nucleotide database using the megablast search tool . To resolve its phylogenetic position, the multilocus dataset containing LSU, ITS, SSU , and rpb2 sequences of representatives of Dothideomycetes used by Maharachchikumbura et al. was obtained. The dataset was, among others, augmented by sequences of representatives of the orders Neodactylariales ( Beaucarneamyces , Neodactylaria , Pseudoarthrographis ) and Oncopodiellales ( Diplocladiella , Oncopodiella ) (Suppl. material 1 ). Sequences were separately aligned for each single-gene dataset using the MAFFT algorithm in Geneious 11.1.5 and concatenated. Phylogenetic relationships were inferred using the concatenated LSU-ITS- SSU - rpb2 alignment by the maximum likelihood ( ML ) analysis using RAxML-NG v. 1.1.1 , with 1000 bootstrap replicates. The best-fit substitution models were selected with ModelTest-NG v. 0.2.0 using the Bayesian Information Criterion ( BIC ) . The final phylogenetic tree was visualized using FigTree v1.4.3. The alignment was deposited at figshare.com . Section title: Phylogenetic analysis Educational score: 4.303217887878418 Domain: biomedical Document type: Study Language: en The concatenated multilocus dataset (LSU, ITS, SSU , and rpb2 ) included sequences of 154 representatives of most of the orders of the class Dothideomycetes , including all representatives of Neodactylariales , and a member of the class Arthoniomycetes ( Schismatomma decolorans ) used as an outgroup. The concatenated alignment contained 4483 characters . The best-fit substitution models selected for single-gene alignments were as follows: GTR+I+G4 for both ITS and LSU, TrNef+I+G4 for SSU , and TPM3uf+I+G4, TPM2uf+I+G4, and TIM2+I+G4 for rpb2 (three codons). The phylogenetic tree resulting from maximum likelihood analysis is shown in Fig. 1 . Representatives of Dothideomycetes formed lineages that correspond to the orders that were well supported, but relationships between orders were mostly not resolved. The strain of a new genus and species, Szaferohypha enigmatica , clustered within the lineage assigned to the order Neodactylariales , but its relationship to the remaining genera of this order ( Beaucarneamyces , Neodactylaria , and Pseudoarthrographis ) was not resolved. The clustering of genera assigned to Neodactylariales was well supported (MLB = 85%), and this order formed a strongly supported (MLB = 98%) sister group to the lineage assigned to the order Oncopodiellales that was fully supported (MLB = 100%). Section title: Neodactylariales Educational score: 1.3918721675872803 Domain: biomedical Document type: Other Language: pl H. Zheng & Z.F. Yu, MycoKeys 73: 74 , emend. Piątek, Stryjak-Bogacka & Czachura Section title: Neodactylariales Educational score: 1.0069527626037598 Domain: biomedical Document type: Other Language: de B462339F-821C-5248-B4D8-13398D9CA787 Section title: Description. Educational score: 3.873689889907837 Domain: biomedical Document type: Other Language: en Asexual morph from human-associated organs or saprobic on plant debris or epiphytic on living leaves . Conidiophores acroauxic, macronematous or micronematous , mononematous, branched or unbranched, or reduced to conidiogenous cells . Conidiogenous cells mono- and polyblastic, sympodially extended or not . Conidia solitary or in branched or unbranched chains , hyaline or pale pigmented, smooth, verrucous, or echinulate. Chlamydospores sometimes present. Sexual morph not observed. Section title: Type genus. Educational score: 1.6686482429504395 Domain: biomedical Document type: Other Language: es Neodactylaria Guevara-Suarez, Deanna A. Sutton, Wiederh. & Gené. Section title: Notes. Educational score: 1.5741275548934937 Domain: biomedical Document type: Other Language: en Because of the inclusion of genera Beaucarneamyces , Pseudoarthrographis , and Szaferohypha in Neodactylariales , the emended description of this order is necessary. The original description of this order is included without changes and emended by crucial characters of these genera given in bold. Section title: Neodactylariaceae Educational score: 1.401848554611206 Domain: biomedical Document type: Other Language: pl H. Zheng & Z.F. Yu, MycoKeys 73: 75 , emend. Piątek, Stryjak-Bogacka & Czachura Section title: Neodactylariaceae Educational score: 1.0024933815002441 Domain: biomedical Document type: Other Language: en B6ABE117-6D66-58A5-B038-493B2ABD34E6 Section title: Description. Educational score: 3.9430060386657715 Domain: biomedical Document type: Study Language: en Mycelium superficial or immersed, composed of branched, septate, hyaline to subhyaline hyphae. Conidiophores macronematous or micronematous , mononematous, straight or flexuous, septate, unbranched, or reduced to conidiogenous cells . Conidiogenous cells terminal or intercalary, monoblastic or polyblastic, sympodial or not , with short-cylindrical denticles or without denticles . Conidial secession schizolytic. Conidia solitary or in branched or unbranched chains , smooth or finely echinulate. Chlamydospores sometimes present. Sexual morph not observed. Section title: Type genus. Educational score: 1.6686482429504395 Domain: biomedical Document type: Other Language: es Neodactylaria Guevara-Suarez, Deanna A. Sutton, Wiederh. & Gené. Section title: Notes. Educational score: 1.9664419889450073 Domain: biomedical Document type: Other Language: en The order Neodactylariales contains one family, Neodactylariaceae , that is emended by features of genera Beaucarneamyces , Pseudoarthrographis , and Szaferohypha . The original description of this family is included without changes, and the emended part is given in bold. Section title: Szaferohypha Educational score: 1.5881973505020142 Domain: other Document type: Other Language: pl Piątek, Stryjak-Bogacka & Czachura gen. nov. Section title: Szaferohypha Educational score: 1.2660876512527466 Domain: biomedical Document type: Other Language: en 3497AF02-D430-58CD-9593-E0740167931A Section title: Szaferohypha Educational score: 0.9979390501976013 Domain: other Document type: Other Language: unknown 856722 Section title: Etymology. Educational score: 0.9271566271781921 Domain: other Document type: Other Language: en Named after Polish botanist and palaeobotanist Professor Władysław Szafer , the first director of the W. Szafer Institute of Botany, Polish Academy of Sciences. Section title: Description. Educational score: 3.991952657699585 Domain: biomedical Document type: Study Language: en Colonies erumpent, spreading, umbonate, grayish-brown, with a velvety surface caused by abundant aerial mycelium, margin undulate. Mycelium composed of branched, septate, hyaline, subhyaline, pale brown, or brown, smooth or verrucose, usually thick-walled hyphae. Conidiophores micronematous, reduced to conidiogenous cells, rarely macronematous. Conidiogenous cells terminal, rarely lateral, monoblastic, hyaline, subhyaline, pale brown, or brown. Conidia globose, subglobose, rarely broadly ellipsoid, hyaline, subhyaline, or brown, aseptate, rarely with 1–2 septa or muriformly septate, smooth or finely verrucose, thick-walled, sometimes produced intercalary. Section title: Type species. Educational score: 1.5916739702224731 Domain: biomedical Document type: Other Language: pl Szaferohypha enigmatica Piątek, Stryjak-Bogacka & Czachura. Section title: Szaferohypha enigmatica Educational score: 1.5492905378341675 Domain: biomedical Document type: Other Language: pl Piątek, Stryjak-Bogacka & Czachura sp. nov. Section title: Szaferohypha enigmatica Educational score: 0.9312447905540466 Domain: biomedical Document type: Other Language: de 48C7118F-FEEF-54F1-9AB3-E5553922377E Section title: Szaferohypha enigmatica Educational score: 1.0010961294174194 Domain: other Document type: Other Language: unknown 856723 Section title: Szaferohypha enigmatica Educational score: 1.3592480421066284 Domain: biomedical Document type: Other Language: da Figs 2 , 3 , 4 Section title: Etymology. Educational score: 1.8409250974655151 Domain: biomedical Document type: Other Language: en Refers to the uncertain taxonomic position of this fungus after the first molecular analyses. Section title: DNA barcodes. Educational score: 2.449439287185669 Domain: biomedical Document type: Other Language: en ITS , LSU , SSU , rpb2 . Section title: Typus. Educational score: 1.1044585704803467 Domain: other Document type: Other Language: en Poland • Małopolska Province, Tarnów County: Tarnów–Piaskówka, municipal greenery, isolated from sooty mould community on Symphoricarpos albus leaves, 1 Oct. 2018, leg. M. Piątek, W. Bartoszek & P. Czachura . Section title: Description. Educational score: 4.238259315490723 Domain: biomedical Document type: Study Language: en Mycelium composed of branched, septate, hyaline, subhyaline, pale brown, or brown, smooth or verrucose, usually thick-walled hyphae, 2–4 µm, sometimes with oil guttules; wall ca. 0.5 µm thick. Conidiophores micronematous, reduced to conidiogenous cells, rarely macronematous. Conidiogeneous cells terminal, rarely lateral, monoblastic, hyaline, subhyaline, pale brown, or brown, 3.5–13.5 × 2.5–4.5 µm. Conidia globose, subglobose, rarely broadly ellipsoid, hyaline, subhyaline, or brown, aseptate, rarely with 1–2 septa or muriformly septate, smooth or finely verrucose, thick-walled, 6.5–15 × 6–13.5 µm, sometimes germinating into hypha or produced intercalary, wall ca. 0.5–1.5 µm thick. Section title: Culture characteristics. Educational score: 2.2432658672332764 Domain: biomedical Document type: Other Language: en Colonies on MEA erumpent, spreading, umbonate, grayish, reaching 1 mm diam. after 4 weeks at 6 °C, 4 mm diam. after 4 weeks at 15 °C, and 8 mm diam. after 4 weeks at 25 °C, with a velvety surface caused by abundant aerial mycelium, margin entire. Reverse black. Colonies on PDA erumpent, spreading, umbonate, grayish-brown, reaching 3 mm diam. after 4 weeks at 6 °C, 4 mm diam. after 4 weeks at 15 °C, and 7 mm diam. after 4 weeks at 25 °C, with a velvety surface caused by abundant aerial mycelium, margin finely undulate. Reverse black. Section title: Notes. Educational score: 2.8978521823883057 Domain: biomedical Document type: Study Language: en Colonies photographed after 15 weeks of growth, depicted in Fig. 2g, h , are radially folded on MEA or folded on PDA, possess abundant aerial mycelium, grayish with white patches on MEA and brown with grayish patches on PDA, and are distinctly undulate at the margin. Section title: Notes. Educational score: 4.5594329833984375 Domain: biomedical Document type: Study Language: en Based on a megablast search of NCBI’s GenBank nucleotide database, the closest hits of the named species using the ITS sequence are Pseudoarthrographis phlogis (strain CPC 32759, GenBank MH327796 ; identities = 370/414 (89%), 11 gaps (2%)), Oncopodiella trigonella (strain FMR 10788, GenBank KY853455 ; identities = 356/408 (87%), 11 gaps (2%)), and Xylographa parallela (voucher Z. Palice 22099 (PRM), GenBank MK778618 ; identities = 344/396 (87%), seven gaps (1%)). The closest hits of the named species using the LSU sequence are Beaucarneamyces stellenboschensis (strain CPC 45687, GenBank PP791445 ; identities = 826/873 (95%), four gaps (0%)), Pseudoarthrographis phlogis (strain CPC 32759, GenBank NG_064540 ; identities = 835/883 (95%), no gaps), and Umbilicaria hypococcinea (strain A12, GenBank JQ739991 ; identities = 876/927 (94%), eight gaps (1%)). The closest hits using the SSU sequence are Cophinforma atrovirens (strain CSM_72, GenBank MF436134 ; identities = 857/870 (99%), no gaps), Gloeopycnis protuberans (specimen DAOM 745762, GenBank NG_067652 ; identities = 857/870 (99%), no gaps), and Botryosphaeria mamane (strain CBS 117444, GenBank KF531821 ; identities = 857/870 (99%), no gaps). The closest hits using the rpb2 sequence are Shiraia bambusicola (voucher SICAUCC 23-0005, GenBank OR424351 ; identities = 237/283 (84%), six gaps (2%)), Lindra obtusa (strain AFTOL-ID 5012, GenBank FJ238382 ; identities = 207/252 (82%), three gaps (1%)), and Natonodosa speciosa (strain CLM-RV86, GenBank MH745150 ; identities = 217/266 (82%), six gaps (2%)). In the case of SSU and rpb2 , the sequences of these two regions are not available for representatives of the most closely related genera, namely Beaucarneamyces and Pseudoarthrographis . Section title: Discussion Educational score: 4.102034568786621 Domain: biomedical Document type: Study Language: en In this study, morphological and phylogenetic analyses were conducted to identify the enigmatic fungal strain isolated from sooty mould biofilm on the surface of Symphoricarpos albus leaves. In consequence, the isolated fungus is described as a new genus and species, Szaferohypha enigmatica , and assigned to the order Neodactylariales . Three genera were previously included in this order, namely Beaucarneamyces , Neodactylaria , and Pseudoarthrographis , and Szaferohypha is morphologically different from all of them. Section title: Discussion Educational score: 4.579002857208252 Domain: biomedical Document type: Study Language: en Beaucarneamyces , with type species B. stellenboschensis , was described from dead leaves of Beaucarnea stricta in South Africa and is characterized by hyaline conidiophores reduced to conidiogenous cells that are polyblastic with several apical denticles. Conidia in this genus are solitary, fusoid-ellipsoid, hooked, 3-septate, hyaline but with pale brown central cells . Neodactylaria is typified with N. obpyriformis described from human bronchoalveolar lavage in the USA . The second species in this genus is N. simaoensis , described from submerged unidentified dicotyledonous leaves in China . Neodactylaria is characterized by having straight or flexuous, brown conidiophores with polyblastic, sympodial, and denticle-like conidiogenous loci, which form solitary, obpyriform or rostrate, 0-1-septate, pale brown conidia . Pseudoarthrographis , with a type species Pseudoarthrographis phlogis described from Phlox subulata in New Zealand, has smooth hyphae and smooth, 0–1-septate cylindrical arthroconidia with truncate ends produced in branched or unbranched chains. In culture it also forms smooth, globose chlamydospores occurring in chains . Section title: Discussion Educational score: 1.917967438697815 Domain: biomedical Document type: Study Language: en Despite numerous strains obtained during our studies of sooty mould communities, only a single strain belonging to Szaferohypha enigmatica was isolated. It is therefore unclear whether this species represents a very rare or accidental inhabitant of these communities and its main habitat is elsewhere. Section title: Discussion Educational score: 4.271823883056641 Domain: biomedical Document type: Study Language: en The order Neodactylariales , with one family Neodactylariaceae , was originally described to accommodate only one genus, Neodactylaria , containing two species in China and the USA . Recently, two monotypic genera, Beaucarneamyces and Pseudoarthrographis , containing species known in South Africa and New Zealand, respectively, were assigned to this order by Crous et al. . Here, yet another monotypic genus, Szaferohypha , known from Poland, is added to Neodactylariales . Thus, currently this order includes fungi living in opposite corners of the world. The order Neodactylariales and family Neodactylariaceae were originally circumscribed based on characters known in the genus Neodactylaria . They therefore need emendation by the features of the genera Beaucarneamyces , Pseudoarthrographis , and Szaferohypha. In our phylogenetic analysis, Neodactylariales is related to the order Oncopodiellales that is going to be described . Two genera, Diplocladiella and Oncopodiella , assigned to two families, Diplocladiellaceae and Oncopodiellaceae , are included in this order . The relationships of Neodactylariales and Oncopodiellales are revealed here for the first time. Section title: Discussion Educational score: 3.9352591037750244 Domain: biomedical Document type: Study Language: en Sooty mould communities are an underexplored source of rare and undescribed species, of which many are probably extremophilic since they live in extreme environments . They were previously studied using classical morphological methods that are, however, inadequate to reveal their true diversity, and only recently started to study in modern ways using morphological and molecular methods, though mainly in tropics and much more rarely in temperate regions . In the course of our ongoing studies of sooty mould communities covering the surface of leaves/needles of ornamental plants in Poland, i.e., in the temperate climate, about 190 species were isolated from these communities (M. Piątek et al. unpublished data), being either as constant or accidental colonizers, of which many are undescribed. These include four new Rachicladosporium species , a new Lapidomyces species , a new Xenoramularia species , a new Pseudopezicula species , and many more that are waiting for description. | Other | biomedical | en | 0.999998 |
PMC11699517 | Section title: Introduction Educational score: 4.205183982849121 Domain: biomedical Document type: Study Language: en The urbanisation process represents one of the main threats to biodiversity , causing fragmentation, modification and loss of habitats and consequently resulting in richness and abundance loss . However, studies quantifying the effects of urbanisation on biodiversity seem to provide conflicting results. Recent works have shown that the urban environment, particularly urban green areas, can even have positive effects on the conservation of biodiversity compared to other ecosystems strongly impacted by anthropogenic activities, such as areas subject to intensive agriculture . Not all species are adversely affected by urbanisation and urban areas can provide unique opportunities for conservation . For example, the positive effects on pollinator biodiversity may be the result of biotic factors, such as a greater diversity of plant species in urbanised areas than in rural areas due to the presence of exotic and ornamental plants that make trophic resources available for a longer period during the year and increase the diversity of habitats and microclimates . Another possible factor can be the degradation in the quality of habitats in rural areas . Abiotic factors can also have important effects. The 'heat island effect' produces higher air temperatures and more stable climatic conditions in cities than in surrounding areas , affecting insect phenology and survival . For this reason, urban green spaces can represent important locations for the conservation of several animal groups of vertebrates and invertebrates , particularly for insect pollinators such as bees. Despite the considerable efforts made in recent years through studies and investments, there is still a lack of knowledge regarding insect urban diversity and ecology. In particular, few studies have focused on pollinators in cities. Italy is a biodiversity hotspot for wild bees . Focusing on the most important and complete papers published on Apoidea biodiversity in Italy in recent decades, 1173 species were reported in Comba . Lhomme et al. reduced the number to 1028 species using data provided by Ascher and Pickering and showing that Italy has the eleventh bee richness in the Mediterranean Basin. Wood et al. , while revising Italian Andrena , subsequently added nine more species to those previously listed, confirming that Italy hosts one of the largest Andrena faunas in the world. This addition resulted from the revision of eight species and the description of a new one. Cornalba et al. recently provided eight new faunistic records for the Italian fauna, taking the number of species to 1045. The diversity of Hymenoptera Apoidea inhabiting Rome was previously discussed in Comba and Comba , in which 450 species were reported for the Latium Region and, in Comba and Comba , 378 wild bee species were recorded within the Grande Raccordo Anulare (GRA; see Materials and Methods). Despite the large number of species listed in these works, unpublished data and specimens housed in the Maurizio Mei collection (Museum of the Institute of Zoology, Sapienza University, Rome, Italy) suggest that the number of species present in the urban area of Rome might exceed 400. One of the most recent papers on this topic is that of Geppert et al. , who studied the functional traits and ecology of wild bees in Rome and provided a list of bee species. The objective of this study is to provide precise and original georeferenced faunistic data on the Apoidea Anthophila inhabiting the urban natural reserves of Rome, making them immediately available in the standard Darwin-core format to facilitate their use in future scientific work focused on the biodiversity of pollinating insects in urban areas. In addition, these data constitute an integration and update of the previously available information, thus setting the new faunistic reference to be used as a starting base. Producing this type of data for a city such as Rome, which is amongst the largest and most urbanised in Europe, is an important starting point for understanding how urbanisation defines and might affect animal biodiversity. Providing precise information on Apoidea distributions and insects in general in large urban green areas is essential for implementing plans to protect and enhance the positive effects on the diversity and abundance of pollinator communities. This type of data and information can also be used to provide guidelines for urban green management actions aimed at encouraging the presence of pollinating insects in the urban environment. Section title: Study area and sites Educational score: 1.620362401008606 Domain: other Document type: Study Language: en Rome is one of the largest cities in Europe and the third most populated city in the European Union, with a population of 2,873,494 . The urban area is conventionally identified with the region delimited by the motorway ring known as the Grande Raccordo Anulare (GRA), with a length of approximately 360 km 2 . All nature reserves sampled in this study are located within the boundaries delimited by the freeway that surrounds the more densely urbanised area of the city (ca. 360 km 2 ). Sampling took place from April to September 2022 in eight large green areas of the city of Rome, Italy . Eight nature reserves were selected for sampling, with an extension ranging from 152 hectares (Riserva Naturale Laurentino-Acqua Acetosa) to 4580 hectares (Parco Regionale dell'Appia Antica). Seven nature reserves are part of the RomaNatura network. In addition to these seven areas, we included the Parco Regionale dell'Appia Antica, which is the largest urban green area in Europe and is located southeast of the city. Section title: Study area and sites Educational score: 1.243906855583191 Domain: other Document type: Other Language: en In the following is reported the list of nature reserves considered in this study, the relative abbreviations, the coordinate of the centroids reported in decimal degrees, the extent and the number of fixed transects performed in each reserve for session: Section title: Study area and sites Educational score: 1.0007219314575195 Domain: other Document type: Other Language: ca Parco Regionale dell'Appia Antica (APP) ; Riserva Naturale dell'Insugherata (INS) (697 hectares/3 transects); Riserva Naturale Valle dei Casali (VDC) (466 hectares/2 transects); Riserva Naturale Valle dell’Aniene (VAN) (650 hectares/2 transects); Riserva Naturale Tenuta dei Massimi (TDM) (774 hectares/2 transects); Riserva Naturale di Monte Mario (MMA) (204 hectares/1 transect); Riserva Naturale Tenuta dell'Acquafredda (ACQ) (249 hectares/1 transect); Riserva Naturale Laurentino-Acqua Acetosa (LAU) (152 hectares/1 transect). Section title: Sampling method Educational score: 3.503392219543457 Domain: biomedical Document type: Study Language: en Sampling was carried out along 250-metre-long fixed transects (the transects remained the same in length and position throughout the study) and was repeated once a month, from April to September, in all eight nature reserves selected for the study. The number of transects performed in each site depended on the size of each nature reserve. In total, we performed 15 fixed transects, one each 250 hectares of extension, for a maximum of three transects per nature reserve. The transect locations were chosen where the vegetation was representative of the area and at least 100 metres apart. In three out of the eight sites, this ratio was not mathematically respected due to some specific characteristics of the area and limits to accessibility. The total extension of TDM is 774 hectares, but we performed two linear transects, due to the ban on access to a large archaeological private area. In VAN, we performed two transects because the park has elongated and narrow shapes that extend to the sides of a part of the Aniene River. It increased greatly the ratio between the perimeter and the surface of the area and meant that a large part of the park is occupied by the watercourse. In addition, a relevant percentage of the remaining walkable surface is made up of roads to move inside the park reducing the areas where a transect could be performed. APP is a huge urban green area. It extends from the centre of the city to the outskirts and presents, within it, a mosaic of inhabited, grazing, recreational and archaeological areas, but also integral reserves, accessible to citizens. Here, we carried out the transects in three areas, homogeneously distributed along the park. The investigations were conducted under sunny, low-wind weather conditions between 10:00 am and 3:00 pm. Wild bees were collected via an entomological net within 2 m on both sides of each sample line . Each transect was carried out by walking at a constant speed for 45 minutes. All the observations were conducted by the same person (LF) walking along the sample lines. All the samples were collected while foraging on flowers along the transects. Section title: Preparation and identification Educational score: 3.8813459873199463 Domain: biomedical Document type: Study Language: en All collected samples were stored in Falcon tubes, exposed to ethyl acetate and dry-pinned. Morphological identification was performed using reference literature on the group and taxonomic keys such as those provided by Michez et al. for genera identification, Amiet et al. for Apidae , Smith for Nomada , Amiet et al. and Pauly for Halictidae , Amiet et al. for Megachilidae , Dathe for Hylaeus , Amiet et al. for Colletes , Wood for Andrena and taking as reference the collection from the Museum of Zoology of La Sapienza University. The identifications have been validated by an expert taxonomist author (MM). The final list of species collected during the study is reported below. The checklist respects the alphabetical order and the latitude/longitude given for each specimen are those of the mid-point of the transect where it was collected. The taxonomic arrangement followed that of Ghisbain et al. , Wood and Orr et al. . Section title: Andrena aeneiventris Educational score: 1.0745211839675903 Domain: other Document type: Other Language: pl Morawitz, 1872 Section title: Andrena aeneiventris Educational score: 0.9890064001083374 Domain: biomedical Document type: Other Language: en 399B9BAB-682B-5C67-8A9F-83AC41D67830 Section title: Andrena agilissima Educational score: 0.9792194366455078 Domain: biomedical Document type: Other Language: en D6D793C9-3127-5FAB-A737-1CBBD3CD4516 Section title: Andrena albopunctata Educational score: 1.0129636526107788 Domain: biomedical Document type: Other Language: de 6810E1D9-8DEF-584C-BC92-ECB81B6DBC66 Section title: Andrena bisulcata Educational score: 1.0783580541610718 Domain: other Document type: Other Language: pl Morawitz, 1877 Section title: Andrena bisulcata Educational score: 1.0419576168060303 Domain: other Document type: Other Language: en 1219BF55-FD87-5C81-BBF4-F836A6DF5839 Section title: Andrena braunsiana Educational score: 1.1772490739822388 Domain: other Document type: Other Language: af Friese, 1887 Section title: Andrena braunsiana Educational score: 0.9892297387123108 Domain: other Document type: Other Language: en 6C3CEEE2-DE9E-5600-8159-6F2D9A24B1E2 Section title: Andrena cinerea Educational score: 1.2358158826828003 Domain: other Document type: Other Language: fr Brullé, 1832 Section title: Andrena cinerea Educational score: 0.8966039419174194 Domain: biomedical Document type: Other Language: de F21B401B-BF5D-52D7-9235-DA986AC6BF88 Section title: Andrena clypella Educational score: 1.0098292827606201 Domain: other Document type: Other Language: de Strand, 1921 Section title: Andrena clypella Educational score: 0.8796014189720154 Domain: biomedical Document type: Other Language: en 2B4A52F7-4CE6-578F-B8EC-2C0D9D92644C Section title: Andrena colletiformis Educational score: 1.090821385383606 Domain: other Document type: Other Language: pl Morawitz, 1874 Section title: Andrena colletiformis Educational score: 1.009856939315796 Domain: biomedical Document type: Other Language: hu A4A0AAE1-1213-50DB-A548-8A69631059D7 Section title: Andrena combaella Educational score: 0.9967527985572815 Domain: other Document type: Other Language: en Warncke, 1966 Section title: Andrena combaella Educational score: 0.9526943564414978 Domain: other Document type: Other Language: de 66BBBF1C-D9FE-5FF9-8092-3D0EA25F8EE7 Section title: Andrena congruens Educational score: 1.0729576349258423 Domain: other Document type: Other Language: de Schmiedeknecht, 1884 Section title: Andrena congruens Educational score: 1.0956767797470093 Domain: biomedical Document type: Other Language: hu 59A6A81E-C7AB-55B6-A70A-8F557679CABE Section title: Andrena decipiens Educational score: 1.1615341901779175 Domain: other Document type: Other Language: de Schenck, 1861 Section title: Andrena decipiens Educational score: 1.1643240451812744 Domain: biomedical Document type: Other Language: hu DB70A8C1-BA0D-5772-A671-2848C8F91AD0 Section title: Andrena distinguenda Educational score: 1.153288722038269 Domain: other Document type: Other Language: de Schenck, 1871 Section title: Andrena distinguenda Educational score: 1.0189144611358643 Domain: other Document type: Other Language: pt 173927D5-3F8A-5D5E-A870-33CF7E95201D Section title: Andrena flavipes Educational score: 1.2332649230957031 Domain: biomedical Document type: Other Language: de Panzer, 1799 Section title: Andrena flavipes Educational score: 1.0780798196792603 Domain: biomedical Document type: Other Language: de 162ABCC9-D4C3-5AC6-AB39-76B86D9EC6F5 Section title: Andrena foeniculae Educational score: 0.9541569948196411 Domain: other Document type: Other Language: so Wood, 2020 Section title: Andrena foeniculae Educational score: 0.9262265563011169 Domain: biomedical Document type: Other Language: de 6D7494E7-C20D-5687-8D18-0D89A4C2F289 Section title: Andrena fulvago Educational score: 0.9592017531394958 Domain: other Document type: Other Language: de Christ, 1791 Section title: Andrena fulvago Educational score: 1.0669792890548706 Domain: biomedical Document type: Other Language: en 4964CB8E-A2D5-57C8-B534-1288A5CE6DAE Section title: Andrena fulvitarsis Educational score: 1.2358158826828003 Domain: other Document type: Other Language: fr Brullé, 1832 Section title: Andrena fulvitarsis Educational score: 0.9512476921081543 Domain: biomedical Document type: Other Language: de 37D2B920-8D76-5490-80BB-A776E6BE1289 Section title: Andrena fuscosa Educational score: 1.1846513748168945 Domain: other Document type: Other Language: de Erichson, 1835 Section title: Andrena fuscosa Educational score: 0.9106069207191467 Domain: other Document type: Other Language: de 39E70B2B-FE11-54DC-8B22-7FF3649D7D23 Section title: Andrena hattorfiana Educational score: 1.37467360496521 Domain: biomedical Document type: Other Language: ca Fabricius, 1775 Section title: Andrena hattorfiana Educational score: 0.994229257106781 Domain: biomedical Document type: Other Language: en CD091253-7DA3-53F2-8C5E-58F8BD489922 Section title: Andrena hesperia Educational score: 1.062803864479065 Domain: other Document type: Other Language: en Smith, 1853 Section title: Andrena hesperia Educational score: 1.0297619104385376 Domain: biomedical Document type: Other Language: de 84C33E8F-1F96-582E-B41F-AEBB32EA2699 Section title: Andrena humilis Educational score: 1.1781362295150757 Domain: biomedical Document type: Other Language: cy Imhoff, 1832 Section title: Andrena humilis Educational score: 0.9217014908790588 Domain: other Document type: Other Language: de F1B83EE0-9851-5EFA-9D03-DCECC68A3540 Section title: Andrena impunctata Educational score: 1.0988930463790894 Domain: other Document type: Other Language: hu Pérez, 1895 Section title: Andrena impunctata Educational score: 0.9623447060585022 Domain: biomedical Document type: Other Language: en 01AA32C3-E3FA-58D6-8D57-FB170C9ACC1F Section title: Andrena labialis Educational score: 1.1152405738830566 Domain: biomedical Document type: Other Language: de 72602B1D-39CC-51FF-8EFF-EAE86BAA97DC Section title: Andrena labiata Educational score: 1.3279292583465576 Domain: other Document type: Other Language: ca Fabricius, 1781 Section title: Andrena labiata Educational score: 0.9429979920387268 Domain: biomedical Document type: Other Language: pt A0C1CF0A-7A58-5815-8029-C1C4CA5C925F Section title: Andrena lagopus Educational score: 1.0969806909561157 Domain: other Document type: Other Language: fr Latreille, 1809 Section title: Andrena lagopus Educational score: 1.1385823488235474 Domain: biomedical Document type: Other Language: de B6B22079-A01D-572F-BAE0-064839642ECB Section title: Andrena limata Educational score: 1.062803864479065 Domain: other Document type: Other Language: en Smith, 1853 Section title: Andrena limata Educational score: 0.9511411190032959 Domain: biomedical Document type: Other Language: de 30C850E6-D152-53CD-B4F3-6189544F4DF3 Section title: Andrena livens Educational score: 1.0988930463790894 Domain: other Document type: Other Language: hu Pérez, 1895 Section title: Andrena livens Educational score: 1.0267888307571411 Domain: biomedical Document type: Other Language: hu 2264FC77-92D9-5ECA-9A1A-141C2CAF54A3 Section title: Andrena minutula Educational score: 1.0464481115341187 Domain: biomedical Document type: Other Language: hu DD54E718-BEDA-57C0-A965-11BDEE244A2E Section title: Andrena morio Educational score: 1.2358158826828003 Domain: other Document type: Other Language: fr Brullé, 1832 Section title: Andrena morio Educational score: 0.9460045099258423 Domain: biomedical Document type: Other Language: pt A1D29955-663A-5BF6-9043-D222E3139C5D Section title: Andrena nana Educational score: 0.9887471199035645 Domain: biomedical Document type: Other Language: en 8B7B3B17-5950-5B28-890A-4F8C6ECE4AFD Section title: Andrena nigroaenea Educational score: 1.016526699066162 Domain: biomedical Document type: Other Language: pt ED9F7C12-73ED-590C-8AA4-9745D04F54A2 Section title: Andrena nigroolivacea Educational score: 1.116115927696228 Domain: other Document type: Other Language: fr Dours, 1873 Section title: Andrena nigroolivacea Educational score: 0.9643633961677551 Domain: other Document type: Other Language: en 54423317-6B5F-5639-B127-37697CE875E0 Section title: Andrena ovata Educational score: 1.1526365280151367 Domain: other Document type: Other Language: de Schenck, 1853 Section title: Andrena ovata Educational score: 1.0724173784255981 Domain: biomedical Document type: Other Language: de 8BE95420-ED50-583C-8B07-B44F8B1E3C71 Section title: Andrena pandellei Educational score: 1.0988930463790894 Domain: other Document type: Other Language: hu Pérez, 1895 Section title: Andrena pandellei Educational score: 1.0692440271377563 Domain: biomedical Document type: Other Language: hu F9ED2F41-74A4-531C-A7F4-921A75FE687A Section title: Andrena pellucens Educational score: 1.0988930463790894 Domain: other Document type: Other Language: hu Pérez, 1895 Section title: Andrena pellucens Educational score: 0.9466824531555176 Domain: other Document type: Other Language: en 51154857-9A8C-5FCD-846B-CC01D2BED5E5 Section title: Andrena pilipes Educational score: 1.3279292583465576 Domain: other Document type: Other Language: ca Fabricius, 1781 Section title: Andrena pilipes Educational score: 0.944516658782959 Domain: biomedical Document type: Other Language: cy 4BB89F81-8CB8-5C72-9384-6C2DFCCC4C82 Section title: Andrena pusilla Educational score: 1.102597713470459 Domain: other Document type: Other Language: hu Pérez, 1903 Section title: Andrena pusilla Educational score: 1.0524945259094238 Domain: biomedical Document type: Other Language: pt 6EFA2D8C-A082-5C82-9C31-F9D5E2EB173C Section title: Andrena ranunculi Educational score: 1.0711437463760376 Domain: other Document type: Other Language: de Schmiedeknecht, 1883 Section title: Andrena ranunculi Educational score: 1.0386677980422974 Domain: biomedical Document type: Other Language: hu 996BD50A-953C-595A-8B83-81429ED989AB Section title: Andrena waschulzi Educational score: 1.0098292827606201 Domain: other Document type: Other Language: de Strand, 1921 Section title: Andrena waschulzi Educational score: 0.9372416138648987 Domain: other Document type: Other Language: de D478449D-4E28-5504-80D2-38625DA76F57 Section title: Andrena senecionis Educational score: 1.0988930463790894 Domain: other Document type: Other Language: hu Pérez, 1895 Section title: Andrena senecionis Educational score: 0.9159714579582214 Domain: biomedical Document type: Other Language: hu 43A73D84-77A8-52A5-BB86-4A193D0A494F Section title: Andrena schencki Educational score: 1.1199051141738892 Domain: other Document type: Other Language: pl Morawitz, 1866 Section title: Andrena schencki Educational score: 0.8672226071357727 Domain: other Document type: Other Language: de D04AC559-85E4-5ED0-9B52-3643A41E2F06 Section title: Andrena schmiedeknechti Educational score: 1.1812858581542969 Domain: other Document type: Other Language: it Magretti, 1883 Section title: Andrena schmiedeknechti Educational score: 1.1829050779342651 Domain: biomedical Document type: Other Language: hu 7C6EAE05-3236-5735-A6D9-088A3C924ABD Section title: Andrena simontornyella Educational score: 1.0845800638198853 Domain: other Document type: Other Language: pl Noskiewicz, 1939 Section title: Andrena simontornyella Educational score: 1.0169538259506226 Domain: biomedical Document type: Other Language: de E8F9FA3D-E621-50B5-A7D1-9D316C045AF0 Section title: Andrena stabiana Educational score: 1.2189416885375977 Domain: biomedical Document type: Other Language: ro Morice, 1899 Section title: Andrena stabiana Educational score: 0.9252398610115051 Domain: other Document type: Other Language: de A8354DAB-FDCA-5C0D-92D8-6F6350E857E4 Section title: Andrena thoracica Educational score: 1.37467360496521 Domain: biomedical Document type: Other Language: ca Fabricius, 1775 Section title: Andrena thoracica Educational score: 1.1330350637435913 Domain: biomedical Document type: Other Language: de 760D156E-4512-5B48-AD68-516577A6D464 Section title: Andrena truncatilabris Educational score: 1.0783580541610718 Domain: other Document type: Other Language: pl Morawitz, 1877 Section title: Andrena truncatilabris Educational score: 1.2776457071304321 Domain: biomedical Document type: Other Language: pt E145D8AD-576F-5A33-A47E-DD48CCA657FB Section title: Andrena ventricosa Educational score: 1.116115927696228 Domain: other Document type: Other Language: fr Dours, 1873 Section title: Andrena ventricosa Educational score: 0.9970521330833435 Domain: biomedical Document type: Other Language: de 493235DC-621E-581D-BD5E-7E6A16BDEDDD Section title: Andrena vetula Educational score: 1.2070327997207642 Domain: biomedical Document type: Other Language: af Lepeletier, 1841 Section title: Andrena vetula Educational score: 1.0380865335464478 Domain: biomedical Document type: Other Language: de D775C0FA-438A-5FD6-B3F3-D6E3C01317D1 Section title: Panurgus canescens Educational score: 1.1463830471038818 Domain: other Document type: Other Language: fr Latreille, 1811 Section title: Panurgus canescens Educational score: 0.980603039264679 Domain: biomedical Document type: Other Language: hu 2B4F389A-AB93-51D2-A1F5-DFF22F6305B5 Section title: Amegilla albigena Educational score: 1.0126922130584717 Domain: biomedical Document type: Other Language: pt DAF543C5-F0FC-5E6E-982B-8C8A9B9A6EAE Section title: Amegilla garrula Educational score: 1.1304494142532349 Domain: biomedical Document type: Other Language: pt E7CAF1DA-94C6-59A1-99E1-82D69AD38985 Section title: Amegilla quadrifasciata Educational score: 1.0311057567596436 Domain: biomedical Document type: Other Language: de B204920E-8966-5C86-8BD4-77D8B89840D1 Section title: Anthophora atroalba Educational score: 1.2070327997207642 Domain: biomedical Document type: Other Language: af Lepeletier, 1841 Section title: Anthophora atroalba Educational score: 1.0478863716125488 Domain: biomedical Document type: Other Language: hu 9AEC3CCF-0B77-5049-BCBC-CCD7A53EBA23 Section title: Anthophora crinipes Educational score: 1.0311297178268433 Domain: other Document type: Other Language: en Smith, 1854 Section title: Anthophora crinipes Educational score: 0.9243758320808411 Domain: other Document type: Other Language: en 7B28C5A1-C994-5D2E-9AFE-11F78EFB340B Section title: Anthophora dispar Educational score: 1.2070327997207642 Domain: biomedical Document type: Other Language: af Lepeletier, 1841 Section title: Anthophora dispar Educational score: 0.997428297996521 Domain: biomedical Document type: Other Language: pt E03A9421-587F-51BE-BFD8-5CC04269EDD2 Section title: Anthophora plumipes Educational score: 0.924491286277771 Domain: biomedical Document type: Other Language: hu 28C062BA-1A52-5A3F-9B0D-E2FAFEA7AD19 Section title: Bombus pascuorum Educational score: 0.9732434153556824 Domain: other Document type: Other Language: en 2E16B104-35CB-527C-ABE0-F1E66DBCD290 Section title: Bombus ruderatus Educational score: 0.969174325466156 Domain: biomedical Document type: Other Language: de 20AFD618-8269-5D34-B69D-2C70E0467BC5 Section title: Bombus terrestris Educational score: 1.1724119186401367 Domain: biomedical Document type: Other Language: hu 05399715-E621-53A6-A71A-D6D42E61EBFE Section title: Ceratina chalcites Educational score: 1.388388991355896 Domain: biomedical Document type: Other Language: de Germar, 1839 Section title: Ceratina chalcites Educational score: 1.1649447679519653 Domain: biomedical Document type: Other Language: hu 567FFA2C-B291-53EC-A777-B282577DBC87 Section title: Ceratina cucurbitina Educational score: 0.963020384311676 Domain: biomedical Document type: Other Language: de F8328E1E-0D24-50DD-8388-B61BD6B38113 Section title: Ceratina cyanea Educational score: 0.9509404301643372 Domain: biomedical Document type: Other Language: de B2533808-2E8D-505A-9221-6BD35E342DAA Section title: Ceratina dallatorreana Educational score: 1.2146435976028442 Domain: other Document type: Other Language: af Friese, 1896 Section title: Ceratina dallatorreana Educational score: 0.9728197455406189 Domain: biomedical Document type: Other Language: en 2CC84ECE-3880-53A0-B739-FC6D91061653 Section title: Ceratina dentiventris Educational score: 1.0834736824035645 Domain: other Document type: Other Language: de Gerstaecker, 1869 Section title: Ceratina dentiventris Educational score: 1.0698292255401611 Domain: biomedical Document type: Other Language: de 7415D5D1-AF77-5EAC-8AC0-988AD4C64AB7 Section title: Ceratina gravidula Educational score: 1.0834736824035645 Domain: other Document type: Other Language: de Gerstaecker, 1869 Section title: Ceratina gravidula Educational score: 1.2471028566360474 Domain: biomedical Document type: Other Language: de 648FF869-14EC-5939-955C-BF779DC5F0E4 Section title: Ceratina nigrolabiata Educational score: 1.2146435976028442 Domain: other Document type: Other Language: af Friese, 1896 Section title: Ceratina nigrolabiata Educational score: 1.0098868608474731 Domain: biomedical Document type: Other Language: en A46BE5F3-F116-522B-86BE-1C4AC5B0FAF2 Section title: Epeolus cruciger Educational score: 0.9318236708641052 Domain: other Document type: Other Language: cy 79D89057-86C4-5425-97CD-C0570C2C1D21 Section title: Eucera clypeata Educational score: 1.1846513748168945 Domain: other Document type: Other Language: de Erichson, 1835 Section title: Eucera clypeata Educational score: 0.9502044320106506 Domain: other Document type: Other Language: pt 884F0703-BDE0-5272-9A7C-F700878DA163 Section title: Eucera grisea Educational score: 1.3647501468658447 Domain: biomedical Document type: Other Language: ca Fabricius, 1793 Section title: Eucera grisea Educational score: 0.9218280911445618 Domain: biomedical Document type: Other Language: de E824886F-0F44-5E56-9201-6F37D63EA317 Section title: Eucera interrupta Educational score: 1.6308884620666504 Domain: biomedical Document type: Other Language: sv Bär, 1850 Section title: Eucera interrupta Educational score: 1.0023231506347656 Domain: biomedical Document type: Other Language: pt 422C0E2A-5588-5757-899C-D2E7ACA110E4 Section title: Eucera nigrescens Educational score: 1.1248141527175903 Domain: other Document type: Other Language: hu Pérez, 1879 Section title: Eucera nigrescens Educational score: 0.9794536232948303 Domain: biomedical Document type: Other Language: cy 615CBC40-92C6-5548-8CC0-F3CC87B85C42 Section title: Eucera nigrifacies Educational score: 1.2070327997207642 Domain: biomedical Document type: Other Language: af Lepeletier, 1841 Section title: Eucera nigrifacies Educational score: 1.0956194400787354 Domain: biomedical Document type: Other Language: pt B1A3613E-CF84-5A62-AA72-BCF6F9E56C6E Section title: Eucera numida Educational score: 1.2070327997207642 Domain: biomedical Document type: Other Language: af Lepeletier, 1841 Section title: Eucera numida Educational score: 1.0793671607971191 Domain: other Document type: Other Language: de FAED67EF-30D5-5C07-871F-F724C5999F5C Section title: Eucera oraniensis Educational score: 1.2070327997207642 Domain: biomedical Document type: Other Language: af Lepeletier, 1841 Section title: Eucera oraniensis Educational score: 1.2338894605636597 Domain: biomedical Document type: Other Language: hu DCBAB724-3582-5BF2-BBDA-89A87965C414 Section title: Eucera pannonica Educational score: 0.8825697302818298 Domain: other Document type: Other Language: hu Mocsáry, 1878 Section title: Eucera pannonica Educational score: 1.0119291543960571 Domain: biomedical Document type: Other Language: hu 6EF3334C-E78D-50A8-9B3A-FDDB9318145B Section title: Eucera pollinosa Educational score: 1.0311297178268433 Domain: other Document type: Other Language: en Smith, 1854 Section title: Eucera pollinosa Educational score: 0.9972627758979797 Domain: biomedical Document type: Other Language: pt FFB307AA-24F8-53FD-A1E8-4E1AFA8C7C1B Section title: Eucera rufa Educational score: 0.9964845776557922 Domain: other Document type: Other Language: de 9F0BEE64-510F-538E-95DA-01F1E2743D45 Section title: Eucera vulpes Educational score: 1.2358158826828003 Domain: other Document type: Other Language: fr Brullé, 1832 Section title: Eucera vulpes Educational score: 1.0750716924667358 Domain: other Document type: Other Language: hu 5686F196-6685-5A07-98AF-9ECE7236FE52 Section title: Eupavlovskia funeraria Educational score: 0.93260258436203 Domain: other Document type: Other Language: pt A17F03F5-576F-5C74-AC04-16E255FE5938 Section title: Eupavlovskia obscura Educational score: 0.9772773385047913 Domain: other Document type: Other Language: de 56093BB5-F954-5B2F-8C95-8145A1D33572 Section title: Habropoda tarsata Educational score: 1.0311297178268433 Domain: other Document type: Other Language: en Smith, 1854 Section title: Habropoda tarsata Educational score: 0.9278436303138733 Domain: other Document type: Other Language: en B084C7E7-F922-5957-9AE6-C4369A0DF513 Section title: Habropoda zonatula Educational score: 1.0311297178268433 Domain: other Document type: Other Language: en Smith, 1854 Section title: Habropoda zonatula Educational score: 1.092401146888733 Domain: biomedical Document type: Other Language: de 1700662D-EF1D-591E-95BB-7E4501DBCA85 Section title: Melecta aegyptiaca Educational score: 1.0048927068710327 Domain: biomedical Document type: Other Language: pt 3989BEE2-C962-56AC-939C-52FCA8EA3114 Section title: Melecta albifrons Educational score: 1.1385512351989746 Domain: other Document type: Other Language: de Herrich-Schäffer, 1839 Section title: Melecta albifrons Educational score: 0.9419370889663696 Domain: biomedical Document type: Other Language: de F31A8554-B599-5CFB-8B89-73B8999B8254 Section title: Nomada argentata Educational score: 1.1385512351989746 Domain: other Document type: Other Language: de Herrich-Schäffer, 1839 Section title: Nomada argentata Educational score: 1.0102503299713135 Domain: biomedical Document type: Other Language: hu 20B2A505-90A2-52C1-951E-C716160DEEC5 Section title: Nomada basalis Educational score: 1.3270329236984253 Domain: biomedical Document type: Other Language: pt 97DE5E9B-E024-5755-A318-1970FF151F86 Section title: Nomada distinguenda Educational score: 1.0976678133010864 Domain: other Document type: Other Language: hu Pérez, 1913 Section title: Nomada distinguenda Educational score: 1.0876904726028442 Domain: biomedical Document type: Other Language: de 03621743-FB53-5158-B420-51FB48D98FA1 Section title: Nomada fallax Educational score: 1.0976678133010864 Domain: other Document type: Other Language: hu Pérez, 1913 Section title: Nomada fallax Educational score: 1.002885103225708 Domain: other Document type: Other Language: hu BA979498-F25A-546F-9967-291DACF328C3 Section title: Nomada femoralis Educational score: 1.066352128982544 Domain: other Document type: Other Language: de Schmiedeknecht, 1882 Section title: Nomada femoralis Educational score: 1.1151326894760132 Domain: biomedical Document type: Other Language: hu 328C02DB-7144-5A42-BAFC-A61B36EA877E Section title: Nomada insignipes Educational score: 0.9945542216300964 Domain: biomedical Document type: Other Language: de 03AB05E1-2CB6-53AA-B9DA-8066C49F76DD Section title: Nomada sexfasciata Educational score: 1.0341018438339233 Domain: biomedical Document type: Other Language: de FBEAAEC3-3D7E-52FA-9C91-7ADCB19B98D4 Section title: Nomada sheppardana Educational score: 1.116115927696228 Domain: other Document type: Other Language: fr Dours, 1873 Section title: Nomada sheppardana Educational score: 1.037634015083313 Domain: biomedical Document type: Other Language: en 3B39654C-CB43-521E-BAFF-8BF56AFDBCE1 Section title: Nomada tridentirostris Educational score: 1.066352128982544 Domain: other Document type: Other Language: de Schmiedeknecht, 1882 Section title: Nomada tridentirostris Educational score: 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title: Hylaeus communis Educational score: 0.9338915944099426 Domain: other Document type: Other Language: de 60BB1E1C-B032-5EF9-B6B9-B954911980E4 Section title: Hylaeus cornutus Educational score: 1.3157912492752075 Domain: biomedical Document type: Other Language: en Curtis, 1831 Section title: Hylaeus cornutus Educational score: 0.9540353417396545 Domain: other Document type: Other Language: en 73739934-1ED7-556A-BD3F-D16C52301853 Section title: Hylaeus difformis Educational score: 0.9956315755844116 Domain: biomedical Document type: Other Language: de 3B1B6B64-6D39-5DE1-88CB-65576E365AB2 Section title: Hylaeus hyalinatus Educational score: 1.0779597759246826 Domain: other Document type: Other Language: en Smith, 1842 Section title: Hylaeus hyalinatus Educational score: 0.9874081015586853 Domain: biomedical Document type: Other Language: de F19DBA4E-20B6-5A6D-9FB5-747C3D75C97A Section title: Hylaeus intermedius Educational score: 1.3275326490402222 Domain: biomedical Document type: 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Educational score: 0.9231741428375244 Domain: biomedical Document type: Other Language: de B6F45F2B-8FBB-5235-A0E9-0118E0EB768B Section title: Osmia caerulescens Educational score: 1.1603392362594604 Domain: biomedical Document type: Other Language: de 6E0D7838-7B3E-5D11-AAB7-EC99525ACB80 Section title: Osmia cephalotes Educational score: 1.078894019126892 Domain: other Document type: Other Language: pl Morawitz, 1870 Section title: Osmia cephalotes Educational score: 0.9225545525550842 Domain: biomedical Document type: Other Language: en 87E51D4D-C6CB-520F-8CC7-A7EFF372663E Section title: Osmia latreillei Educational score: 1.1353379487991333 Domain: biomedical Document type: Other Language: en C5F0FFD2-FB4B-56CA-9626-79E4B485CB5A Section title: Osmia leaiana Educational score: 1.0674487352371216 Domain: biomedical Document type: Other Language: hu 469E6903-A9AA-5819-B254-705A155CFC3E Section title: Osmia ligurica Educational score: 1.0849908590316772 Domain: other Document type: Other Language: pl Morawitz, 1868 Section title: Osmia ligurica Educational score: 1.0323294401168823 Domain: biomedical Document type: Other Language: en 06CD8F17-C564-5853-8241-4AABDD036EB6 Section title: Osmia melanogaster Educational score: 1.3637175559997559 Domain: biomedical Document type: Other Language: it Spinola, 1808 Section title: Osmia melanogaster Educational score: 1.0367180109024048 Domain: biomedical Document type: Other Language: en 32C2FFEB-779F-5900-8F42-4CEFE2E4F199 Section title: Osmia niveata Educational score: 1.3862406015396118 Domain: biomedical Document type: Other Language: ca Fabricius, 1804 Section title: Osmia niveata Educational score: 0.8646796941757202 Domain: biomedical Document type: Other Language: hu 16A74B2B-A10C-58E3-A2F4-F45B33C79490 Section title: Osmia rufohirta Educational score: 1.1463830471038818 Domain: other Document type: Other Language: fr Latreille, 1811 Section title: Osmia rufohirta Educational score: 1.0617529153823853 Domain: other Document type: Other Language: de 838DA931-157A-51CE-B05B-D5420531ED2D Section title: Osmia scutellaris Educational score: 1.0849908590316772 Domain: other Document type: Other Language: pl Morawitz, 1868 Section title: Osmia scutellaris Educational score: 0.9267370700836182 Domain: other Document type: Other Language: hu A68435FB-78CA-5684-BE0B-1399AF225617 Section title: Osmia spinulosa Educational score: 1.0251624584197998 Domain: other Document type: Other Language: de 3647201E-83B2-5C24-AE5E-B917E2DD591E Section title: Pseudoanthidium melanurum Educational score: 1.043630838394165 Domain: biomedical Document type: Other Language: hu 3F2FA00E-0265-51AA-BD29-F26B5A11B13A Section title: Rhodanthidium septemdentatum Educational score: 0.9817398190498352 Domain: biomedical Document type: Other Language: en 841C539D-62BA-5467-BD38-7182242394CF Section title: Trachusa integra Educational score: 0.9260791540145874 Domain: biomedical Document type: Other Language: hu 3A09A79D-7AFA-599C-8117-7F1F912E2BE6 Section title: Trachusa interrupta Educational score: 1.1271964311599731 Domain: biomedical Document type: Other Language: de 4EB49B09-39F8-5165-8807-3794028CACD8 Section title: Dasypoda hirtipes Educational score: 1.1280220746994019 Domain: biomedical Document type: Other Language: en 176C8443-7866-5D55-B133-8C3C59A05BFA Section title: Analysis Educational score: 4.07944917678833 Domain: biomedical Document type: Study Language: en The survey allowed the collection of 2139 specimens (spc.) of Apoidea belonging to 208 species (spp.) in 36 genera and six different families. Amongst the areas we investigated, APP presented both the greatest number of specimens and the greatest number of species collected (448 spc., 110 spp.), followed by INS (329 spc., 93 spp.), VDC (278 spc., 84 spp.), VAN (313 spc., 83 spp.), TDM (269 spc., 73 spp.), LAU (145 spc., 68 spp.), MMA (186 spc., 58 spp.) and ACQ (171 spc., 53 spp.). LAU presented the smallest number of specimens, whereas ACQ presented the lowest number of species (Table 1 ). The most abundant species were Halictus scabiosae (148 spc.), Lasioglossum malachurum (110 spc.) and Bombus pascuorum (63 spc.). The species we recorded in the nature reserves selected for the study represent approximately 74% of the known bee species for the area within the GRA of Rome since 1997, 46% of the Latium Region and approximately 20% of Italy. In April, the first month of sampling, we collected the greatest number of bees compared to the other months, while we observed a clear decrease in abundance in August and September compared with the previous months. We found the highest number of species in May (114 spp.), while we found the lowest number of species in September (22 spp.) . Section title: Analysis Educational score: 3.7932605743408203 Domain: biomedical Document type: Study Language: en Halictidae is the most common family, with 788 spc. representing 36% of the samples belonging to 40 species and three genera (19% of the total species) . This bee family is the most abundant in six out of the eight nature reserves, except for INS and LAU . Apidae counts 505 spc., representing 24% of the samples and belonging to 48 species and 11 genera (23% of the total species) . Apidae is the most abundant family in INS and LAU . Andrenidae represents 17% of the samples , belonging to 48 species and two genera (23% of the total species) . Megachilidae represents 15% of the samples , belonging to 52 species and 14 genera (25% of the total species) . Colletidae counts 148 spc., representing 7% of the samples and belonging to 19 species and two genera (10% of the total species) . Mellittidae is represented just by Dasypoda hirtipes . It represents 1% of the total samples . We did not collect Mellittidae from APP, MMA or VDC, whereas, in ACQ, it represented 11% of all the samples . We found eight species included in the European Wild Bees Red List. One species, Trachusa interrupta , is classified in the category Endangered (EN) and seven species are in the category Near Threatened (NT) (Table 2 ). Twenty-four species amongst those collected are new records for the urban area of Rome (Table 3 ). Section title: Discussion Educational score: 4.172015190124512 Domain: biomedical Document type: Study Language: en Although the entire experiment and sampling took place over a single year, carried out in eight green areas, using only one collection method (entomological net along fixed transects) and performed by only one operator (LF), we collected 208 species, representing 74% of the wild bee species reported within the urban area of Rome , 46% of the Latium Region and 20% of the species reported for Italy . The percentage of species detected in this experiment, relative to all those cumulative and historically reported for the study area and the addition of twenty-four new taxa represents, in our opinion, a good achievement, showing that an intensive sampling effort may yield new results regarding the distribution of wild bees in southern Europe. To highlight the effectiveness of the work and show with as little approximation as possible the state of the diversity of bees, it is possible to compare the methodological strategy and faunal results reported with the recent article by Geppert et al. , which represents a landmark study on the topic of wild bees in Rome from an ecological point of view. The two sampling strategies are not comparable in terms of collection effort, but they provide an idea of what may be the best approach to use if the goal is to study the alpha diversity of an area. The work of Geppert et al. resulted in the collection of 3280 wild bees belonging to 96 species, representing 34.5% of the species reported in the urban area of Rome. Geppert's data were collected during four months of sampling, which was carried out at thirty-six sites of different sizes within the GRA and using yellow pan traps. Compared with passive sampling methods such as yellow pan traps, active sampling using entomological netting more exhaustively captures alpha diversity, but results in a bias in the abundances of the groups collected because it is highly dependent on sampler capabilities . In this study, we collected data over two more months (April and May), but in only eight large green areas and we collected more than twice the number of species recorded by Geppert et al. . In this way, we provide a recent snapshot of the presence and distribution of wild bee species in the nature reserves of Rome, representing a starting point for monitoring them in the future. In addition, not all the species collected were reported for the area; in fact, twenty-four species (11% of the spp. collected) are new reports for the urban area of Rome. Section title: Discussion Educational score: 3.763556480407715 Domain: biomedical Document type: Study Language: en In terms of the abundance data, April was the month in which the greatest number of bees were collected overall . As we expected, from April to July, wild bees were more abundant than they were in August and September, the months in which a significant decrease in abundance was evident. The peak in the species diversity was detected during the May session (114 spp.) and declined steadily until September when only 22 species were detected. In April, the first month of sampling, the number of species detected already amounted to 82. Flower plants provide sustenance to bees and almost all herbaceous plants observed during sampling had completed their flowering period by August. The sampling period was selected according to the guidelines given in the literature for sampling pollinating insects in the Mediterranean environment , but in April, the peak of abundance for wild bees may have already been reached and a relatively good number of species is already active. The “heat island effect” produced by large urban centres and the average warmer and drier climate cause pollinators to be active early. For this reason, it would probably be correct to start sampling for pollinator insect studies in early March or even mid-February to obtain more complete information on the presence and abundance of species with early phenology. Section title: Discussion Educational score: 3.462822675704956 Domain: biomedical Document type: Study Language: en Amongst those species we collected, eight are included in the European Red List of bees . The presence of Red-listed species within the city shows that large urban green areas can be a refuge not only for wild bee species with a generalist ecology, but also for species with a more restricted ecology and that the nature reserves of Rome allow the survival of rarer species. The work of Fusco et al. , for example, studied ground spider diversity in APP itself and showed that it is true for other groups of arthropods. Focusing on APP, it is the largest urban nature reserve in Europe. It represents a unique case, being a single continuous green area that extends from the city centre to the area outside the city. In this way, APP offers ecological continuity and hosts an exceptionally high biodiversity of botanical species . The diversity of plants in the area is due to the presence of spontaneous, non-native and ornamental herbaceous and shrubby species in villas and home gardens distributed throughout the Reserve. Many plant species can have many different flowering periods that overlap. It can ensure continuity in the supply of resources for pollinators and spontaneous plants, promoting more functionally diverse bee communities . Section title: Discussion Educational score: 4.129014492034912 Domain: biomedical Document type: Study Language: en This study revealed that, given its unique combination of natural and semi-natural elements embedded in the urban matrix, Rome hosts and offers shelter to a remarkable variety of Apoidea Anthophila , including multiple threatened species. Notably, the bee community was probably not subject to any particular erosion in terms of richness between 1997 and 2022, indicating that the current complexity and repartition of green areas within or at the margin of Rome possibly buffered some of the drivers causing pollinators to decline. However, from a conservation perspective, the protection of urban fauna has to pass through the adoption of correct green management and planning strategies. Furthermore, faunistic surveys aimed at investigating bee diversity in urban ecosystems should consider the ‘heat island effect’ that occurs in large urban centres, a phenomenon that affects bee phenology and enhances flowering in plants in an urban context. In this regard, an earlier sampling period in February and March would be desirable to intercept the more precocious species, which we probably did not encounter in the present survey. | Study | biomedical | en | 0.999997 |
PMC11699519 | Section title: Introduction Educational score: 3.9078824520111084 Domain: biomedical Document type: Other Language: en Interventional radiology (IR) plays a pivotal role in the management of complex urological conditions, particularly in cases where traditional approaches prove challenging . This technical note introduces an IR technique that addresses recurrent urinary tract infections (UTIs) and discomfort experienced by patients with nephroureteral stents with an ileal conduit urostomy. This technique demonstrates a practical solution to internalizing nephroureteral stents to ureteral stents in a patient with an ileal conduit. Our patient also highlights the clinical utility of this technique to avoid complications with external nephroureteral stents. Section title: Case presentation Educational score: 3.7574164867401123 Domain: clinical Document type: Clinical case Language: en A 61-year-old female patient was presented with a challenging medical history of a hysterectomy, lymph node dissection, and adjuvant radiotherapy in 2009 for curative treatment of stage IB squamous cell carcinoma of the cervix. Unfortunately, this resulted in a right ureteral stricture. Complications further ensued with the need for nephrostomy placement, ureteral stent insertion, and an ileal conduit urinary diversion in August 2018. The patient had long-term bilateral nephroureteral stents, which over prolonged time, were associated with multiple urinary tract infections . The patient also developed skin irritation from urinary leaks at the skin entrance site and significant discomfort from the nephroureteral stents. Section title: Case presentation Educational score: 3.955028533935547 Domain: biomedical Document type: Study Language: en We hypothesized that if the nephroureteral stents were long enough, they could easily be changed retrogradely every three months, allowing the patient to live without the external nephrostomy component of the stents. An alternative approach used at our institution involves retrogradely cannulating the ureters from the ileal conduit and placing pigtail stents, similar to the study by Maher et al. . However, retrograde placement without endoscopic guidance is technically more challenging and time-consuming than antegrade stent placement, particularly in the setting of distal ureteric strictures. Section title: Case presentation Educational score: 1.765831470489502 Domain: clinical Document type: Clinical case Language: en The patient's current status included bilateral flank pain and skin excoriation, worse on the left side, necessitating bilateral nephroureteral stent exchange and internalization of the ureteral stents. Prior imaging was reviewed. Section title: Case presentation Educational score: 1.0910969972610474 Domain: biomedical Document type: Other Language: fr Patient consent Section title: Case presentation Educational score: 1.3834549188613892 Domain: clinical Document type: Clinical case Language: en Comprehensive discussions and informed consent were obtained from the patient. Risks included losing access requiring nephrostomy reinsertion, displacement of the ureteral stents, and hematuria . Written informed consent was also obtained from the patient for publication of this technique. Section title: Case presentation Educational score: 1.7357739210128784 Domain: biomedical Document type: Other Language: en Patient preparation and procedure Section title: Case presentation Educational score: 3.7144932746887207 Domain: clinical Document type: Clinical case Language: en Antibiotic prophylaxis was administered and pre-procedure imaging, blood tests, and medications were reviewed. The patient was positioned prone on the fluoroscopic imaging table in the interventional radiology suite. The bilateral nephroureterostomies were prepped and draped with Betadine. Periprocedural sedation was given by the nursing staff using fentanyl and midazolam. The right nephroureterostomy was approached first, followed by the left. Injection of contrast into the stent under fluoroscopic guidance was performed. The position of the nephroureteral stent was confirmed. A 0.35", 180 cm stiff Terumo wire (Somerset, NJ: Terumo Medical) was inserted into the nephroureteral stent and into the ileal conduit . Section title: Case presentation Educational score: 3.4684829711914062 Domain: clinical Document type: Other Language: en The nephroureteral stent was removed over the wire and exchanged for a 65 cm Berenstein catheter (Bloomington, IN: Cook Medical). The Terumo wire was removed and contrast injection via the Berenstein catheter outlined the ileal conduit . A 0.35" super stiff Amplatz wire (Marlborough, MA: Boston Scientific) was placed through the catheter and into the ileal conduit stoma bag . Section title: Case presentation Educational score: 3.8570749759674072 Domain: clinical Document type: Clinical case Language: en An 8 Fr angiographic percutaneous sheath was placed into the renal pelvis to secure the wire access to the kidney. Bilateral Amplatz wires were secured for change of position with tape. The patient turned into the supine position and the ileal conduit stoma bag was removed and prepped with povidone-iodine (Betadine). Lidocaine hydrochloride gel (Instillagel; Melsungen, Germany: CliniMed) was administered to the stoma. The super-stiff Amplatz wires were secured at the ileal conduit site with a 10 Fr angiographic sheath. New 8 Fr × 26 cm regular double-J ureteral stents (Boston Scientific) were sequentially placed over the Amplatz wires under fluoroscopic guidance and the pigtails formed in each renal pelvis . Section title: Case presentation Educational score: 3.272603988647461 Domain: clinical Document type: Other Language: en Contrast injection via the angiographic sheath within the renal pelvis was used to confirm the position of the proximal left ureteral stent . The wire was removed and ureteral stents were confirmed to be in a satisfactory position. The proximal and distal sheaths were then removed . The distal pigtails were formed external to the ileal conduit. Section title: Case presentation Educational score: 2.2057416439056396 Domain: clinical Document type: Clinical case Language: en The patient tolerated the procedure well. The procedure resulted in the successful internalization of nephroureteral stents to internalized ureteral stents with the distal portions of the stent within the ileal conduit. Section title: Discussion Educational score: 3.4900858402252197 Domain: biomedical Document type: Other Language: en This technique of internalizing nephroureteral stents to internalized ureteral stents within an ileal conduit represents a helpful advancement in the management of complex urological cases . The successful application and safety of this technique depend on the patient (e.g., type of ileal conduit, anatomy, and BMI) and technical, environmental, and device factors . Section title: Discussion Educational score: 1.2231782674789429 Domain: biomedical Document type: Other Language: fr Patient factors Section title: Discussion Educational score: 2.8945136070251465 Domain: biomedical Document type: Other Language: en Patient-related variables are instrumental in determining the appropriateness and success of the internalization technique. Patient selection remains critical, encompassing overall health, comorbidities, and urinary tract history. Patient willingness to actively participate in post-procedure care is essential for successful outcomes. Adequate patient counseling is vital to establish realistic expectations and ensure active patient engagement. Section title: Discussion Educational score: 1.4302679300308228 Domain: biomedical Document type: Other Language: en Technical factors Section title: Discussion Educational score: 3.661710262298584 Domain: biomedical Document type: Other Language: en Technical proficiency plays a paramount role in the execution of the internalization procedure. Interventional radiologists must possess the requisite skills and experience to navigate the intricate steps involved, which include wire insertion, stent removal, and precise stent placement. A commitment to maintaining meticulous sterile technique and fostering effective communication among the procedural team, particularly when switching from prone to supine, is equally indispensable. Section title: Discussion Educational score: 1.638961911201477 Domain: biomedical Document type: Other Language: en Device factors Section title: Discussion Educational score: 4.030235290527344 Domain: biomedical Document type: Review Language: en The choice of devices and equipment employed during the procedure significantly influences its outcome. Employing high-quality wires, catheters, and stents is crucial for facilitating smooth wire passage, stent exchange, and secure placement. Ongoing advancements in device technology, with a focus on tailoring equipment to the internalization technique, can enhance procedural efficiency and safety . Bander stents are an alternative specialized ureteral diversion stent used in ureteroileal conduit construction which can be 6 or 7 Fr and are polytetrafluoroethylene (PTFE)-covered stents . Similar to other devices, these facilitate drainage from the kidney to an external stoma, and they include color-coded radiopaque markers to assist in accurate positioning. The design ensures the stents are secure and can be easily replaced. Another option is to use biliary stents depicted in Figure 8 , which are held in place by a gastrostomy disc. Close collaboration between interventional radiologists and device manufacturers holds promise for driving innovations that can further enhance patient outcomes and reduce procedural complexity . Section title: Discussion Educational score: 2.024717092514038 Domain: biomedical Document type: Other Language: en Types of ileal conduit Section title: Discussion Educational score: 3.780144453048706 Domain: biomedical Document type: Other Language: en The type of ileal conduit employed also influences the procedure's application and success. The standard ileal conduit (Bricker procedure) is the most common, but variations include the Studer orthotopic neobladder and cutaneous ureteroileostomy . Patients with different conduit types may require tailored approaches for effective stent management. While the internalization technique may not be directly applicable in some cases, the consideration of how to manage stents within these various conduit types remains a crucial aspect of patient care. Section title: Discussion Educational score: 1.7486364841461182 Domain: biomedical Document type: Other Language: en Anatomical factors Section title: Discussion Educational score: 3.9005348682403564 Domain: biomedical Document type: Other Language: en Anatomical or physiological complexities pose challenges to the procedure's execution. Factors such as ureteral strictures, anatomical variations, peri-conduit adhesions, active infection, inflammation, and obesity can complicate wire passage, stent placement, and overall procedural success . A meticulous pre-operative assessment and procedural planning are essential for addressing challenging anatomical factors, minimizing risks, and ensuring patient safety. Section title: Discussion Educational score: 1.3194478750228882 Domain: biomedical Document type: Other Language: ca Environmental factors Section title: Discussion Educational score: 2.2546684741973877 Domain: biomedical Document type: Other Language: en Environmental factors, including the availability and expertise of support staff, sterility of the operating environment, and accessibility of necessary equipment, play a critical role in the procedure's success. Section title: Conclusions Educational score: 3.915208339691162 Domain: biomedical Document type: Other Language: en In conclusion, the technique of internalizing nephroureteral stents to ureteral stents within an ileal conduit is useful in the management of complex urological cases. Success depends on a nuanced understanding of patient, technical, environmental, and device factors. Additionally, an awareness of the types of ileal conduit and anticipation of challenging anatomical factors is crucial in tailoring the procedure to individual patient needs. As interventional radiology continues to evolve, ongoing collaboration between clinicians and device manufacturers holds promise for further enhancing the technique's safety and efficacy. This technique holds promise for similar cases and highlights the importance of patient-centered approaches in interventional radiology. | Other | biomedical | en | 0.999997 |
PMC11699541 | Section title: Introduction and background Educational score: 3.4020180702209473 Domain: biomedical Document type: Other Language: en Bone defects can be caused by numerous reasons; the most significant ones in dental medicine include periodontal disease, trauma, peri-implantitis, and atrophy. In these cases, bone loss not only affects the esthetic appearance but also diminishes the structural integrity and function of the dental and facial framework. In order to overcome these issues, the demand for advanced treatment is increasing, and the search for innovative materials and techniques to facilitate bone regeneration has become extremely important . Section title: Introduction and background Educational score: 4.159430503845215 Domain: biomedical Document type: Review Language: en Guided bone regeneration (GBR) and guided tissue regeneration (GTR) have been recognized as gold standards among the numerous techniques employed. These methods use barrier membranes in a strategic manner to create a protective environment that prevents the infiltration of unwanted cell populations such as fibroblasts and epithelial cells while also supporting osteogenesis . However, traditional membranes, including polytetrafluoroethylene (PTFE) and collagen-based alternatives, are often limited by insufficient mechanical strength, a high risk of soft tissue invasion, and in some cases, the need for surgical removal on non-resorbable variants . The introduction of the cortical laminar bone membrane (CLBM), derived from xenogeneic bone tissue has revolutionized regenerative dentistry. CLBM's unique properties, including high mechanical stability, controlled degradation, and osteoconductivity, enable it to overcome the limitations of traditional membranes. Particularly, in the context of large or complex defects, CLBM has demonstrated remarkable clinical outcomes. The present review probes into the structural characteristics, biological mechanism, clinical applications, and challenges associated with CLBM supported by evidence from recent studies. Section title: Review Educational score: 2.1629271507263184 Domain: biomedical Document type: Other Language: en Key properties of cortical laminar bone membrane Section title: Review Educational score: 4.620889663696289 Domain: biomedical Document type: Study Language: en CLBM is characterized by its rigidity, which significantly surpasses the mechanical strength of traditional collagen membranes. This rigidity ensures that the membrane effectively maintains space within bone defects, preventing collapse under soft tissue pressure. This property is critical when regenerating extensive or irregular defects, where maintaining structural integrity is essential for predictable outcomes . The integrity of the membrane is maintained by adapting it onto the site and fixing it in place using stainless steel mini screws . A study by Rossi et al. highlights the use of CLBM in cases of severe bone atrophy . The ability of the membrane to stabilize graft materials and maintain defect morphology was instrumental in achieving successful regeneration. Such mechanical stability is particularly important in demanding clinical scenarios, such as lateral ridge augmentation or large vertical defects, where traditional membranes often fail to provide adequate support . Since CLBM is derived from xenogeneic bone, it undergoes stringent processing to eliminate antigenic proteins while preserving its osteoconductive properties. This ensures compatibility with human tissues and creates an optimal surface for osteoblast attachment and mesenchymal stem cell differentiation . Tumedei et al. emphasized that CLBM not only supports the adhesion and proliferation of osteogenic cells but also facilitates their integration into the host bone tissue. This compatibility with surrounding tissues is a crucial factor in achieving long-term stability and functionality in regenerated bone . Unlike rapidly degrading collagen membranes, CLBM has a much slower and more controlled resorption profile. This property ensures that the membrane provides prolonged protection against soft tissue invasion while supporting the healing process over an extended period . Schuh et al. investigated the use of CLBM in multilayer regenerative techniques and found that its longevity significantly improved outcomes, particularly in esthetically demanding areas. The controlled degradation rate allows for sufficient time for bone remodeling, thus reducing the chances of complications such as soft tissue intrusion or incomplete regeneration . Section title: Review Educational score: 2.503251791000366 Domain: biomedical Document type: Other Language: en Biological mechanism of action Section title: Review Educational score: 2.7340123653411865 Domain: biomedical Document type: Other Language: en CLBM acts through various mechanisms to induce bone formation . The manner in which bone is laid down following the use of CLBM differs from other membranes, wherein unlike the latter, it is similar to natural bone remodeling. Section title: Review Educational score: 1.7094162702560425 Domain: biomedical Document type: Other Language: no Osteogenesis Support Section title: Review Educational score: 4.138413906097412 Domain: biomedical Document type: Study Language: en CLBM functions as a scaffold that mimics the natural extracellular environment of bone. Its surface promotes the adhesion of osteogenic cells, facilitating the migration and differentiation of mesenchymal stem cells into osteoblasts. This process is essential for the formation of new bone and the eventual remodeling of the defect site . Furthermore, the membrane supports osteoclastic activity, which is vital for remodeling and integration with the host bone. This dual support for osteogenesis and remodeling distinguishes CLBM from purely barrier-focused materials, such as PTFE membranes. Section title: Review Educational score: 1.7594212293624878 Domain: biomedical Document type: Other Language: en Growth Factor Modulation Section title: Review Educational score: 4.045153617858887 Domain: biomedical Document type: Study Language: en CLBM acts as a reservoir for bioactive molecules, including bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF). BMPs are critical for osteogenesis, while VEGF stimulates angiogenesis, ensuring the delivery of oxygen and nutrients to the regenerating site . Studies have shown that the synergistic effects of BMPs and VEGF enhance both bone and vascular tissue regeneration, making CLBM particularly effective in complex defect management. For example, Lee and Kim demonstrated how CLBM's ability to modulate these growth factors contributed to accelerated healing and improved clinical outcomes . Section title: Review Educational score: 1.4548894166946411 Domain: biomedical Document type: Other Language: en Barrier Effectiveness Section title: Review Educational score: 3.8970720767974854 Domain: biomedical Document type: Other Language: en The dense structure of CLBM prevents the infiltration of epithelial and fibroblastic cells into the defect site. This barrier function is crucial in maintaining an undisturbed environment for bone regeneration, particularly in GTR procedures where the regeneration of periodontal ligament and connective tissue is prioritized . Section title: Review Educational score: 1.1994376182556152 Domain: biomedical Document type: Other Language: it Clinical applications Section title: Review Educational score: 3.4065682888031006 Domain: biomedical Document type: Review Language: en CLBM has proven effective in treating both vertical and horizontal periodontal bone defects. Recent studies have shown its potential to augment the ridge laterally . When combined with bone grafts, the membrane enhances clinical attachment levels and reduces probing depths, making it a valuable tool in managing periodontitis . Section title: Review Educational score: 3.7357656955718994 Domain: biomedical Document type: Review Language: en In cases of peri-implantitis, CLBM has demonstrated its ability to regenerate bone around failing implants. Clinical studies have reported substantial bone gain and improved implant stability when CLBM is paired with xenografts, highlighting its utility in restoring compromised implant sites . Section title: Review Educational score: 3.876680612564087 Domain: biomedical Document type: Review Language: en For ridge augmentation procedures, CLBM provides the necessary mechanical strength to stabilize graft materials and create adequate bone volume for implant placement. Deepika-Penmetsa et al. reported successful outcomes in lateral ridge augmentation, with the membrane ensuring graft stability and promoting long-term bone regeneration . Also, a recent review highlighted the superior vertical bone gains achieved with cortical lamina compared to traditional membranes . Section title: Review Educational score: 3.43953537940979 Domain: biomedical Document type: Other Language: en In sinus lift procedures, CLBM prevents graft displacement while promoting new bone formation. High success rates for implant placement have been documented in sites treated with CLBM, making it a reliable choice for sinus augmentation procedures . CLBM holds several advantages over conventional membranes, which are listed in Table 1 . Section title: Review Educational score: 2.0496251583099365 Domain: biomedical Document type: Study Language: en A list of published literature on cortical laminate bone membrane is shown in Table 2 . Section title: Review Educational score: 1.4401935338974 Domain: biomedical Document type: Other Language: en Challenges and limitations Section title: Review Educational score: 4.401992321014404 Domain: biomedical Document type: Review Language: en The rigidity of CLBM is advantageous for maintaining space, but at the same time, it poses a challenge to adapt the membrane to irregular or complex defects such as morphologies . Unlike more flexible collagen-based membranes, CLBM requires precision in trimming and shaping to ensure optimal coverage of the defect. This inflexibility may increase surgical time and the potential for errors during placement. The use of cortical lamina requires precise handling and customization, making it technique-sensitive . Deepika-Penmetsa et al. emphasized that achieving proper adaptation demands advanced surgical expertise, which may limit its use among less experienced clinicians or in high-stress clinical scenarios . Due to its slower degradation rate, CLBM is prone to exposure if not adequately covered by soft tissue. Proper flap management is critical to minimizing this risk . Membrane exposure not only compromises the regenerative outcome by increasing the risk of infection but can also necessitate additional surgical interventions to address complications . Rossi et al. noted that poor flap management or insufficient soft tissue coverage could lead to exposure, particularly in cases involving thin gingival biotypes or significant defect dimensions . This highlights the need for careful case selection and advanced soft tissue management techniques. The production of CLBM involves stringent processing to remove antigenic proteins while preserving its osteoconductive properties. This complex manufacturing process contributes to the high cost of the material, limiting its availability and adoption, particularly in resource-constrained settings . Tumedei et al. pointed out that these costs could present a barrier to widespread clinical use, particularly when compared to more economical alternatives such as resorbable collagen membranes . As cost efficiency becomes an increasingly important consideration in healthcare, this limitation highlights the need for continued innovation to make CLBM more accessible. Section title: Review Educational score: 3.852874279022217 Domain: biomedical Document type: Review Language: en Although numerous studies have demonstrated the short- to mid-term success of CLBM in regenerative procedures, there is a relative lack of long-term clinical trials comparing its outcomes with traditional membranes or other advanced biomaterials. As Schuh et al. noted, long-term follow-up studies are essential to assess the durability and predictability of CLBM in diverse clinical contexts . The absence of such data makes it challenging for clinicians to fully evaluate its advantages and risks over extended periods. Section title: Review Educational score: 0.9416666626930237 Domain: other Document type: Other Language: ro Future perspective Section title: Review Educational score: 3.6914427280426025 Domain: biomedical Document type: Other Language: en To expand the use of CLBM, ongoing research aims to enhance its handling properties, reduce production costs, and enhance antimicrobial properties. Additionally, incorporating bioactive molecules or growth factors could further enhance its regenerative potential. Long-term clinical trials and comparative studies are essential to validate its superiority over other advanced biomaterials. Section title: Conclusions Educational score: 3.831346035003662 Domain: biomedical Document type: Review Language: en CLBM represents a significant step forward in regenerative dentistry. With their unique combination of mechanical stability, biocompatibility, and osteoconductive properties, CLBM addresses many of the limitations associated with traditional membranes. While challenges such as handling difficulties and costs remain, the transformative potential of CLBM positions it as a cornerstone in modern regenerative techniques. Ongoing innovations and research will continue to refine its applications, ensuring its role in achieving predictable and successful clinical outcomes. | Other | biomedical | en | 0.999997 |
PMC11699556 | Section title: Introduction Educational score: 3.9107580184936523 Domain: biomedical Document type: Review Language: en Endometrial cancer (EC) is a gynecological tumor occurring in the endometrium . In China, the incidence of EC and its mortality are on the rise every year, posing a serious threat to society . Several factors, including tumor stage, histological types, and age, determine the prognosis of EC . Based on statistics, the 5-year survival rate of EC patients that are diagnosed and treated early exhibits a range of 70–90%, whereas those diagnosed late typically experience 20–60% due to tumor metastasis . Recent advancements in gene-targeted therapy for EC have expanded treatment options, offering patients more precise and efficacious interventions, with the potential to enhance future survival rates and quality of life [ 6 – 8 ]. Section title: Introduction Educational score: 4.174404144287109 Domain: biomedical Document type: Study Language: en Microfibril-associated protein 5 (MFAP5), an important extracellular matrix (ECM) protein, regulates the structure and function of ECM by mainly binding to microfibrils . In cancer, MFAP5 was found to function in tumor growth, metastasis, and angiogenesis . For example, MFAP5 may activate the epithelial–mesenchymal transition (EMT) program to promote basal-like breast cancer growth and aggressiveness . MFAP5 modulated EMT-related pathways, which led to a reduction in cervical cancer cell migration and invasion . MFAP5 facilitated the proliferation and metastasis of breast cancer cell, as reflected by the elevated levels of matrix metalloproteinase 2 (MMP2) and MMP9 after MFAP5 overexpression . However, there is no clear understanding of MFAP5’s role in EC. Section title: Introduction Educational score: 3.8140406608581543 Domain: biomedical Document type: Study Language: en Herein, down-regulated MFAP5 was found by retrieving the TCGA database ( https:// tcga -data.nci.nih.gov/ tcga ). Subsequently, a series of in vitro experiments was conducted to investigate the effect of MFAP5 expression on the malignant behavior of EC cells. Section title: Cell culture Educational score: 3.8817765712738037 Domain: biomedical Document type: Study Language: en Normal human cervical epithelial cells (HcerEpic) and EC cell lines (JEC, KLE, HEC-1A, and Ishikawa) were purchased from ATCC (Rockville, MD, USA). Cells were grown in DMEM medium with 10% FBS at 37°C with 5% CO 2 . Section title: Cell transfection Educational score: 3.643371820449829 Domain: biomedical Document type: Study Language: en Adenovirus vector overexpressing MFAP5 (ad-MFAP5) and adenovirus-negative control (ad-NC) were prepared by Genepharma (Shanghai, China). Then, transfection of ad-NC or ad-MFAP5 was conducted with 50 multiplicity of infection for 48 h. The blank group (Control) was taken as a control for the experiment. Section title: CCK8 assay Educational score: 4.068357467651367 Domain: biomedical Document type: Study Language: en Cells (1,000 cell/well) were planted into 96-well plate for 48 h. Whereafter, each well was injected with 10 µL of CCK8 reagent (Dojindo, Shanghai, China). A 2 h incubation was followed by the measurement of absorbance at OD 450 nm and the determination of cell viability. Section title: Colony formation assay Educational score: 3.9039511680603027 Domain: biomedical Document type: Study Language: en Cells (2,000 cell/well) were inoculated into six-well plates and grown at 37°C for about 2 weeks. Afterward, a 30 min fixation with methanol was followed by staining the cells with 0.2% crystal violet. Photographs of the colonies were captured with a camera. Section title: Flow cytometry Educational score: 3.870396852493286 Domain: biomedical Document type: Study Language: en After collecting the cells, they were washed with buffer containing PBS. Following that, according to Annexin V-FITC detection kit (Dojindo), cells were stained with FITC-conjugated Annexin V and PI. A flow cytometry (FC500, BECKMAN COULTER, USA) was employed to analyze cell apoptosis within 1 h. Section title: Transwell assays Educational score: 4.121660232543945 Domain: biomedical Document type: Study Language: en Cells were suspended in serum-free medium and added to the upper compartment of a transwell. The lower compartment contained medium with 10% FBS. After cultivating for 24 h at 37°C, the upper chamber was removed, fixed in methanol, and stained with Giemsa. After cleaning, unmigrated cells were removed using a cotton swab. Next, the cells were visualized under the microscope (Leica Microsystems, Wetzlar, Germany) and counted. For cell invasion assay, a layer of Matrigel matrix was applied to the bottom of the transwell chamber. Section title: Western blot Educational score: 4.038293838500977 Domain: biomedical Document type: Study Language: en Cell lysates were prepared using RIPA buffer, then centrifuged and denaturated. Protein samples were then run on a 10% SDS-PAGE and transferred to PVDF membranes. After blocking, incubation of primary antibodies on the membranes at 4°C overnight was followed by secondary antibody incubation for 1 h. Table 1 lists the primary antibodies used. An ECL luminescent solution was utilized to render the color, and the protein bands were analyzed and quantified using Image J. Section title: Statistical analysis Educational score: 2.974839448928833 Domain: biomedical Document type: Study Language: en Each experiment was conducted three times in triplicate. Data were analyzed using GraphPad Prism 8.0, presented as mean ± SD, and compared among groups using one-way ANOVA with a significance level of p < 0.05. Section title: MFAP5 is down-regulated in EC Educational score: 3.80707049369812 Domain: biomedical Document type: Study Language: en Based on TCGA analysis, the results revealed that MFAP5 expression was lower in EC tissues compared to normal tissues . Subsequent results from western blot showed that MFAP5 protein expression was observably decreased in EC cell lines . These data suggest that MFAP5 is expressed at low levels in EC. Section title: MFAP5 overexpression inhibits proliferation and promotes apoptosis of EC cells Educational score: 4.1257405281066895 Domain: biomedical Document type: Study Language: en An overexpression of MFAP5 in HEC-1-A and Ishikawa cells was conducted to study the function of the protein in EC. Western blot results showed a significantly elevated protein level of MFAP5 in cells after MFAP5 overexpression . Furthermore, CCK8 assay and colony formation assay indicated significantly reduced cell viability and clonogenic activity in cells overexpressing MFAP5 compared to the control cells . Moreover, results of flow cytometry showed that the MFAP5 overexpression group displayed a significant increase in apoptosis rates . These data demonstrate that overexpression of MFAP5 suppressed proliferation and facilitated apoptosis of EC cells. Section title: MFAP5 overexpression attenuates migration and invasion of EC cells Educational score: 4.078271865844727 Domain: biomedical Document type: Study Language: en Further, MFAP5 was examined for its impact on cell migration and invasion by transwell assays. As shown in Figure 3a , comparing the overexpressing MFAP5 group with the control and ad-NC groups, the number of migrating and invading cells was both significantly reduced. In addition, western blot results showed that MMP2 and MMP9 protein levels were observably decreased in the MFAP5 overexpression group . These data indicate that overexpression of MFAP5 in EC cells inhibited their migration and invasion capabilities. Section title: MFAP5 overexpression suppresses EMT in EC cells Educational score: 4.076424598693848 Domain: biomedical Document type: Study Language: en Using western blot analysis, we further quantified the levels of EMT markers expressed in cells overexpressing MFAP5. As shown in Figure 4 , MFAP5 overexpression resulted in an increase in E-cadherin and a decrease in N-cadherin and α-SMA. These findings suggest that EMT in EC cells may be inhibited by overexpressing MFAP5. Section title: MFAP5 overexpression inactivates the AKT/mTOR pathway Educational score: 4.055340766906738 Domain: biomedical Document type: Study Language: en Next, the phosphorylation of AKT and mTOR was monitored to explore whether overexpressing MFAP5 in EC cells mediated the activity of the AKT/mTOR pathway. Western blot analysis revealed that phosphorylations of AKT and mTOR were reduced when MFAP5 was overexpressed in EC cells . These data suggest that MFAP5 overexpression decreased AKT/mTOR activity. Section title: Discussion Educational score: 4.137123107910156 Domain: biomedical Document type: Study Language: en Poor prognosis of EC is largely determined by tumor metastasis . Patients with cancer cells spreading to other organs make treatment more challenging, and the prognosis worsens . Therefore, investigating the mechanisms of tumor metastasis in EC may help to develop effective treatment strategies to improve patient outcomes. In this study, reduced level of MFAP5 was tested in EC cells, and cell proliferation was suppressed while cell apoptosis was promoted by overexpressing MFAP5. Importantly, our results demonstrate that MFAP5 overexpression inhibited migration, invasion, and EMT of EC cells. These data show a vital role of MFAP5 in EC cell malignant behaviors. Section title: Discussion Educational score: 4.483061790466309 Domain: biomedical Document type: Study Language: en MFAP5, a matrix glycoprotein consisting of microfibers, serves as a multifunctional protein implicated in endothelial cell behavior and mediated cell adhesion . Recently, most research studies indicated that MFAP5 expression is high in various tumor types, leading to enhanced tumor proliferation, endothelial cell motility, and angiogenesis [ 11 – 13 , 20 , 21 ]. For example, MFAP5 has been reported to enhance the stem cell features of non-small cell lung cancer cells, while the knockdown of MFAP5 has been shown to exert the opposite effect . Another study has found that MFAP5 silencing resulted in apoptosis and growth inhibition in cervical cancer cells . Herein, our TCGA database analysis revealed contrasting findings, showing underexpression of MFAP5 in EC tumor tissues, which differs from previous studies. Later, we detected diminished MFAP5 expression in EC cells. The invasion and metastasis of tumor cells are intricately regulated by the ECM, with MMPs, particularly MMP2 and MMP9, playing a crucial role in facilitating tumor formation and metastasis by degrading matrix proteins . Dysregulation of MMPs can compromise the integrity of tissue barriers, facilitating tumor invasion . This process promotes the degradation of the ECM and basement membrane, thereby enhancing tumor penetration, invasion of adjacent tissues, and metastasis to distant sites . Our findings demonstrate that MFAP5 overexpression led to a decrease in MMP2 and MMP9 levels, thereby confirming the inhibitory effect of MFAP5 on EC cell migration and invasion. However, how MFAP5 affects MMPs expression should be investigated further. EMT is a biological phenomenon in which cancer cells undergo a phenotypic change from epithelial to mesenchymal cells, resulting in enhanced abilities for proliferation, migration, and resistance to apoptosis [ 26 – 28 ]. MFAP5 has been shown to play a role in EMT process in cancer progression . Similar to previous results, our further data found an increased level of E-cadherin and a decreased N-cadherin and α-SMA in cells overexpressing MFAP5, indicating that MFAP5 overexpression inhibited EMT in EC cells. Section title: Discussion Educational score: 4.170172691345215 Domain: biomedical Document type: Study Language: en AKT, a protein kinase, can be activated by various growth factors, subsequently activating mTOR, thus promoting cellular proliferation and growth . In addition, EMT can be accelerated by activating AKT . Research indicates that dysregulated activation of the AKT/mTOR pathway in EC tissues can enhance cancer proliferation, invasion, and metastasis, while suppressing apoptosis and accelerating tumor malignancy [ 34 – 36 ]. Consequently, the modulation of the AKT/mTOR pathway has emerged as a prominent focus in the realm of EC therapy. In this study, the activity of the AKT/mTOR pathway was found to be reduced in EC cells overexpressing MFAP5, manifested by the inhibition of AKT and mTOR phosphorylation levels. However, further experiments will be required to determine whether MFAP5 exerts its biological role in EC by mediating the AKT/mTOR pathway. In addition, whether MFAP5 has the same biological function in vivo and the further regulatory mechanisms need in-depth investigation. Section title: Discussion Educational score: 4.0645976066589355 Domain: biomedical Document type: Study Language: en In conclusion, our findings confirmed that MFAP5 regulates the EMT and malignancy in EC cells, and inactivation of the AKT/mTOR pathway may be one of the mechanisms , suggesting that targeting MFAP5 may represent an attractive strategy for EC treatment. | Review | biomedical | en | 0.999997 |
PMC11699559 | Section title: Introduction Educational score: 4.28087854385376 Domain: biomedical Document type: Review Language: en Retinal diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), and uveitis are major causes of vision impairment and blindness worldwide . These conditions involve complex processes such as chronic inflammation, oxidative stress, and abnormal angiogenesis. Inflammation, in particular, plays a crucial role in the progression of these diseases by disrupting the delicate balance of pro- and anti-inflammatory cytokines, thus leading to tissue damage and abnormal blood vessel growth . Cytokines are small signaling proteins that regulate various aspects of the immune response, including inflammation, cell proliferation, and cell death. Among these, the interleukin-6 (IL-6) family of cytokines, including Oncostatin M (OSM), has received significant attention due to their dual role in both promoting and resolving inflammation. These cytokines are implicated in the development of various inflammatory and autoimmune diseases, including those affecting the retina . Section title: Introduction Educational score: 4.060971260070801 Domain: biomedical Document type: Review Language: en This review aims to explore the potential therapeutic effects of OSM and the broader IL-6 family of cytokines in retinal diseases. By examining the molecular mechanisms through which these cytokines influence the health and disease of the retina, as well as by reviewing preclinical and clinical studies, this article aims to provide a comprehensive understanding of their potential as targets for novel therapies. Additionally, this review will discuss the challenges and future directions for translating these findings into clinical practice. Section title: OSM and the IL-6 family Educational score: 4.997342109680176 Domain: biomedical Document type: Study Language: en OSM is a significant member of the IL-6 cytokine family, characterized by the presence of the gp130 signal receptor subunit in their receptor complexes. This family includes various cytokines such as IL-6, IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine factor 1, and IL-27 . OSM was first discovered by Zarling et al. in the U-937 human lymphoma cell line . The gene encoding human OSM is located on chromosome 22q12.2. The OSM gene encodes a polypeptide that initially consists of 252 amino acid residues. This polypeptide includes an N-terminal sequence of 25 amino acids and a C-terminal sequence of 32 amino acids. After proteolytic processing, the mature OSM protein consists of 195 amino acid residues. The mature human OSM protein has a molecular weight of approximately 28 kDa. Structurally, OSM is characterized by four α-helices arranged in an “up–up–down–down” topology [ 6 – 8 ]. This structural arrangement is typical of cytokines in the IL-6 family and is critical for their biological activity. OSM is widely expressed in vivo and plays a crucial role in various physiological and pathological processes. Several types of immune cells are known to produce and secrete OSM [ 9 – 12 ]. The receptor complexes for OSM are heterodimers, and their composition defines the type of signaling they mediate. Two main types of OSM receptor (OSMR) complexes exist: type I and type II . The type I OSMR complex consists of the α subunit gp130 and the β subunit leukemia inhibitory factor receptor (LIFR) β subunit and the type II OSMR complex is composed of the α subunit gp130 and OSMR β subunit . The receptor complex diversity mediates the different biological functions of OSM through the activation of multiple signaling pathways. The OSMR complex operates in a dynamic and species-specific manner, with its activation dependent on the presence of its subunits and ligand (OSM) . In the absence of OSM, the two subunits of the OSMR complex, gp130 and either OSMR or LIFR, exist separately in a non-associated state. This separation prevents spontaneous activation of the downstream signaling pathways. When OSM is present, it first forms a low-affinity heterodimer with gp130. This initial interaction is crucial for the subsequent recruitment of the second subunit. After forming a heterodimer with gp130, the complex then recruits either the OSMR or LIFR subunit, depending on the specific receptor complex involved. The recruitment leads to the formation of a high-affinity receptor complex that is now capable of signaling . Computational techniques including homology modeling, protein–protein docking, and molecular dynamics simulations were used by Du et al. to identify several critical amino acid residues involved in the binding process between OSM and OSMR . These residues are crucial for the stability and specificity of the interaction, ensuring that OSM can effectively bind and activate its receptor, revealing potential “hot spots” that could serve as targets for inhibitor design to block this pathway. Moreover, it has been showed the OSM/OSMR signaling module exhibits a unique microenvironment-restricted expression pattern, suggesting that targeting this pathway could mitigate the side effects associated with anti-IL-6 therapies, strengthening the demonstrated safety and tolerability of humanized anti-OSM antibodies as a potential therapeutic strategy for inflammatory diseases and cancer treatment . Also, it has been revealed that OSM and LIF share structural similarities, which explains why both cytokines can bind to the LIFR receptor . Despite these similarities, OSM and LIF have distinct biological functions, attributed to differences in their overall structure and the specific receptor subunits they recruit. In mice, OSM predominantly binds to type II OSMR complexes, which include gp130 and OSMR. Mouse OSM generally does not activate LIFR, except at very high concentrations, where it can weakly stimulate LIFR. There is a single report that mouse-derived OSM can activate LIFR specifically in mouse osteoblasts, suggesting a degree of context-dependent flexibility . OSM from rats and humans can bind to both type I (gp130 and LIFR) and type II (gp130 and OSMR) receptor complexes within their respective species. This dual-binding capacity allows OSM from these species to participate in a broader range of biological functions and signaling pathways compared to mouse OSM . The interaction between OSM and its receptor complexes is a finely tuned process that depends on the presence of specific receptor subunits and the structural characteristics of OSM. The formation of the OSMR complex, particularly the role of OSMR in type II complexes, enables OSM to activate unique signaling pathways that differentiate its functions from other cytokines in the IL-6 family. The species-specific nature of OSM binding further underscores the complexity and specificity of its biological activities, making OSM a critical cytokine in both normal physiology and disease states Section title: Role of OSM and IL-6 family in immune response, inflammation, angiogenesis, and cell survival Educational score: 4.826746463775635 Domain: biomedical Document type: Study Language: en Abnormal levels of OSM are indeed linked to various inflammatory diseases. Compared to healthy controls, OSM levels are elevated in patients with pulmonary fibrosis . It has been demonstrated that increased the expression of OSM and OSMR in many inflammatory bowel disease lesions . Elevated levels of Oncostatin M receptor beta subunit (OSMRβ) in fibroblasts and dermal endothelial cells have been observed in patients with scleroderma, a chronic autoimmune disease characterized by fibrosis, vascular abnormalities, and immune system dysregulation . Studies have indicated that cytokines such as OSM and interleukin-31 (IL-31) play a critical role in promoting itch and inflammation in this condition, particularly through their interaction with dermal cells expressing IL-31RA (IL-31 receptor A) and OSMRβ . Elevated levels of OSM in gingival crevicular fluid have been observed in patients with periodontal disease, and research indicates that OSM concentrations increase in correlation with the severity of the disease, progressing from early-stage periodontal disease to chronic periodontitis [ 26 – 28 ]. In patients with coronavirus disease 2019 (COVID-19), OSM levels have been found to be elevated in peripheral blood plasma compared to healthy controls. These elevated levels of OSM are associated with the severity of the disease, suggesting that OSM plays a significant role in the immune response and inflammation characteristic of severe COVID-19 cases . The role of OSM in promoting angiogenesis, particularly in the context of cancer, is supported by accumulating evidence . OSM, a cytokine in the IL-6 family, can significantly influence angiogenesis through its effects on capillary endothelial cells and its activation of various signaling pathways. The recombinant human OSM has been shown to activate the STAT3 signaling pathway in endothelial cells. This activation leads to increased angiogenesis, particularly in tumor environments where new blood vessels are required to supply nutrients and oxygen to the growing tumor mass . In osteosarcoma, a highly aggressive bone cancer, the activation of the OSM/JAK2/STAT3 axis has been linked to both increased angiogenesis and the invasive behavior of cancer cells. The upregulation of MMP2 and vascular endothelial growth factor (VEGF) by this pathway not only promotes the formation of new blood vessels but also enhances the cancer cell’s ability to invade surrounding tissues, contributing to the aggressive nature of osteosarcoma . Neutrophils, which are often recruited to the tumor microenvironment, can produce OSM. Neutrophil-derived OSM interacts with the type II OSMR (composed of gp130 and OSMRβ) on breast cancer cells, leading to the induction of VEGF expression . Indeed, while OSM and other IL-6 family cytokines share the ability to activate the STAT3 signaling pathway, their effects on angiogenesis can be inconsistent. This variability is influenced by several factors, including the specific cytokine involved, the cellular context, and the microenvironment. CNTF exhibits anti-angiogenic effects in vascular endothelial cells and the retina, suggesting that CNTF-induced STAT3 signaling can inhibit rather than promote angiogenesis in certain contexts. This contrasts with the generally pro-angiogenic effects of other IL-6 family cytokines and highlights the complexity of cytokine signaling pathways. This study sheds light on the nuanced role of STAT3 in angiogenesis and reveals the complex and context-dependent role of STAT3 signaling in angiogenesis, particularly in response to different IL-6 family cytokines like CNTF and OSM . The findings underscore that the effects of STAT3 on angiogenesis are not uniform but are influenced by the specific cytokine involved, the concurrent activation of other signaling pathways, and the overall cellular environment. IL-6, a key cytokine in the IL-6 family, has been shown to induce the expression of VEGF mRNA in various cancer cell lines, including A431 cells (a human epidermoid carcinoma cell line) and C6 cells (a rat glioma cell line). This induction of VEGF expression highlights IL-6’s role in promoting angiogenesis, particularly in the context of tumor growth . The study by Wei et al. using nude mice demonstrated that IL-6 plays a significant role in promoting tumor growth in human cervical cancer, specifically in the C33A cell line, through a mechanism that is dependent on VEGF-mediated angiogenesis . In several cancers, including hepatocellular carcinoma, renal cell carcinoma, colorectal cancer, and glioblastoma, increased levels of circulating IL-6 have been linked to a poor response to therapies that target the VEGF/VEGFR pathway. These therapies include sunitinib, a tyrosine kinase inhibitor, and bevacizumab, an anti-VEGF antibody . Section title: Molecular mechanisms and preclinical studies Educational score: 4.741199493408203 Domain: biomedical Document type: Study Language: en Animal models have played an essential role in advancing our knowledge of retinal diseases and assessing potential treatments like OSM and the IL-6 family of cytokines. Among these models, rodents, particularly mice and rats, are favored due to their genetic adaptability and close resemblance to human retinal structures. One notable model is the oxygen-induced retinopathy (OIR) model in mice, which replicates the abnormal blood vessel growth seen in DR and ROP . By subjecting mice to high oxygen levels followed by a return to normal levels, this model induces retinal ischemia and new blood vessel formation, mirroring the processes observed in human diseases. This model has been used to investigate the roles of various cytokines, including OSM and IL-6, in retinal inflammation and abnormal blood vessel growth and researchers have been trying to unveil the intricate and multifaceted roles of OSM and the IL-6 family in the context of retinal diseases. The IL-6 cytokine family is a diverse group of inflammatory and pleiotrophic cytokines, encompassing IL-27, IL-11, IL-6, LIF, CT-1, CLC, CNTF, and OSM. These cytokines utilize similar receptor complexes and commonly activate the STAT3 signaling pathway within cells. The angiomodulatory effect of CNTF-driven STAT3 signaling in different mouse models of vasoproliferative eye diseases involves both a direct anti-angiogenic impact on vascular endothelial cells and an indirect effect mediated by Müller cells . Also, intravitreal injection of LIF can increase the vascular density in contrast to the OSM injection through changes in the cathepsin B and L expression . OSM, in particular, exhibits a duality in its effects, displaying both protective and detrimental impacts contingent on the disease stage and the specific retinal microenvironment. Other preliminary studies showed that OSM treatment promotes the proliferation of choroidal and retinal endothelial cells, in contrast to its effect on aortic endothelial cells . Rapp et al. observed a positive angiomodulatory effect of OSM following its intravitreal injection in the OIR model. The treatment activated STAT3 signaling in various retinal cell types, including Müller and vascular endothelial cells. In vitro studies indicate that OSM influenced the secretory profile of Müller cells. OSM promotes the regeneration of rods and cone photoreceptors in the early stage of degeneration via interacting with muller cells through activating STAT3. Also, it has been the regenerative role of OSM reported in the muscle, bone , and heart . Additionally, RNA sequencing of isolated retinal vascular endothelial cells from OIR P17 mice revealed a transcriptomic shift resulting from OSM treatment at P12. These findings highlight the critical role of Müller cells in vasoproliferative eye diseases and suggest that the effects of angiogenic agents may vary significantly between in vitro and in vivo environments. Also, it suggests that angiomodulatory agents like OSM might produce unanticipated and occasionally opposing effects in vivo versus in vitro , depending on the cell types involved within the intricate retinal environment. OSM shows a positive angiomodulatory influence on retinal angiogenesis, with Müller cells being key contributors to vascular homeostasis. Another study highlights the intricate role of STAT3 signaling in vascular endothelial cells in response to cytokine treatment, leading to diverse angiogenic outcomes . The angiomodulatory effects of STAT3 appear to be influenced by the activity of other intracellular signaling pathways and its specific localization within the cytosol, mitochondria, or nucleus. OSM and CNTF differ in their intracellular signaling mechanisms and STAT3 specificity, resulting in distinct transcriptomic profiles and metabolic activities . Section title: Molecular mechanisms and preclinical studies Educational score: 4.382778644561768 Domain: biomedical Document type: Review Language: en Another significant model is the streptozotocin (STZ)-induced diabetic mouse model, which allows for the study of the long-term impacts of high blood sugar on the retina, particularly in the context of DR. Through STZ administration, the destruction of pancreatic β cells occurs, leading to elevated blood sugar levels and consequent damage to retinal blood vessels . Robinson et al. demonstrated that inhibiting IL-6 trans-signaling significantly reduces diabetes-induced oxidative damage systemically and in the retina in an STZ-induced diabetic mouse model . Another study discovered that high levels of IL-6, a proinflammatory cytokine in the eye, were significantly linked to the advancement of proliferative diabetic retinopathy (PDR) and poor outcomes after eye surgery. This indicates that IL-6 within the eye may play a crucial role in the development of abnormal blood vessel growth associated with PDR, possibly by increasing VEGF expression . Mason et al. systematically review and analyze the changes in cytokine levels in the eyes of patients with nonproliferative DR. The meta-analysis highlights significant alterations in inflammatory cytokines, which could potentially contribute to the pathophysiology of the disease and may inform future therapeutic strategies . Moreover, other constituents of the IL-6 family, such as LIF and CNTF, have exhibited potential as neuroprotective agents in diverse models of retinal degeneration. For instance, LIF has demonstrated the ability to shield and protect the choriocapillaris, and yet, the photoreceptors, in a mouse model of dry-AMD, induced with sodium iodate . Yang et al. showed that a single intravitreal injection of CNTF-NP or OSM-NP provided protection for retinal ganglion cells in an acute glaucoma model and safeguarded photoreceptors and vision for over 70 days in a rat model of retinitis pigmentosa . Section title: Clinical studies and trials Educational score: 4.024231910705566 Domain: biomedical Document type: Study Language: en The investigation of OSM and the IL-6 family as potential therapeutic targets in retinal diseases has progressed from animal experiments to clinical trials, summarized in Table 1 . Clinical studies have mainly focused on the role of IL-6 in retinal diseases. One important clinical trial examined the use of tocilizumab, a monoclonal antibody targeting IL-6, in patients with macular edema related to non-infectious uveitis. The trial demonstrated that tocilizumab (at both 4 and 8 mg/kg doses) effectively enhances visual acuity, reducing vitreous haze and central macular thickness in eyes with noninfectious uveitis. Tocilizumab is generally well-tolerated but requires careful patient monitoring due to risks such as neutropenia and gastrointestinal perforations. It offers a new avenue for managing challenging cases of retinal inflammatory diseases . Section title: Clinical studies and trials Educational score: 4.076891899108887 Domain: biomedical Document type: Study Language: en Sarilumab is a fully human anti-IL-6Rα mAb that blocks the IL-6 signaling pathways by binding to membrane and soluble forms of IL-6Rα. After receiving approval for rheumatoid arthritis, it is undergoing clinical trials for use in the management of posterior segment non-infectious uveitis. In the SATURN study, patients with non-infectious uveitis showed reduced vitreous haze and lower steroid dosing with 200 mg subcutaneous dose every 2 weeks. The treatment improved visual acuity and central macular thickness with fewer side effects than other therapies, but adverse events like neutropenia and elevated alanine aminotransferase levels were reported . Section title: Clinical studies and trials Educational score: 3.087444305419922 Domain: biomedical Document type: Other Language: en Designed specifically for intravitreal delivery, EBI-031 blocks both free IL-6 and the IL-6 receptor complex. Clinical trials have been conducted to assess the effectiveness and safety of EBI-031 in patients with diabetic macular edema . Section title: Clinical studies and trials Educational score: 4.006646633148193 Domain: biomedical Document type: Other Language: en The current Phase I clinical trial involving KSI-501 marks a significant leap forward in the medical field. This trial aims to combat neovascular AMD and Diabetic Macular Edema (DME) by targeting both IL-6 and VEGF. KSI-501 is an innovative bispecific inhibitor specifically engineered to concurrently address IL-6 and VEGF, with the goal of overcoming the limitations encountered in prior studies by combining anti-inflammatory and anti-angiogenic effects. Initial findings from this trial have displayed encouraging results in terms of safety and preliminary effectiveness, and more comprehensive data are anticipated to unveil the therapeutic potential of this dual-target approach . Section title: Clinical studies and trials Educational score: 4.078749656677246 Domain: biomedical Document type: Study Language: en Many trials are currently underway to evaluate the effectiveness of blocking the activation of OSM pathways, but none are focused on the retinal field. For instance, a humanized anti-OSM monoclonal antibody developed by GSK for rheumatoid arthritis showed limited efficacy in clinical trials due to poor binding affinity and was eventually discontinued . Another GSK anti-OSM monoclonal antibody demonstrated safety in early trials for systemic scleroderma but showed no significant efficacy, and all patients in the highest dose group experienced adverse effects . Researchers have developed a specific binding protein targeting OSM to inhibit its interaction with gp130 or LIFR , potentially applicable to inflammatory skin diseases and cancer . Boise State University developed small molecule inhibitors targeting Site III of OSM , particularly SMI-10B, to reduce tumor cell detachment and metastasis, confirmed through molecular dynamics simulation . Section title: Clinical studies and trials Educational score: 3.961350917816162 Domain: biomedical Document type: Review Language: en Upcoming clinical trials will explore the combination of cytokine modulation with established treatments, such as anti-VEGF therapies, to enhance therapeutic results. Moreover, there is growing interest in personalized medicine strategies, where patient-specific cytokine profiles could guide the selection and combination of therapies to optimize treatment effectiveness and minimize adverse effects. The potential for therapies targeting OSM in retinal diseases also continues to be an area of intense study, with preclinical data suggesting the potential neuroprotective benefits of OSM modulation in certain retinal conditions. Section title: Therapeutic strategies and future perspectives Educational score: 4.37064266204834 Domain: biomedical Document type: Review Language: en Modulating the activity of OSM and the IL-6 family offers a promising approach to treating retinal diseases. The cytokine network in the retina is complex, and strategies to modulate this network must be carefully designed to achieve therapeutic benefits without unintended adverse effects. One approach to cytokine modulation involves using inhibitors targeting the IL-6 receptor (e.g., tocilizumab) are being assessed for their ability to halt disease progression by blocking IL-6 signaling pathways . These therapies aim to reduce the inflammatory and angiogenic processes that contribute to retinal degeneration. Another potential strategy involves using agonists that activate protective cytokine pathways. For instance, recombinant forms of neuroprotective cytokines like CNTF have been investigated for their ability to slow down the degeneration of light-sensitive cells in conditions like retinitis pigmentosa . CNTF works by activating survival pathways that counteract signals that lead to cell death in the retina, thereby preserving vision. Despite promising preclinical results, challenges exist in ensuring the effective and targeted delivery of such therapies to the retina. One of the key challenges in cytokine modulation is achieving precise control over cytokine activity. Over-inhibition of cytokines like IL-6 could impair the retina’s natural defense mechanisms, leading to unintended consequences. Conversely, excessive activation of protective cytokines could potentially trigger unintended effects or lead to immune system imbalances. Therefore, the development of cytokine-based therapies must take into account the balance between efficacy and safety . Section title: Therapeutic strategies and future perspectives Educational score: 4.323780536651611 Domain: biomedical Document type: Review Language: en Combination therapies that target multiple pathways simultaneously represent a promising approach to enhancing treatment efficacy in retinal diseases. For example, an ongoing Phase I clinical trial is investigating a bispecific inhibitor that targets both IL-6 and VEGF . By simultaneously addressing the inflammatory and angiogenic components of retinal diseases, this dual-target approach has the potential to provide superior therapeutic outcomes compared to monotherapy. Additionally, combining cytokine modulation with existing treatments, such as anti-VEGF therapies, may offer synergistic effects. For instance, in AMD and DME, where VEGF plays a central role in pathological angiogenesis, adding cytokine-targeting agents could further suppress disease progression by modulating the inflammatory environment that supports neovascularization. Personalized medicine also holds great potential in the context of cytokine modulation for retinal diseases. By tailoring treatments based on individual patient profiles, including specific cytokine expression patterns and genetic predispositions, clinicians could optimize therapeutic efficacy while minimizing side effects. For example, patients with elevated levels of IL-6 or OSM may benefit more from therapies targeting these cytokines, while others may require different approaches. Future research will likely focus on identifying biomarkers that can guide personalized treatment strategies and on developing advanced delivery systems that ensure precise targeting of cytokine modulators to the retina. As our understanding of the cytokine network in retinal diseases deepens, the potential for personalized and combination therapies to transform the management of these conditions will continue to grow. Section title: Conclusion Educational score: 4.114533424377441 Domain: biomedical Document type: Review Language: en In this review, we have examined the various roles of OSM and the IL-6 family of cytokines in relation to retinal diseases. The evidence emphasizes the significant influence of these cytokines on inflammation, neovascularization, and cell survival within the retina. Preclinical studies show the potential effectiveness of targeting these pathways while emerging clinical data indicate encouraging avenues for therapeutic intervention. The increased expression of IL-6 and OSM in retinal diseases, such as DR and AMD, may reflect a failure of anti-inflammatory pathways to properly regulate chronic low-grade inflammation, which is a key feature in the progression of these diseases. This disruption could lead to a compensatory yet maladaptive overproduction of pro-inflammatory cytokines, contributing to ongoing retinal damage. More studies need to determine whether this cytokine upregulation is a primary mechanism or a secondary response to deficiencies in anti-inflammatory signaling, which could offer new insights into therapeutic approaches targeting inflammation regulation in retinal diseases. Section title: Conclusion Educational score: 4.123152256011963 Domain: biomedical Document type: Review Language: en To address both inflammation and vascular remodeling, targeting the OSM pathways may offer a promising alternative to current therapies for retinal diseases. While potential challenges like antibody formation exist, strategies to minimize immunogenicity and combine these treatments with anti-VEGF agents may enhance therapeutic efficacy. The optimal treatment regimen will depend on patient-specific responses, and further studies are needed to refine these approaches and ensure long-term effectiveness. Moreover, there are challenges, such as the requirement for specific and safe delivery methods and the management of potential side effects. Future research should concentrate on refining these therapeutic approaches, including exploring combination therapies and personalized medicine strategies tailored to individual patient profiles. Continued clinical investigation is essential to fully realize the potential of OSM and IL-6 family cytokine modulation, which could revolutionize therapeutic approaches for retinal diseases and enhance patient outcomes . Section title: Conclusion Educational score: 3.5416910648345947 Domain: biomedical Document type: Review Language: en Table 1 summarizes various anti-IL-6, anti-OSM, and therapeutic agents under investigation or clinical use, highlighting their molecular structure, targeted pathways, current FDA-approved indications, and potential future applications. | Review | biomedical | en | 0.999995 |
PMC11699560 | Section title: Introduction Educational score: 4.2516188621521 Domain: biomedical Document type: Review Language: en Gestational diabetes mellitus (GDM) is a prevalent metabolic disorder that occurs during pregnancy, characterized by glucose intolerance that typically manifests in the second and third trimesters . This condition is associated with several adverse outcomes, including abnormal embryonic development, insulin resistance, hyperinsulinemia, and hyperglycemia . The incidence of GDM has been rising, primarily due to increasing maternal obesity and advanced maternal age . GDM significantly increases the risk of severe complications for both mother and infant, such as macrosomia, dystocia, and neonatal hypoglycemia . Despite ongoing research, the pathogenesis of GDM remains incompletely understood. Known etiological factors include oxidative stress, insulin resistance, and inflammation, which are triggered by placental hormones and strongly linked to the progression of GDM . Therefore, the identification of novel and effective therapeutic agents is critical for managing GDM. Section title: Introduction Educational score: 4.466329097747803 Domain: biomedical Document type: Review Language: en Baicalein is a flavonoid compound extracted from the roots of Scutellaria baicalensis Georgi, known for its diverse pharmacological effects . Recent studies have revealed various biological functions of Baicalein, including its role in regulating different diseases. For instance, Baicalein can alleviate oxidative stress and promote autophagy, and was shown to improve cardiac hypertrophy in mice . Additionally, Baicalein targets the GPX4/ACSL4/ACSL3 axis to inhibit ferroptosis, thereby reducing cerebral ischemia-reperfusion injury . In hyperlipidemic pancreatitis, Baicalein modulates the miR-192-5p/TXNIP axis to inhibit the NLRP3/Caspase-1 pathway, thereby mitigating pyroptosis and inflammation . Moreover, Baicalein regulates the AhR/IL-22 pathway to enhance the intestinal epithelial barrier, which alleviates ulcerative colitis . Notably, Baicalein has been shown to target the miR-17-5p-Mfn1/2-NF-κB pathway in trophoblast cells, reducing high glucose-induced inflammation and apoptosis . Despite these findings, the regulatory effects and mechanisms of Baicalein in GDM progression in vivo remain unexplored. Section title: Introduction Educational score: 4.027211666107178 Domain: biomedical Document type: Study Language: en In this study, we demonstrate that Baicalein inhibits the NLRP3 inflammasome and alleviates placental inflammation and oxidative stress in a GDM mouse model, suggesting that Baicalein holds potential as a therapeutic agent for managing GDM. Section title: Animal model Educational score: 4.1396355628967285 Domain: biomedical Document type: Study Language: en C57BL/KsJ +/+ (wild-type) and C57BL/KsJ db/+ (db/+) mice (10 weeks old, n = 24 per sex) were obtained from the Nanjing Model Animal Center (Nanjing, China). All animal procedures were conducted in accordance with the guidelines of the Ethics Committee of Zhejiang Provincial People’s Hospital. Female mice were individually paired with male mice of the same genotype. The presence of copulatory plugs the following morning was used to confirm mating and designate gestation day (GD) 0. Pregnant female mice were then divided into four groups: Control (normal pregnancy, C57BL/KsJ +/+ ), GDM (C57BL/KsJ db+ (db/+) mice treated with DMSO), GDM + 20 mg/kg Baicalein (C57BL/KsJ db+ (db/+) mice treated with 20 mg/kg Baicalein), and GDM + 40 mg/kg Baicalein (C57BL/KsJ db+ (db/+) mice treated with 40 mg/kg Baicalein). Each group consisted of six mice. Baicalein was dissolved in DMSO and administered orally at doses of 20 or 40 mg/kg daily for 20 days starting from pregnancy. On GD 10, glucose and insulin tolerance were assessed using intraperitoneal glucose tolerance tests (IPGTT) and intraperitoneal insulin tolerance tests (IPITT). On GD 20, fasting blood glucose levels were measured with a glucometer (Roche Diagnostics, Risch-Rotkreuz, Switzerland), and insulin levels were quantified using an Ultra-Sensitive Mouse Insulin ELISA kit (ALPCO Diagnostics, Salem, NH). Cesarean sections were performed on GD 20 to collect placentas and fetuses for further analysis. Litter size and birth weight were recorded. Section title: Detection of glucose and insulin tolerance Educational score: 4.03215217590332 Domain: biomedical Document type: Study Language: en For the IPGTT, after a 6 h fast, the mice received an intraperitoneal injection of glucose (2.0 g/kg). Blood glucose levels were measured at 0, 30, 60, 90, and 120 min using an ACCU-CHEK Advantage glucometer (Roche Diagnostics, Risch-Rotkreuz, Switzerland). Section title: Detection of glucose and insulin tolerance Educational score: 3.9228241443634033 Domain: biomedical Document type: Study Language: en For the IPITT, after a 6 h fast, insulin (0.75 U/kg) was administered intraperitoneally. Blood glucose levels were then measured at 0, 30, 60, 90, and 120 min. Section title: ELISA Educational score: 3.744006872177124 Domain: biomedical Document type: Study Language: en The levels of TNF-α , IL-1β , and IL-6 were quantified using commercial ELISA kits according to the manufacturer’s protocols. Section title: Detection of oxidative stress Educational score: 3.9486441612243652 Domain: biomedical Document type: Study Language: en Oxidative stress markers, including malondialdehyde , glutathione , myeloperoxidase , and superoxide dismutase , were measured using commercial kits in placental tissue samples. Section title: Western blot Educational score: 3.9751980304718018 Domain: biomedical Document type: Study Language: en Proteins from mice placental tissues were extracted using RIPA lysis buffer (Thermo Fisher Scientific, Inc.), separated using 10% SDS-PAGE, and transferred to PVDF membranes (Beyotime, Shanghai, China). After blocking, membranes were incubated with primary antibodies at 4°C for 12 h. Secondary antibodies were then applied. Protein detection was performed using a chemiluminescence detection kit (Thermo Fisher Scientific, Inc.). Section title: Western blot Educational score: 2.6309449672698975 Domain: biomedical Document type: Study Language: en The primary antibodies used in this study were as follows: NLRP3 , IL-1β , IL-18 , and β-actin . Section title: Statistical analysis Educational score: 2.6512482166290283 Domain: biomedical Document type: Study Language: en Data are presented as mean value ± standard deviation (SD) and analyzed using GraphPad Prism 9 (GraphPad Inc, La Jolla, CA, USA). Comparisons were performed using one-way analysis of variance with Tukey’s post hoc test. A p -value < 0.05 was considered statistically significant. Section title: Baicalein improves metabolic symptoms in GDM mice Educational score: 4.074517726898193 Domain: biomedical Document type: Study Language: en Our results showed that the glucose tolerance in GDM mice was impaired, but this impairment could be reversed using Baicalein (40 mg/kg) . Similarly, insulin tolerance was reduced in GDM mice, and similarly, this reduction could be mitigated using Baicalein (40 mg/kg) . Additionally, fasting blood glucose and plasma insulin levels were elevated in GDM mice, and these elevations could be normalized following Baicalein (40 mg/kg) treatment . Overall, our findings indicate that Baicalein could improve metabolic symptoms in GDM mice. Section title: Baicalein enhances reproductive outcomes in GDM mice Educational score: 4.00286340713501 Domain: biomedical Document type: Study Language: en In the GDM group, the number of offspring per litter was reduced, but this reduction was alleviated by Baicalein (40 mg/kg) treatment . Furthermore, the birth weight of the offspring was increased in the GDM group; this effect was attenuated with Baicalein (40 mg/kg) administration . Thus, Baicalein positively influenced reproductive outcomes in GDM mice. Section title: Baicalein reduces inflammation and oxidative stress in GDM mice Educational score: 4.107527732849121 Domain: biomedical Document type: Study Language: en ELISA results showed that levels of IL-6, IL-1β, and TNF-α were elevated in GDM mice, but these increases were significantly reduced following Baicalein (40 mg/kg) treatment . Additionally, markers of oxidative stress, including MDA and MPO, were elevated, while SOD and GSH levels were decreased in GDM mice. Baicalein (40 mg/kg) treatment normalized these oxidative stress markers . Taken together, these findings indicate that Baicalein could effectively alleviate inflammation and oxidative stress in GDM mice. Section title: Baicalein attenuates NLRP3 inflammasome activation in placentae of GDM mice Educational score: 4.114445209503174 Domain: biomedical Document type: Study Language: en NLRP3 protein expression was elevated in the placentae of GDM mice, but this elevation was reduced following Baicalein (40 mg/kg) treatment . Similarly, levels of IL-1β and IL-18 were increased in GDM mice, but these increases were diminished after Baicalein (40 mg/kg) administration . Collectively, Baicalein decreased NLRP3 inflammasome activation in the placentae of GDM mice. Section title: Discussion Educational score: 4.128475666046143 Domain: biomedical Document type: Study Language: en Numerous extracts from Chinese herbs have been shown to influence the progression of gestational diabetes mellitus (GDM). For instance, Polyphyllin I modulates the AMPK pathway to reduce inflammatory damage in GDM . Similarly, Astragaloside IV alleviates placental inflammation and oxidative stress in GDM mice , and Resveratrol activates the AMPK pathway to improve GDM progression in mice . Baicalein, a flavonoid with diverse biological functions, has been implicated in the management of various diseases . However, its regulatory roles and mechanisms in the context of GDM have not been fully elucidated. This study demonstrates that Baicalein improves metabolic symptoms in GDM mice by enhancing glucose and insulin tolerance, and normalizing fasting blood glucose and plasma insulin levels. Additionally, Baicalein positively affected reproductive outcomes, including the number of offspring per litter and birth weight. Section title: Discussion Educational score: 4.120509624481201 Domain: biomedical Document type: Study Language: en Inflammation and oxidative stress play pivotal roles in the progression of GDM . Consequently, many researchers have concentrated on modulating these factors to slow the progression of GDM. For instance, cryptotanshinone has been shown to alleviate placental inflammation and oxidative stress in GDM mice . Similarly, N-acetyl-L-cysteine helps reduce both inflammation and oxidative stress associated with GDM . Additionally, miR-875-5p targets TXNRD1, thereby influencing oxidative stress and inflammation in GDM rats . Myrtenol has also been demonstrated to modulate the TLR4/MyD88/NF-κB pathway, thereby mitigating inflammation and oxidative stress in GDM rats . In alignment with these findings, our study observed that inflammation and oxidative stress were elevated in GDM mice. However, these effects were significantly mitigated following treatment with Baicalein (40 mg/kg), suggesting that Baicalein can also effectively alleviate inflammation and oxidative stress in the context of GDM. Section title: Discussion Educational score: 4.272754669189453 Domain: biomedical Document type: Study Language: en Inflammatory factors such as TNF-α, IL-6, and C-reactive protein are known to contribute to insulin resistance in GDM . The nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, a macromolecular complex consisting of NLRP3, caspase-1, and ASC, plays a critical role in this process . The interaction between caspase-1 and pathogen-associated molecular patterns or damage-associated molecular patterns activates the NLRP3 inflammasome, leading to the secretion of IL-1β and IL-18 from immune cells and promoting inflammation . Previous studies have shown that Baicalein can target the miR-17-5p-Mfn1/2-NF-κB pathway in trophoblast cells, reducing high glucose-induced inflammation and apoptosis . Despite these findings, the effects of Baicalein on the NLRP3 inflammasome in the context of GDM progression in vivo have not been well characterized. In this study, we observed that protein levels of NLRP3, IL-1β, and IL-18 were increased in GDM mice. However, these effects were attenuated following Baicalein treatment, indicating that Baicalein inhibits the NLRP3 inflammasome. Section title: Discussion Educational score: 4.01966667175293 Domain: biomedical Document type: Study Language: en In conclusion, Baicalein effectively inhibited the NLRP3 inflammasome and mitigated placental inflammation and oxidative stress associated with GDM in mice. Nevertheless, this study has certain limitations, including the need for further investigation using human samples, additional cell models, and a broader range of phenotypes. Future research should focus on these areas to provide a more comprehensive understanding of Baicalein’s effects and potential therapeutic applications. | Review | biomedical | en | 0.999995 |
PMC11699562 | Section title: Introduction Educational score: 4.603982925415039 Domain: biomedical Document type: Review Language: en TAU is a major microtubule-associated protein (MAP) in the human brain, where it contributes to microtubule (MT) stabilization and regulates MT dynamics. Normally, TAU is sorted and restricted mainly to the axon, where it is associated with MTs. In pathological conditions, it can dissociate from MTs, mislocalize into the somatodendritic compartment, and form insoluble aggregates, known as neurofibrillary tangles (NFTs). These NFTs are the hallmark lesions of tauopathies, which include Alzheimer’s disease (AD), frontotemporal dementia, atypical Parkinson’s syndromes like corticobasal degeneration, progressive supranuclear palsy, and other tauopathies . Missorted TAU leads to or coincides with the breakdown of the MT system, compromised axonal transport, synaptic dysfunction, and neurodegeneration . Much is known about the downstream effects of missorted TAU, but the upstream events that lead to TAU missorting or the mechanisms enabling physiological sorting under healthy conditions are poorly understood. Section title: Introduction Educational score: 4.314881324768066 Domain: biomedical Document type: Review Language: en In the case of AD, we know that the loss of TAU’s MT-stabilizing function and its downstream effects constitute a distinct cause leading to neurodegeneration, aside from the toxic gain-of-function of TAU aggregates . Besides AD, many other neurodegenerative diseases display the effects of a disrupted MT network – for example, intracellular protein aggregates disturb axonal transport in synucleinopathies, ALS or Huntington’s disease , and defects of motor proteins lead to intellectual disabilities . Moreover, mutations in MT-related genes cause defective cell migration, as seen in lissencephaly, or hinder MT-cytoskeletal rearrangements during neuronal development, like in autism spectrum disorders . Section title: Introduction Educational score: 4.325560569763184 Domain: biomedical Document type: Study Language: en Importantly, MTs in neurons have a particular organization that differs significantly from other cell types and also within the compartments of the neuron itself . The minus end is anchored in the MTOC, which is classically the centrosome in most dividing eukaryotic cells. In spherical somatic cells, e.g., fibroblasts or unpolarized neuronal progenitors, MTs would classically grow out centrifugally from a central centrosome. In postmitotic neurons, the centrosome loses its function as an MTOC during development, and instead, the MTs grow out from Golgi outposts in dendrites or by non-centrosomal MTOC proteins, e.g., on behalf of augmin from pre-existing MTs or from the MAP CAMSAP2 at the proximal axon . Section title: Introduction Educational score: 4.6357269287109375 Domain: biomedical Document type: Study Language: en Aside from atypical growth origins, MTs have particular features differing between axons and dendrites, including their orientation, stability, bundling, and chemical tubulin modifications . MTs in the axon are uniformly orientated with their plus end outwards, thus growing anterogradely from the soma, while MTs in the dendrite show a mixed polarity, with a varying fraction of anterograde and retrograde growing MTs depending on the species and neuronal cell type . In the dendrite, anterograde MTs make up a portion of approximately 65% in mice in vivo , 11% in Drosophila , 55% in hippocampal rat neurons , and 85% in human . The different orientation in axons and dendrites seems to be important for two mechanisms: (a) the uniform orientation of MTs in the axon is important for the axonal outgrowth and specification and (b) the different polarity of MTs directly drives polarized cargo transport. Kinesin-driven cargo is directed towards the plus end and targeted primarily to the axon, whereas dynein-driven vesicles drive toward the minus end, preferentially on dendritic MTs . This is important because the cargos themselves contain proteins that determine the axonal or dendritic fate, e.g., synaptic receptors for the axonal synapse. Section title: Introduction Educational score: 4.326060771942139 Domain: biomedical Document type: Study Language: en Neuronal MT dynamics are being studied i.a. in primary neurons derived from rodents, the standard model for mammalian neurobiology in vitro . Unfortunately, human disease is only partially recapitulated in rodents and rodent-based human disease models, and there are notable differences in gene and protein sequences and isoform composition of disease-associated players, which is in particular true for TAU . This makes it difficult to draw conclusions from rodent models, but primary human neurons are very rarely available (e.g., from abort material or from neurosurgically removed peripheral ganglia in case of injury) and unsuitable for research purposes. With biotechnological progress over the past two decades, inducible pluripotent stem cells (iPSCs) can now be differentiated into various types of human cells, including neurons and their numerous subtypes . Somatic cells are genetically reprogrammed into pluripotent iPSCs, which can be transformed into neuronal precursor cells and finally neurons, by exposing them to external morphogens – a method called “directed differentiation” – or via genetic fating usually achieved by induced expression of a neurotransmitter driving differentiation into specific neuronal subtypes . Section title: Introduction Educational score: 4.327314376831055 Domain: biomedical Document type: Study Language: en An alternative human neuronal cell system, besides induced pluripotent stem cell (iPSC)-derived neurons (iN), are secondary neuronal cell lines derived from neuronal tumors. An extensively used type is the human neuroblastoma cell line SH-SY5Y. Treatment of this cell line with several chemicals, including retinoic acid (RA), brain-derived neurotrophic factor (BDNF), neuronal growth factor, or others, results in the generation of excitatory neuronal cells . SH-SY5Y cells can be differentiated within 2–3 weeks in cultures showing neuronal morphology and neuronal markers . Undifferentiated SH-SY5Y neuroblastoma cells show catecholaminergic characteristics but during differentiation, they develop into neurons inheriting cholinergic, noradrenergic, and dopaminergic properties, of which all three neuronal subtypes are present in AD-affected loci . As pre-mitotic cells, SH-SY5Y cells undergo cell division and can provide a vast quantity of neuronal cells for experimental arrays. Their differentiation is less time- and cost-consuming than those of iN and yields cultures of higher homogeneity and robustness. Section title: Introduction Educational score: 4.409574031829834 Domain: biomedical Document type: Study Language: en Here, we set out to test MT dynamics in iN and SH-SY5Y-derived neurons (SHN) and in different neuronal subcompartments. We first tested proper neuronal compartmentalization, i.e., axodendritic development, axon-initial-segment (AIS) establishment, and polarized sorting of TAU and MAP2. We compared the efficiency of axonal sorting of TAU and MAP2, and expression of the AIS-marker proteins AnkG and TRIM46 in iN to SHN and the standard model of neurobiology, mouse primary neurons (mPN). We found that iN and SHN showed remarkably similar TAU sorting properties, which were also comparable to mPN, but SHN lacked expression of AnkG and showed reduced expression of TRIM46. We then investigated MT dynamics via EB3-live-imaging and found that i.a. MTs are orientated anterogradely in axons of both iN and SHN, but more retrogradely in iN dendrites. MT polymerization speed was much alike in the three subcompartments of SHN and iN, whereas in SHN, we observed generally more growth events. Conclusively, overall cell polarization and neuronal compartmentalization do take place in SHN and iN, but only iN express and enrich AnkG and TRIM46 at the AIS and their MT-dynamics appear more reminiscent of mature neurons. Section title: Cultivation Educational score: 4.087120056152344 Domain: biomedical Document type: Study Language: en Both SH-SY5Y cells and iPSCs followed a differentiation protocol into neuronal cell cultures with a duration of 2–3 weeks. Both cell models were cultivated in 6-well, 12-, and 24-well plates (VWR) or in imaging dishes (ibidi). Well plates have partly been equipped with coverslips (VWR) for microscopy experiments. If not stated otherwise, the amount of solutions and media was 2 ml for a well of a six-well plate, 1 ml for a well of a 12-well plate and 0.5 ml for a well of a 24-well plate. Media reagents were prewarmed at 37°C and cells were kept in a humidified incubator at 37°C with 5% CO 2 . Section title: SY-SY5Y cultivation Educational score: 4.14695930480957 Domain: biomedical Document type: Study Language: en Undifferentiated SH-SY5Y cells were cultivated in DMEM/F-12 GlutaMAX™ supplemented with 10% fetal bovine serum (FBS, Biochrom AG), and Antibiotic–Antimycotic (referred to as SHM-10 medium) in uncoated T75 flasks (VWR). SHM-10 medium was changed twice per week and SH-SY5Y cells were passaged when reaching 80% confluence. For passaging cells were washed with phosphate-buffered saline (PBS, TFS), trypsinized (0.05% trypsin/EDTA, TFS) for 3–5 min, and centrifuged for 3 min at 1,000 × g . The cell pellet was re-suspended in 10 ml SHM-10 medium, diluted to 1:10, and seeded for further cultivation. Section title: SY-SY5Y cultivation Educational score: 4.099933624267578 Domain: biomedical Document type: Study Language: en For differentiation, SH-SY5Y was seeded onto plates or imaging dishes, previously coated with 20 µg/ml poly- d -lysine in PBS. The PDL coating solution was added to the well plates and incubated for at least 3 h at 37°C. The coating solution was then discarded and wells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS). For imaging experiments, glass coverslips (VWR) were placed into the wells (25 mm for a 6-well plate, 12 mm for a 24-well plate) before adding the coating solution. Seeding density was 8,000 or 5,500 cells/cm 2 when seeding one, respectively, 2 days prior to the start of differentiation. Section title: SH-SY5Y differentiation Educational score: 4.177577972412109 Domain: biomedical Document type: Study Language: en SH-SY5Y cells were differentiated into SHN within 2 weeks performing sequential medium exchanges with the supplements RA and BDNF. SHM-10 medium was exchanged to SHM-10 with 10 μM of RA on days 0, 3, and 5. On day 7 of differentiation, SHM-10 + RA (Sigma-Aldrich) medium was removed, cells were washed once with DPBS and SHM-10 medium without FBS (referred to as “SHM-0”), supplemented with 10 ng/ml BDNF (Peprotech) was added. On day 10, another medium exchange with SHM-0 + BDNF was carried out. On day 14, cell medium was again changed to SHM-10 without supplements. Differentiation was finished at this point with a differentiation efficiency of about 75% and SHN could be used for further experiments. Cultivation continued in SHM-10 medium with medium exchange every 3 days. Section title: Human iPSC (hiPSC)-derived neurons Educational score: 4.046017646789551 Domain: biomedical Document type: Study Language: en hiPSCs were transformed into cultures of cortical glutamatergic neurons following a differentiation protocol, adapted from . The used iPSC cell line WTC11 carries a Neurogenin 2 (Ngn2) transgene, inducible by doxycycline, a transcription factor that rapidly converts iPSCs into neurons (for detailed protocol including troubleshooting guide, see ). Section title: iPSC cultivation Educational score: 4.2103657722473145 Domain: biomedical Document type: Study Language: en For routine cultivation, undifferentiated iPSC colonies were grown in cBrew with medium exchanges every 2–3 days on Geltrex-coated (TFS) 6-well plates. Plates were coated on the day of use with 200 μg/ml Geltrex for 30 min at 37°C. Habitually, certain iPSCs differentiated spontaneously before starting induced differentiation and were hence removed manually with a P200 pipette under a sterile hood. iPSCs were passaged when they reached a confluence of about 80%. For passaging, iPSCs were washed with PBS and dissociated with Versene (TFS) for 3–5 min. Versene was aspirated and 1 ml of cBrew was added. Then, the colonies were carefully detached with a cell scraper and the cell suspension was transferred to a 15 ml tube (Falcon). Cell clumps within the suspension were dissolved by trituration with a 5 ml Pasteur pipette. The cell suspension was diluted in cBrew to a routine passaging ratio of about 1:10. Additionally, Thiazovivin (Axon Med Chem) was added in a concentration of 1:5,000 to the cell suspension, which was then distributed to the culture wells. On the next day, the medium was changed to fresh cBrew without Thiazovivin. Cells would typically reach 80% confluency again after 3–4 days. Section title: Differentiation of iPSCs into iN Educational score: 4.18367862701416 Domain: biomedical Document type: Study Language: en iPSCs were transformed into cortical glutamatergic neuronal cultures following a 3-day pre-differentiation (d3 to d0), followed by a multiple-week-long differentiation (d0 to d21 or further) protocol. Three days before starting differentiation (d3), iPSCs were passaged onto Geltrex-coated plates. Cells were washed once with DPBS and then dissociated with Accutase (Merck). Cells were incubated in Accutase for 5–8 min at 37°C. Dissociation was stopped by adding 3 ml of DPBS, then cells were collected in a 15 ml tube (Falcon) and centrifuged for 5 min at 400 × g . The supernatant was discarded and the cell pellet was re-suspended in 1 ml of cBrew. iPSCs were diluted in a pre-differentiation medium, supplemented with Thiazovivin (1:5,000), and seeded in a density of 1.5–2 × 10 5 cells/cm 2 . The medium was changed on days 2 and 1 to a fresh pre-differentiation medium without Thiazovivin. Section title: Differentiation of iPSCs into iN Educational score: 4.108554840087891 Domain: biomedical Document type: Study Language: en For differentiation (d0), pre-differentiating iPSCs were passaged onto PDL/Cultrex-coated plates. Plates were first coated with 20 μg/ml PDL in DBPS 1 day before the start of differentiation (d–1), incubated overnight at 37°C, washed once with DBPS, and then coated with 20 μg/ml Cultrex in DPBS for another hour (d0). Plates were used within 24 h and washed twice with DPBS before seeding the iPSCs. Section title: Differentiation of iPSCs into iN Educational score: 4.140105724334717 Domain: biomedical Document type: Study Language: en For seeding, dissociation with Accutase, collection, and centrifugation were performed as described during the pre-differentiation, except for re-suspending the cell pellet in 1 ml of neuronal maturation medium (NMM), instead of using cBrew. Cells were diluted in NMM containing freshly added Geltrex (1:100) to reach a density of 0.5–0.8 × 10 6 cells/well and 2.5–4 × 10 6 cells/well in a 24-well plate and 6-well plate or imaging dish, respectively. Half of the amount of NMM was exchanged every weeks (d7, d14, d21) while iPSCs differentiated progressively into iN. Section title: Media recipes/medium ingredients Educational score: 3.3840880393981934 Domain: biomedical Document type: Other Language: it Pre-differentiation medium: KO DMEM/F-12 1× neuropan-2 supplement 1× non-essential amino acids 10 ng/ml BDNF 10 ng/ml NT-3 1.5 μg/ml laminin 2 μg/ml doxycycline 1× Antibiotic–antimycotic solution Section title: Media recipes/medium ingredients Educational score: 3.124795913696289 Domain: biomedical Document type: Other Language: it NMM: 50% neurobasal medium 50% DMEM/F-12 0.5× neuropan-2 supplement 1× non-essential amino acids 0.5× B27 supplement 10 ng/ml BDNF 10 ng/ml NT-3 1.5 μg/ml laminin 2 μg/ml doxycycline 1× antibiotic–antimycotic solution Section title: Transfection Educational score: 4.141336917877197 Domain: biomedical Document type: Study Language: en SHN were transfected using the polymer-based PolyJet DNA transfection reagent (SignaGen) at d7 of differentiation. Transfection was performed according to the manufacturer’s protocol with the following modifications: prior to transfection, 1/2 of the conditioned medium was collected from each well and stored at 37°C with 5% CO 2 . For 1 well of a 24-well plate, 0.33 μg of plasmid DNA and 1 μl PolyJet reagent were separately mixed with 25 ml DMEM each, then joined, incubated for 10–15 min, and added dropwise to the culture medium. After 3 h, cells were washed once with DMEM and then cultivated in a previously collected medium filled up with fresh SHM-10. The transfection efficiency ranged widely depending on the used plasmid between 5 and 20%. Section title: Transfection Educational score: 4.1198344230651855 Domain: biomedical Document type: Study Language: en iN were transfected using the LipoStem transfection reagent (TFS) 2–4 days before experimental use, according to the manufacturer’s protocol. Prior to transfection, 1/2 of the conditioned medium was collected from each well and stored at 37°C with 5% CO 2 . For 1 well of a 24-well plate, 0.5 μg plasmid DNA was mixed with 25 μl of Opti-MEM (mix A) and 0.5 μl LipoStem was mixed with 25 µl DMEM (mix B). Mixes A and B were joined, incubated for 10 min, and added dropwise to the cell culture. After 24 h, cells were washed with warm KO-DMEM and cultivated in a previously collected medium filled up with fresh NMM. Section title: Immunofluorescence experiments Educational score: 4.144371032714844 Domain: biomedical Document type: Study Language: en Cells were fixed with 3.7% formaldehyde (FA) in PBS. After incubation for 18 min, cells were stored in 55% (v/v) glycerol in PBS (storage solution) at −20°C for later analysis or washed three times with PBS for direct staining. For immunofluorescence staining, FA or storage solution was aspired and cells were washed three times in PBS. Cells were permeabilized and blocked in 5% BSA (Carl Roth) and 0.2% Triton X-100 (Carl Roth) in PBS for 10 min. Afterward, cells were washed once in PBS and then stained with primary antibodies at 4°C overnight. On the following day, coverslips were washed three times in PBS and incubated with the secondary antibody for 3 h. Finally, coverslips were washed three times with PBS and stained with Hoechst 33342 (NucBlue™, TFS) in PBS for another 25 min at room temperature. Cells were washed twice with deionized water and then mounted on objective slides with a Poly-Mount mounting medium (Polysciences). Mounted slides were dried for at least 24 h before imaging and kept at 4°C for long-term storage. Section title: Immunofluorescence experiments Educational score: 1.7587894201278687 Domain: biomedical Document type: Other Language: en Antibodies used were Rabbit Anti-TRIM46 (Synaptic System), Mouse Anti-Ankyrin G (Neuromab), Chicken Anti-MAP2 (Abcam), and Rabbit Anti-TAU (SantaCruz). Section title: Fluorescence microscopy Educational score: 4.063072681427002 Domain: biomedical Document type: Study Language: en Microscopy images were taken with a widefield fluorescence microscope (Zeiss), equipped with an LED lamp (Colibri 7, Zeiss) and a fluorescence camera (Axiocam 503 mono, Zeiss). Images were acquired using the Zen imaging software (Blue pro, Zeiss). Images were taken with magnifications of 200×, 400×, or 630×, using 10×, 20×, and 40× objectives air based and the 63× objective with immersion oil (Immersol 518F, Zeiss). Exposure time and laser power were adjusted so that the fluorescence signal was not oversaturated. Within the same experiment, all images were taken with identical LED intensity and exposure time to ensure statistical comparability of the protein expression levels. Section title: Live cell imaging Educational score: 4.12900447845459 Domain: biomedical Document type: Study Language: en For investigation of MT dynamics by live imaging, SHN and iN were transfected with a plasmid expressing tdTomato-tagged EB3 (ptdTomato-EB3) following the transfection protocol described above and before . SY5Y-cells were transfected on day 7 while iN were transfected on days 12–14 post-differentiation. Generally, the neurons expressed the EB3-tdTomato 2–3 days after transfection. The movement of fluorescently tagged EB3 particles was recorded with a Leica DMi8 S Platform microscope equipped with a Leica-DFC9000 fluorescence camera and the image acquisition software Las X Life Sciences (Leica Microsystems). The cells were continuously covered with a CO 2 incubation unit and a microscope chamber to protect the cells from external light radiation and were left to accommodate 15 min before imaging. The time-lapse video was composed of images taken at a rate of 1 image per 2 s over a period of 2 min (for in-depth protocols, see also ). Section title: qPCR Educational score: 4.120550155639648 Domain: biomedical Document type: Study Language: en A quantitative RT-qPCR was performed to assess the expression of TRIM46 and AnkG in different cell types. Cells were harvested and lysed and total RNA was isolated using PureLink RNA Mini Kit (TFS). Reverse transcription into cDNA was carried out with ProtoScript II First Strand cDNA Synthesis Kit (NEB). Quantitative RT-qPCR was performed with SYBR green I (TFS) on the following cycling protocol: denaturation at 95°C for 10 s, annealing at 58.5°C for 30 s, and elongation at 68°C for 60 s, 30 cycles. The HPRT gene was used as a reference gene for relative quantification. Used primers were the following: TRIM46 Primer 1 (GCAGCTGCACAACAGGATTG) and Primer 2 (ATCATAGGCAAAGGTGCGCT); AnkG Primer 1 (GTCTGAGCAAAAGCAGGGAGA) and Primer 2 (ACCGTTCGCTGTTACGAGTG). Section title: EB3 MT analysis Educational score: 4.154782295227051 Domain: biomedical Document type: Study Language: en The acquired movie files were registered and exported with LasX Image acquisition software. The movie file was then analyzed with ImageJ/Fiji software (OpenSource) as follows: a 30 μm line region of interest (ROI) was drawn along the proximal axon and the dendrite, respectively, and a 50–200 μm line ROI along the distal axon (at least 400 μm away from soma) with the segmented line tool. The ImageJ plugin Kymograph Reslice Wide generated kymographs from the EB3 comets moving along this linear ROI, which were then read out for (comet number/30 μm/min), comet direction (anterograde/retrograde), and comet speed (μm/min). The comet speed was calculated from the gradient of the comet track in the kymograph. All experiments were conducted in 3 experimental replicates of 20–60 cells each. To compare EB3 dynamics in the different neuronal compartments, it was essential to distinguish axons from dendrites. Axons were distinguished from dendrites using established morphological characteristics (Bell et al., ; Tjiang and Zempel, ; Zempel et al., ), e.g., a constant small diameter, longer outgrowth than dendrites (>300 μm), and branching pattern of 90°. In case of uncertain morphological discrimination, we fixed the neurons after live imaging, re-identified the imaged cells on the gridded imaging chamber, and stained for TAU, MAP2, and AnkG/TRIM46 to ascertain that we analyzed/imaged the appropriate neuronal subcompartments . Section title: Axonal enrichment factor (AEF) Educational score: 4.345224380493164 Domain: biomedical Document type: Study Language: en To quantify the enrichment of TAU and MAP2 in the axon, relative to the soma, we calculated an AEF (AEF Tau , AEF MAP2 ). For both proteins, we measured the mean fluorescent intensity (MFI) in ROIs drawn in the soma (MFI S ), the axon (MFI A ) (>100 µm distal from the AIS), as well as in the empty space next to the soma (MFI bgS ) and axon (MFI bgA ), to normalize for background noise. The ROI in the soma was drawn, so it would not overlap with the nuclear signal. We subtracted MFI S – MFI bgS (=MFI Soma ) and MFI A –FI bgA (=MFI Axon ) to exclude background fluorescent noise. Next, an axon-to-soma ratio was calculated, for TAU and MAP2 each (MFI Axon /MFI Soma ). Finally, to account for volume bias in the fluorescent intensities, we normalized the TAU/MAP2 axon-to-soma ratios to the axon-to-soma ratio of the randomly distributed volume markers tdTomato or GFP e.g., AEF Tau = ( MFI Axon / MFI Soma ) Tau ( MFI Axon / MFI Soma ) tdTomato \documentclass[10pt]{article}\usepackage{wasysym}\usepackage[substack]{amsmath}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{amsbsy}\usepackage[mathscr]{eucal}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\[\left(\phantom{\rule[-0.75em]{}{0ex}},\text{e}\text{.g}\text{.,}\hspace{.25em}{\text{AEF}}_{\text{Tau}}=\frac{{({\text{MFI}}_{\text{Axon}}/{\text{MFI}}_{\text{Soma}})}_{\text{Tau}}}{{({\text{MFI}}_{\text{Axon}}/{\text{MFI}}_{\text{Soma}})}_{\text{tdTomato}}}\right)]\]\end{document} . ROIs for tdTomato, GFP, MAP2, and TAU were identical. Quantitative data for AEF of SHN from Figure 1b (fourth bar of quantification) was obtained from Michael Bell-Simons from Bell et al. and replotted for comparison. Section title: AnkG/TRIM46 enrichment factor Educational score: 4.113954544067383 Domain: biomedical Document type: Study Language: en To quantify the localized enrichment of TRIM6 and AnkG (AIS-EF TRIM46 , AIS-EF AnkG ) at the beginning of the axon, a ratio between the peak fluorescence intensity (all values >70% of maximum value) and the baseline fluorescence intensities along the AIS (first 80 μm) was calculated. The peak intensity ( y ) was determined as the mean value of all values greater than 70% of the maximum fluorescence signal, along the peak zone of a certain length ( x ). The baseline intensity was determined as the mean value of all fluorescence intensities over the same distance of ( x ) μm at the end of the 80 μm ROI. AIS enrichment factor (AIS EF) was calculated from three experimental replicates for all cell types. Quantitative data for AIS plot profile and AIS enrichment factor of SHN and mPN were obtained from Michael Bell-Simons from Bell et al. and replotted for comparison. Section title: Successful establishment of neuronal cell polarity and TAU distribution in human neurons in vitro Educational score: 4.139405250549316 Domain: biomedical Document type: Study Language: en In AD and related neurodegenerative tauopathies (neurodegenerative diseases [NDD]/tauopathy), cell polarity is impaired, reflected by TAU mislocalization into the somatodendritic compartment . To have a better understanding of the cell culture systems, we first tested which human neuronal cell model is able to recapitulate neuronal cell polarity in terms of successful axonal targeting of TAU and somatodendritic retention of MAP2 similar to primary rodent neurons. Section title: Successful establishment of neuronal cell polarity and TAU distribution in human neurons in vitro Educational score: 4.13861608505249 Domain: biomedical Document type: Study Language: en We differentiated SH-SY5Y cells to neurons (SHN) using our established protocol, which resulted in a relatively high yield of differentiated cells (∼75%), successful axodendritic polarization, and convincing TAU enrichment in the axon after 14 days of differentiation using RA and BDNF, as detailed previously . We also differentiated our iN as established previously using doxycycline-dependent expression of Ngn2, resulting in a differentiation efficiency of >90%, axodendritic polarization, and successful TAU sorting . Finally, we used the established standard model of mammalian neurobiology, mouse primary forebrain neurons, which shows excellent axonal targeting of endogenous TAU and even allows to discriminate subtle differences of axodendritic targeting of, e.g., different isoforms of pseudophosphorylated TAU . Section title: Successful establishment of neuronal cell polarity and TAU distribution in human neurons in vitro Educational score: 4.408973217010498 Domain: biomedical Document type: Study Language: en We found that all neuron types investigated, after sufficient differentiation as appropriate and necessary (i.e., 14 days for human neurons, >7 days for murine neurons, for details, see ), developed axons and dendrites. When stained for the axonal marker TAU and the somatodendritic marker MAP2 and subsequently imaged via immunofluorescence microscopy (IF), we found that TAU was strongly enriched in the axons, but not MAP2, which remained largely in the soma and the dendrites . Morphologically, dendrites were larger, both in diameter and length, in iN and primary neurons compared to SHN . Quantification of the AEF, a measure of enrichment of a protein in the axon compared to the soma (see Section 2 for details) revealed that in all cell types, MAP2 is only slightly enriched in the axon compared to the soma, while TAU is distinctly enriched in the axon. Axonal TAU enrichment is strongest in iN and increases during maturation from ∼5-fold at day 7 after differentiation (d7) to ∼18-fold at d21. Axonal TAU enrichment is equally strong in mPN (∼15-fold) and moderately strong in SHN (∼9-fold). Nonetheless, this means that both SHN and iN show strong axonal enrichment of TAU, but not of MAP2, comparable to murine primary neurons, indicative of successful axodendritic development and establishment of neuronal cell polarity. Section title: Pronounced enrichment of AnkG and TRIM46 in iN, but not in SH-SY5Y neurons Educational score: 4.231646537780762 Domain: biomedical Document type: Study Language: en Next, we compared the AIS development in three neuronal cell systems: primary mouse neurons, iN, and SHN. We conducted AnkG and TRIM46 immunostainings and measured the fluorescent intensities, localization, and local enrichment using IF and calculation of the AIS enrichment factor (AIS-EF), as well as their mRNA amounts via qPCR. Both iN and primary neurons showed pronounced expression of AnkG as well as TRIM46 along the proximal axon and displayed the typical morphology of an AIS in IF . The length and location of their AIS, situated approximately 5–40 μm distal to the soma in the proximal axon, were identical: The proximal enrichment of AnkG (mPN: 9.7-fold, iPSC: 7.3-fold) and TRIM46 (mPN: 9.1-fold, iN: 7.8-fold) was pronounced in both mPN of age DIV9 and in iN at day 17 of differentiation. Moreover, the maximal AnkG and TRIM46 fluorescence were situated ±10 μm distally from the soma and had the same spatial distance of ±2 μm from each other in both cell models . In contrast, IF showed no immuno-labeling of AnkG in SHN. Nevertheless, TRIM46 was expressed in this cell line after differentiation, however, relatively moderate in comparison to iN. Moreover, the local enrichment of TRIM46 (1.6-fold) was much less pronounced than in iN (7.8-fold) and primary neurons (9.1-fold) . Secondary antibody control showed no unspecific binding . Section title: Pronounced enrichment of AnkG and TRIM46 in iN, but not in SH-SY5Y neurons Educational score: 4.12507963180542 Domain: biomedical Document type: Study Language: en To confirm the lower expression level of the AIS-related proteins in SHN, we conducted qPCR to assess the mRNA levels of TRIM46 and AnkG in iN, differentiated SHN, undifferentiated naive SH-SY5Y cells, and HEK cells. The HEK cells served as a negative control for qPCR since no expression of AIS proteins is expected in these cells derived from human embryonic kidney cells. In line with the findings from the IF, there was no expression of AnkG in undifferentiated, naive SH-SY5Y cells (<1% compared to iN). But notably, also AnkG mRNA levels in differentiated SHN were negligible (<1% compared to iN). However, we detected expression of TRIM46 in SHN, which augmented from 10.7% (naive) to 39.9% (differentiated neurons, both values compared to iN) during their differentiation, which is consistent with the moderate signal of TRIM46 in the immunostainings. As expected, we found no expression (<1% of iN) of neither AnkG nor TRIM46 in HEK cells ( n = 1–2 experiments). Section title: Pronounced enrichment of AnkG and TRIM46 in iN, but not in SH-SY5Y neurons Educational score: 4.1173481941223145 Domain: biomedical Document type: Study Language: en Taken together, these data demonstrate that iN develop an AIS, morphologically alike of that in primary mouse neurons. On the other hand, SHN only express TRIM46 in the axon upon neuronal differentiation but lack AnkG, which likely results in a differently organized AIS structure. Section title: Anterograde MT orientation in axons of both iN and SHN, more retrograde MT orientation in iN dendrites, growth events in SHN Educational score: 3.9814343452453613 Domain: biomedical Document type: Study Language: en After observing the absence of a classic AIS, in terms of no detected AnkG expression and low TRIM46 levels, in SHN compared to iN, we investigated if this influences the MT dynamics, which are of great importance for neuronal polarization as well as the pathophysiology of AD and NDD in humans. Section title: Anterograde MT orientation in axons of both iN and SHN, more retrograde MT orientation in iN dendrites, growth events in SHN Educational score: 4.261770248413086 Domain: biomedical Document type: Study Language: en To investigate the dynamics and orientation of MTs in different compartments and cell types, we transfected both our human SHN and human iN with tdTomato-tagged EB3 on day 7, respectively, days 12–14 (Seq. S1). The protein EB3 binds to the plus tips of growing MTs and, when conjugated with a fluorescent protein like tdTomato, enables live-tracing of MT growth events on MT plus ends . These moving EB3 accumulations appear as comet-like structures in the time-lapse recordings, whose direction, speed, and quantity can be read out from kymographs, generated from the time-lapse videos (Movie S1). Kymographs are imaging tools that visualize dynamic processes in biological systems by capturing time-lapse images along a specified axis. Each pixel represents a position and time, creating a two-dimensional graph where the x -axis denotes space and the y -axis indicates time. This technique is particularly effective for studying the movement of cellular structures along cytoskeletal elements, revealing insights into transport mechanisms and motility. We recorded and analyzed EB3 movements in the proximal axon, distal axon, and dendrites of the neurons on days 14–18 of differentiation when cell polarity is well-established. We used morphological characteristics and/or post-imaging immunostaining to distinguish axons from dendrites (see Section 2.9.1 ). While in both cell types identification of the axon is relatively simple (as the axon is smaller and constant in diameter, there are different branching patterns of axons and dendrites, and very different lengths of axons and dendrites as described before , we nonetheless fixed the cells, re-identified the imaged cells, and stained for TAU, MAP2, and ANKG/TRIM46 in initial experiments to ascertain that we analyzed/imaged the appropriate neuronal subcompartments . Section title: Anterograde MT orientation in axons of both iN and SHN, more retrograde MT orientation in iN dendrites, growth events in SHN Educational score: 4.258932113647461 Domain: biomedical Document type: Study Language: en Counting the EB3 comets, we found that in all cell compartments and in both cell types, the majority of MTs was orientated anterogradely (thus with the growing plus end directed to the periphery), represented by EB3 moving outwards from the soma; however, there were notable differences: In SHN, the proportion of anterograde comets was equally high in the proximal axon (94.9% ± 2.0) and distal axon (95.8% ± 0.8), whereas the proportion of anterograde comets was lower in the dendrites (79.8% ± 3.6). Consequently, the percentage of retrograde comets was ∼4–5 times higher in the dendrites (20.2% ± 3.6) than in the axonal compartments (proximal: 5.1% ± 2.0, distal: 4.2 ± 0.8%). A similar distribution was observed in iN, where anterograde comets accounted for 89.0% ± 4.1 in the proximal and 94.3% ± 1.3 in the distal axon of all moving comets. However, the proportion of anterograde comets in the dendrites (72.4 ± 3.4%) was even lower than in the dendrites of SHN, resulting in a significantly higher portion of retrograde comets (27.6% ± 4.7) compared to the other compartments and cell types . Section title: Anterograde MT orientation in axons of both iN and SHN, more retrograde MT orientation in iN dendrites, growth events in SHN Educational score: 4.150789737701416 Domain: biomedical Document type: Study Language: en We also quantified the number of moving comets (comet number/30 μm/min), representing the amount of dynamic MTs and thus the overall growth dynamics . Generally, in both cell models, the highest amount of total moving comets was observed in the dendrites (SHN: 6.0 ± 0.4, iN: 6.1 ± 2.6), followed by those in the proximal axon (SHN: 4.8 ± 1.0, iN: 4.3 ± 1.5) whereas the number of moving comets in the distal axon was significantly lower (SHN: 3.1 ± 0.7, iN: 1.9 ± 0.6). Strikingly, the number of moving comets in the SHN exceeded those in the iN in all three compartments for anterogradely moving comets, but not for retrogradely moving comets. Section title: Anterograde MT orientation in axons of both iN and SHN, more retrograde MT orientation in iN dendrites, growth events in SHN Educational score: 4.170408725738525 Domain: biomedical Document type: Study Language: en Finally, we analyzed the speed of the moving comets from the gradient of the comet traces within the kymographs . The average speed of all moving comets was 12.9 ± 1.1 μm/min in SHN and 12.1 ± 1.1 μm/min in iN. Broadly, the average speed was similar between the two cell types and also between the different compartments, with differences of only 1–2 μm/min. In the SHN, comets moved fastest in the distal axon (14.1 ± 2.1 μm/min), whereas comets of iN had the highest speed (13.9 ± 1.1 μm/min) in the dendrites. Interestingly, the speed of anterograde comets was always ∼1–2 µm/min higher than the retrograde ones in both cell types with the exception of the dendritic compartment in iN, where retrograde comets moved faster than the anterograde ones. However, the differences appeared overall marginal and not statistically significant. Section title: Anterograde MT orientation in axons of both iN and SHN, more retrograde MT orientation in iN dendrites, growth events in SHN Educational score: 4.023743152618408 Domain: biomedical Document type: Study Language: en In summary, MTs in SHN and iN behaved remarkably similarly regarding orientation, dynamics, and speed of growth, but with a higher proportion of retrogradely moving comets particularly in iN dendrites. Section title: Neurons acquire axon formation and polarized TAU sorting without AnkG-organized AIS Educational score: 4.137617588043213 Domain: biomedical Document type: Study Language: en In this study, we set out to evaluate whether and which type of polarized human neurons would be a suitable cell model to investigate compartment-specific MT dynamics, in order to test human model systems also for future studies in the context of axodendritic TAU (mis-)sorting, AD, and associated tauopathies/neurodegeneration (NDD). We used the SH-SY5Y cell line and neurons derived thereof (SHN), which are cost-efficient and which we had judged to be potentially suitable for TAU-based research , and iPSC that can be differentiated via doxycycline-induced expression of Ngn2 into forebrain neurons (iN), which are also valuable for studying, e.g., neuronal activity in disease settings, all of which we have described previously . We used as a reference the standard model of cellular neurobiology, mouse primary neurons (mPN) . Section title: Neurons acquire axon formation and polarized TAU sorting without AnkG-organized AIS Educational score: 4.175382614135742 Domain: biomedical Document type: Study Language: en We assessed if the three neuronal models acquired neuronal polarity, which includes the structural separation of axons and dendrites with compartment-specific enrichment of MAP2 in dendrites and TAU in axons . Our findings indicate that both SHN and iN models show strong axonal enrichment of TAU, comparable to murine primary neurons. Specifically, axonal TAU enrichment was strongest in iN, increasing from approximately 5-fold at day 7 to 18-fold at day 21 post-differentiation. This enrichment was equally strong in mPN (∼15-fold) and moderately strong in SHN (∼9-fold). These results suggest that both SHN and iN are suitable models for studying TAU sorting and neuronal polarity, with iN showing the highest degree of TAU enrichment. Section title: Neurons acquire axon formation and polarized TAU sorting without AnkG-organized AIS Educational score: 4.29850435256958 Domain: biomedical Document type: Study Language: en Strikingly, architectural properties of axons and dendrites were obtained and axonal outgrowth occurred extensively not only in the AIS-positive iN and primary neurons but also in the AnkG-deficient SHN. Additionally, immunostainings for MAP2 and TAU revealed a clearly polarized distribution of the two proteins in all cell types. We also compared AIS development in iN, primary mouse neurons, and SHN, whereas the AIS composition was much alike in primary neurons and iN, we found that SHN did not express AnkG, but only TRIM46 in moderate amounts . Importantly, TRIM46 expression augmented during differentiation, which is in line with its reported role in initial neuronal polarization . AnkG was reported to maintain neuronal polarity and gate the axonal identity , but SHN showed both axon formation as well as polarized protein sorting despite AnkG deficiency. This is in line with AnkG as the sole actor governing the axonal identity being questioned in various studies . So what is the interaction and the role of TRIM46 and AnkG? Section title: Functional crosstalk between AnkG and TRIM46 Educational score: 4.524137496948242 Domain: biomedical Document type: Study Language: en We found that while AnkG and TRIM46 are both involved in establishing neuronal polarity, they play distinct roles. TRIM46 appears to act earlier to initiate axon formation and polarized protein sorting, while AnkG is important for maintaining the axon-dendrite separation later in development . Several lines of evidence suggest that TRIM46 can localize and function independently of AnkG at the axon initial segment (AIS): TRIM46 clusters at the proximal axon earlier than AnkG during development, with 70% of neurites in stage 2 neurons being TRIM46+/AnkG− . In dorsal root ganglion (DRG) neurons that lack AnkG, TRIM46 is still confined to the proximal axon. AnkG knockdown in young hippocampal neurons caused only moderate mislocalization of TRIM46 from the proximal axon . Treating mature hippocampal neurons with AnkG shRNA for over a week reduced TRIM46 levels by only 30% . This suggests that TRIM46 may be recruited to the AIS by binding partners other than known AIS components like ankyrin, spectrins, or sodium channels. The data indicate that TRIM46 can autonomously localize to establish the initial MT organization and polarity at the nascent AIS, independent of AnkG. Section title: Functional crosstalk between AnkG and TRIM46 Educational score: 4.231680870056152 Domain: biomedical Document type: Study Language: en A key finding is that axonal TAU enrichment correlates with the uniform MT orientation established by TRIM46, even in AnkG-deficient SHN. This could suggest that TAU axonal sorting depends on the MT lattice organization rather than a physical diffusion barrier at the AIS, as proposed previously . Yet, TRIM46 may be sufficient to generate this polarized MT array conducive for axonal TAU sorting, independent of AnkG. Section title: MT dynamics and impact of MT orientation on TAU sorting Educational score: 4.542939186096191 Domain: biomedical Document type: Study Language: en MT dynamics, crucial for neuronal polarization and function, were investigated using EB3-comet tracking. Both SHN and iN showed an axodendritic polarity in MT orientation: while MTs were orientated anterogradely in 90–95% in axons of both SHN and iN, dendrites showed MTs of mixed polarity with only 70% (iN) to 80% (SHN) of MT plus-ends directed anterogradely. Importantly, this polarized organization occurred independently of AnkG, which is absent in SHN, supporting the hypothesis that TRIM46 is sufficient to establish axonal MT polarity . The extent of MT reorientation from mixed/multipolar in stage 2 neurons to uniform in stage 3 axons matched previous reports in rodent neurons . Recent investigations in iN displayed a similar trend: in unpolarized stage 2 neurons, 80% of MTs had anterograde orientation which increased to 90% in axons of stage 3 neurons. The fraction of plus-end-out MTs in the dendrites decreased from 80% to 60% in stage 3 neurons . The higher proportion of retrograde dendritic MTs in iN in comparison to SHN could thus hint towards a higher grade of maturity, consistent with structural differences between the two neuronal cultures. Nevertheless, both SH-SY5Y and iN show the MT cytoskeleton of a polarized neuron. Section title: MT dynamics and impact of MT orientation on TAU sorting Educational score: 4.536553382873535 Domain: biomedical Document type: Study Language: en Can we now hypothesize that axonal TAU sorting depends on uniform MT orientation in the axon? Indeed, polarized protein sorting correlated with MT directionality in iN and SHN. In both neurons, MAP2 accumulated in regions with mixed MT polarity, whereas TAU was enriched in axons with unidirectional MTs. Consistently, TAU was distributed evenly throughout unpolarized stage 2 iN (correlating well to our d7 iNs), which have multipolar MTs in their developing neurites . Hence, axonal MT orientation may drive anterograde TAU mobility, e.g., as suggested previously by kinesin motors . Additionally, MT properties – orientation, PTM, spacing – in the AIS may enforce TAU binding and thereby a diffusional gradient of soluble TAU from the soma to the axon . Contrarily, MAP2 might be unable to couple to axonal MT motors, as described for other somatodendritic cargo , and therefore be hindered to drive along axonal MTs. It is thus possible that differential MT orientation in axons and dendrites drives polarized protein sorting by selective acquisition of motors. These results would be compatible both with TAU being simply an MT binder, but also with a functional role of TAU modifying MT liability as proposed previously . Section title: Differences in MT dynamics in different model systems Educational score: 4.509058475494385 Domain: biomedical Document type: Study Language: en Interestingly, SHN exhibited more growth events in all compartments compared to iN, indicating higher overall MT dynamics. This might be due to a higher immaturity of SHN. The speed of EB3 comets was consistent among the compartments as well as among the two cell models, with an average speed of 12.1 μm/min in SHN and 12.9 μm/s in iN. Anterograde growth events were slightly faster than retrograde, except for dendritic MTs in iN. Brown-Handerson et al. reviewed MAPs that regulate MT growth, i.e., catastrophe frequency, and rescue frequency and also growth speed. The differential composition of accelerating MAPs on plus and minus tips may result in a faster growth rate of anterograde MTs. Indeed, Jakobs et al. propose that faster polymerization of anterograde MTs contributes to uniform MT orientation in the axon . Nevertheless, as the polymerization rate is not significantly faster in axons than in dendrites (here and elsewhere ), the extensive outgrowth and specification of the axon likely results from differential MT orientation, reduced catastrophe frequency, or increased rescue frequency – as mediated by for TRIM46 – and not by faster growth rates. Consistently, silencing of TRIM46 does not decrease the MT polymerization rate but the portion of anterograde MTs and thereby axonal outgrowth . Section title: Differences in MT dynamics in different model systems Educational score: 4.2271270751953125 Domain: biomedical Document type: Study Language: en Interestingly, growth speed in human neurons was substantially faster than in numerous other neurons observed in former studies, e.g., 0.12 μm/s (7 μm/min) in Drosophila dendrites , 5 μm/min in axons of Drosophila , 0.1 μm/s (6 μm/min) in Caenorhabditis elegans , 0.1 μm/s (6 μm/min) in cortical mouse neurons , or 0.08 μm/s (4.8 μm/min) , respectively, and 0.2–0.3 μm/s (13–19 μm/min) in hippocampal rat cultures . Importantly, the growth rate appears not related to the fluorescent + TIP used. Thus, these differences could hint toward species-dependent regulation of MT growth: possibly, MT polymerization is regulated faster in humans, since MTs have to accomplish a much longer neurite growth than in neurons of rodents or non-vertebrates. Section title: Differences in MT dynamics in different model systems Educational score: 4.109372138977051 Domain: biomedical Document type: Study Language: en Additionally, we investigated the absolute number of MT growth events within a defined distance of 30 µm along the neurites. Both in SHN and iN, we observed most MT growth events in the dendrites, followed by the proximal axon, and least in the distal axon . This can be explained either by a higher density of MT in the dendrites or alternatively by a greater number of dynamic MTs compared to stable MTs in dendrites, as reported before . Section title: Differences in MT dynamics in different model systems Educational score: 4.286301612854004 Domain: biomedical Document type: Study Language: en How neurons establish a polarized MT network is a broadly debated question in neurobiology. Likely a combination of parallel bundling by TRIM46, local MT nucleation sites, translocation of anterograde and retrograde MTs, de novo MT generation from pre-existing MTs, differential growth behaviors of anterograde and retrograde MTs controlled by associated MAPs, is responsible for unipolar MTs in axons. We can now hypothesize that besides axonal outgrowth and neuronal transport, polarized MT orientation is essential for axonal TAU sorting, but further studies are needed to elucidate the regulators of MT polymerization and its impact on TAU sorting in humans. Section title: Conclusion Educational score: 4.157077789306641 Domain: biomedical Document type: Study Language: en In sum, in this study, we show that while SHN compared to iN lack a proper AIS as defined by the presence of AnkG, axonal targeting of TAU and axodendritic MT polarization is comparable, making both human neuron cell culture types suitable for studying neuronal cell polarity and further understanding of MT-related features. This holds true in particular for SHN, if the focus is not the AIS. Finally, to assess both the influence of the AIS and MT-associated factors on TAU sorting, iN are the more appropriate model to gain a holistic understanding of pathomechanisms in AD/NDD and associated dysfunction of neuronal cell polarity. | Review | biomedical | en | 0.999998 |
PMC11699588 | Section title: Introduction Educational score: 1.4850393533706665 Domain: other Document type: Other Language: en Interest in dental hygiene has recently increased in Japan. Accordingly, the Basic Policy on Economic and Fiscal Management and Reform 2024 promotes lifelong dental checkups (universal dental checkups). Section title: Introduction Educational score: 2.9738638401031494 Domain: biomedical Document type: Other Language: en Patients with diabetes are at a high risk of developing periodontal disease , which exacerbates poor glycemic control . According to the 2011 Survey of Dental Diseases , > 60% of adults show some signs of periodontal disease and the number of patients with periodontal disease is estimated at approximately 70 million, with approximately only four million receiving ongoing treatment . Therefore, improving the dental consultation rate among patients with diabetes who are at high risk of periodontal disease presents an urgent issue. Section title: Introduction Educational score: 3.905541181564331 Domain: biomedical Document type: Study Language: en Several collaborative efforts involving physicians, dentists, and pharmacists have been implemented to encourage dental consultations; however, reports on the involvement of community pharmacists in these initiatives remain limited. Takaki et al. examined the impact of a periodontal disease awareness campaign conducted at pharmacies on the dental consultation rate among patients with diabetes and reported that 8.2% of patients sought dental care after promotion. However, > 50% of the patients did not consult a dentist, highlighting the need for continuous guidance. Additionally, despite the short three-month duration of the study, approximately 20% of pharmacists reported it as a burden, and 35% stated that the three-month period was too long, raising concerns regarding the continuity of such efforts at pharmacies. Section title: Introduction Educational score: 2.152050733566284 Domain: biomedical Document type: Other Language: en Under the Pharmaceutical and Medical Device Act, pharmacists are legally required to provide follow-up after dispensing medications and to report medication adherence to physicians. The effectiveness of follow-up in patients with diabetes has been demonstrated in several studies, including the present one . However, there are no reports focusing specifically on follow-up interventions related to oral health. Section title: Introduction Educational score: 1.6279515027999878 Domain: biomedical Document type: Other Language: en Various approaches such as telephone and Information and Communication Technology (ICT) have been proposed to conduct follow-ups. LINE ® , a widely used communication platform in Japan, has gained recognition as a follow-up tool , with the potential to reduce the burden on both patients and pharmacists. Section title: Introduction Educational score: 3.551119089126587 Domain: biomedical Document type: Study Language: en Thus, we developed follow-up content on periodontal disease for patients with diabetes using the "FollowNavi ® " platform (Unike Software Research Co., Ltd., Minato, Japan), a LINE ® -based medication follow-up support tool. We aimed to investigate whether follow-up by community pharmacists could improve dental consultation rates, enhance the understanding of dental care, and raise awareness of oral hygiene among patients with diabetes. Section title: Materials and methods Educational score: 0.926092803478241 Domain: other Document type: Other Language: en Effectiveness survey Section title: Materials and methods Educational score: 1.3672782182693481 Domain: biomedical Document type: Other Language: da Study Design Section title: Materials and methods Educational score: 2.9936139583587646 Domain: biomedical Document type: Study Language: en This was a multicenter longitudinal before-after study. We evaluated the efficacy of dental follow-ups used by “FollowNavi ® ” for six months. In total, 113 individuals with type 2 diabetes mellitus (T2DM) were recruited from three pharmacies in Japan between November 2022 and January 2023. Section title: Materials and methods Educational score: 1.5506600141525269 Domain: biomedical Document type: Study Language: ca Participants Section title: Materials and methods Educational score: 2.192896604537964 Domain: biomedical Document type: Study Language: en Eligible participants were adults aged ≥20 years with T2DM who had a history of taking medication for more than two months at each pharmacy. Written informed consent was obtained from all the participants. Section title: Materials and methods Educational score: 2.232708215713501 Domain: biomedical Document type: Study Language: en The first questionnaire was conducted before the participants participated in this study. Participants were excluded if they had type 1 diabetes mellitus, had no information on HbA1c within the last two months, or could not use a mobile phone. We also excluded participants who did not calculate the following guidance fees in the Japanese dispensing fee system: drug record administration, guidance fees from family pharmacists, and guidance fees related to home consultation services. Section title: Materials and methods Educational score: 1.9329532384872437 Domain: biomedical Document type: Study Language: en All pharmacists employed at the stores that were the subject of this study were eligible to participate, and informed consent was obtained when they completed the questionnaire. Section title: Materials and methods Educational score: 1.6089894771575928 Domain: biomedical Document type: Other Language: en Procedure Section title: Materials and methods Educational score: 2.253499746322632 Domain: biomedical Document type: Study Language: en Patients who consented to follow-up were asked to register with the pharmacy’s LINE ® account, which was considered their registration for this study. A period of two weeks after registration, Content (A) - "Symptom check for suspected periodontal disease and encouragement to seek dental care" - was sent to participants, followed by Content (B) - "Educational information on diabetes and periodontal disease" - two months later. These contents were then alternated, with (A) and (B) sent every two months thereafter. To prevent follow-ups from becoming purely mechanical, free messages were also made available, allowing for personalized communication when necessary. Details of the follow-up are shown in Figure 1 . Section title: Materials and methods Educational score: 2.1552557945251465 Domain: biomedical Document type: Study Language: fr Outcome Measures Section title: Materials and methods Educational score: 4.034144878387451 Domain: biomedical Document type: Study Language: en The primary outcomes were changes in dental consultation and checkup rates from baseline to six months. Data were collected at baseline and six months later from all participants. HbA1c levels of the patients were determined from the blood test results in the most recently recorded medical history during the six-month follow-up. Information regarding age, sex, and medication use was obtained from the medical history records. Furthermore, information regarding the duration of diabetes, dental consultations (for therapeutic purposes), participation in dental checkups (for cavity and periodontal disease screening, plaque removal, and brushing instruction), oral care status, willingness to prevent periodontal disease, and understanding of periodontal disease were evaluated using questionnaire surveys administered to patients. Section title: Materials and methods Educational score: 0.9565811157226562 Domain: other Document type: Other Language: fr Questionnaire survey Section title: Materials and methods Educational score: 1.8868032693862915 Domain: biomedical Document type: Study Language: en Questionnaires were administered to patients who received follow-ups and pharmacists who conducted the follow-ups. To assess the usability and acceptability of the system, both groups were asked regarding the clarity of the content, whether the number and frequency of questions were appropriate, the time required to respond, future expectations for the system, and any additional requests. Questionnaires were conducted at the end of the follow-up period. Consent was obtained from the respondents before they completed the questionnaire. Section title: Materials and methods Educational score: 1.4600969552993774 Domain: biomedical Document type: Other Language: en Statistical analysis Section title: Materials and methods Educational score: 2.7792251110076904 Domain: biomedical Document type: Study Language: en Data are presented as medians with interquartile ranges (IQR) or percentages. Missing data were imputed using the last-observation-carried-forward method. The Fisher’s exact test or McNemar’s test was used to compare categorical data. p < 0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 27 . Section title: Materials and methods Educational score: 1.149239420890808 Domain: other Document type: Other Language: en Ethical considerations Section title: Materials and methods Educational score: 0.8584032654762268 Domain: biomedical Document type: Other Language: en The study was approved by the ethics committee of the Tokyo University of Science . Section title: Results Educational score: 0.9849379062652588 Domain: biomedical Document type: Other Language: en Follow-up effectiveness Section title: Results Educational score: 1.456114649772644 Domain: biomedical Document type: Other Language: en Patient Characteristics Section title: Results Educational score: 4.144231796264648 Domain: biomedical Document type: Study Language: en This study included 113 patients (55 men and 56 women; 2 unspecified). The median age at baseline was 60 years (interquartile range [IQR]: 52-68), and the median HbA1c level was 6.7% (IQR: 6.3-7.0%). Overall, 26 patients (23.0%) had been diagnosed in less than 5 years, 85 patients (75.2%) had been diagnosed more than five years, and two patients (1.8%) did not provide an answer. The median follow-up period was 182 days (IQR: 125-266), and all patients underwent four follow-ups. The median number of responses to these follow-ups was 3.0 (IQR: 1.0-4.0). The median number of diabetes medications used was 3.0 (IQR: 2.0-4.0). The usage rates of diabetes medications were as follows: biguanides, 62.8%; sodium-glucose cotransporter 2 inhibitors, 51.3%; dipeptidyl peptidase IV inhibitors, 44.2%; glucagon-like peptide 1 receptor agonists, 31.0%; sulfonylureas, 16.8%; thiazolidinediones, 15.9%; insulin, 15.0%; α-glucosidase inhibitors, 13.3%; meglitinides, 12.4%; and imeglimin, 3.5% (multiple responses allowed) (Table 1 ). Section title: Results Educational score: 1.3206722736358643 Domain: biomedical Document type: Other Language: en Changes in the Dental Consultation and Checkup Rates Section title: Results Educational score: 3.959867238998413 Domain: biomedical Document type: Study Language: en The dental consultation and checkup rates at baseline and after six months are shown in Table 2 . A total of 98 patients were included in the analysis, after excluding 15 patients for whom data at either baseline or six months were unavailable. The dental consultation rate increased from 40.8% to 41.8%, and the dental checkup rate increased from 57.1% to 59.2%; however, the changes were not statistically significant (P = 1.000, 0.824). HbA1c levels after six months were 6.8% (IQR: 6.4-7.2), with no significant change (P = 0.062). Section title: Results Educational score: 1.6450132131576538 Domain: biomedical Document type: Other Language: en Changes in Periodontal Disease Understanding Section title: Results Educational score: 3.932636260986328 Domain: biomedical Document type: Study Language: en In the questionnaire, the question "Do you know that having periodontal disease can worsen blood sugar levels?," the number of patients who answered "Yes" increased from 64 (65.3%) at baseline to 85 (86.7%) (P < 0.001). In response to the question "Do you know that diabetes is a risk for periodontal disease?," 72 patients (73.5%) answered "Yes" at baseline, but 93 (94.9%) after six months (P < 0.001) (Table 3 ). Section title: Results Educational score: 1.472759485244751 Domain: biomedical Document type: Other Language: en Changes in Awareness of Oral Care Section title: Results Educational score: 3.695479154586792 Domain: biomedical Document type: Study Language: en In response to the question, "Do you want to prevent periodontal disease (by consultation, the dentist, undergoing checkups, and brushing your teeth properly)?," the number of patients who answered "Strongly agree" or "Agree" increased significantly from 84 (86.6%) at baseline to 92 (94.8%) after six months (P = 0.039). In response to the question "Do you try to keep your teeth healthy?," 62 people (63.3%) answered "Yes" at baseline, and 64 people (65.3%) answered "Yes" after six months (P = 0.845) (Table 4 ). Section title: Results Educational score: 1.4638930559158325 Domain: biomedical Document type: Other Language: en Changes in Pharmacist Consultations Regarding Dental-Related Matters Section title: Results Educational score: 2.026167154312134 Domain: biomedical Document type: Study Language: en In response to the question "Have you ever consulted a pharmacist about your teeth or mouth?", three people (2.8%) answered "Yes" at baseline, and 11 people (11.1%) answered "Yes" after six months (P = 0.024) . Section title: Results Educational score: 0.9565811157226562 Domain: other Document type: Other Language: fr Questionnaire survey Section title: Results Educational score: 1.3461772203445435 Domain: biomedical Document type: Other Language: en Results of the Patient Questionnaire Survey Section title: Results Educational score: 2.6561286449432373 Domain: biomedical Document type: Study Language: en The results of the patient survey conducted at the end of the follow-up period are presented in Table 5 . In response to the question, "Did you consider visiting a dentist by message?," 46 patients (40.7%) answered "Strongly agree" and 39 patients (34.5%) answered "Agree." For the question, "Did you understand the text messages well?," 52 patients (46.0%) responded "Well understood," and 12 (10.6%) said "Understood.” Meanwhile, 2 (1.8%) responded "Slightly difficult, 6 (5.3%) said "Difficult," and 23 (20.4%) did not respond. When asked, "Did you solve your question with FollowNavi ® ," 71 patients (62.8%) stated they "No questions,” 11 (9.7%) answered that "Solved in another way" 6 (5.3%), and 3 (2.7%) were unable to resolve their questions, with 21 (18.6%) providing no answer. Section title: Results Educational score: 2.095402240753174 Domain: biomedical Document type: Study Language: en Regarding the number and frequency of the questions, 80 (70.8%) and 81 (71.7%) patients, respectively, responded that they were "Appropriate." As for the time required to answer the questions, 67 (59.3%) indicated that it took "Within 5 min," and 20 (17.7%) answered "5-10 min." In response to the question, "Do you want to continue using FollowNavi ® ?," 64 patients (56.6%) answered "Yes," 25 (22.1%) said "No," and 24 (21.2%) did not respond. Section title: Results Educational score: 1.2720218896865845 Domain: biomedical Document type: Other Language: en Results of the Pharmacist Questionnaire Survey Section title: Results Educational score: 1.9873814582824707 Domain: biomedical Document type: Study Language: en The results of the pharmacist questionnaire surveys are presented in Table 6 . Surveys were collected from eight pharmacists who implemented follow-ups using the FollowNavi ® . When asked, "Have you been asked questions about dentistry before this trial?," three participants (37.5%) answered "Yes." In response to the question "Did you have any questions about dentistry during this trial?," two pharmacists (25.0%) answered "Yes." Section title: Results Educational score: 1.770013451576233 Domain: biomedical Document type: Study Language: en When asked, "Do you think the text messages are easy for patients to understand?," six pharmacists (75.0%) said "Slightly difficult" and two (25.0%) said, "Difficult.” When asked, "Did you get sufficient patient information?," five (62.5%) answered "Yes," and three (37.5%) said "No." The specific information they were unable to obtain included "patients' feelings, thoughts, and other non-text-based information." Section title: Results Educational score: 1.9162092208862305 Domain: biomedical Document type: Study Language: en Regarding the appropriateness of the number of inquiries and frequency of follow-ups, seven pharmacists (87.5%) responded that both were "appropriate." When asked about the usability of FollowNavi ® , one (12.5%) responded "Easy,” three (37.5%) said "Slightly difficult," one (12.5%) answered "Neutral," and three (37.5%) said "Slightly easy." Section title: Results Educational score: 2.516875982284546 Domain: biomedical Document type: Study Language: en When asked, "How long did it take to check the patient responses?," five pharmacists (62.5%) answered less than 5 min, two (25.0%) answered between 5-10 min, and one (12.5%) answered between 10-20 min. Regarding the expected effects of FollowNavi ® (multiple responses allowed), all eight pharmacists (100%) cited "Confirmation of patient symptoms," five (62.5%) cited "Improvement of adherence," and five (62.5%) mentioned "early detection of adverse drug reactions." Section title: Results Educational score: 1.3760664463043213 Domain: other Document type: Other Language: en Finally, when asked, "Do you want to continue FollowNavi®," five pharmacists (62.5%) answered "Yes." Section title: Discussion Educational score: 2.83088755607605 Domain: biomedical Document type: Study Language: en The dental consultation rate at baseline in this study was 40.8%, which is comparable to that in previous reports indicating 47.8% and 44.0%. Similarly, the dental checkup rate was 58.6%, which was slightly higher than the previously reported rate of 46.8% , although not substantially different. Based on these comparisons, the population in this study is representative of a broad population of patients with diabetes. Section title: Discussion Educational score: 3.9381487369537354 Domain: biomedical Document type: Study Language: en Munenaga et al. reported that in patients with T2DM moderate to severe periodontal disease, HbA1c levels improved significantly three months after periodontal treatment. Engebretson et al. showed that in a meta-analysis of nine RCTs, periodontal treatment significantly reduced HbA1c levels by 0.36%. These studies were conducted on periodontal patients and on patients who had undergone dental treatment. In our study, the rate of dental consultation did not improve significantly, and not all patients had periodontal disease, so it seems that there was no improvement in HbA1c levels. Section title: Discussion Educational score: 3.991102457046509 Domain: biomedical Document type: Study Language: en Regarding the questionnaire survey, the patients' understanding of the relationship between periodontal disease and glycemic control increased significantly, as well as their awareness that diabetes is a risk factor for periodontal disease. At baseline, 65.3% and 73.5% of the patients had this knowledge, and these rates increased significantly to 86.7% and 94.9%, respectively, after six months. This suggests that the follow-up of periodontal content by community pharmacists effectively enhances patient understanding. Section title: Discussion Educational score: 3.927945137023926 Domain: biomedical Document type: Study Language: en In terms of motivation for oral care, the proportion of patients who answered "Strongly agree" or "Agree" to the question, "Do you want to prevent periodontal disease (by visiting the dentist, undergoing checkups, and brushing your teeth properly)?" significantly increased from 86.6% at registration to 94.8% after six months (P = 0.039). Additionally, in response to the question after follow-up "Did you consider visiting a dentist by message?", 46 patients (40.7%) answered "Strongly agree" and 39 (34.5%) answered "Agree." These results confirm that follow-ups improved patients' awareness and motivation for oral care. Section title: Discussion Educational score: 4.122381210327148 Domain: biomedical Document type: Study Language: en Despite these changes in awareness, the actual consultation rate did not improve, possibly because behavioral changes take time. According to the transtheoretical model of health behavior change , there are five stages of behavior change: pre-contemplation (not yet acknowledging that there is a problem behavior that needs to be changed), contemplation (acknowledging that there is a problem but not yet ready, sure of wanting, or lacks confidence to make a change), preparation (getting ready to change), action (changing behavior), and maintenance (maintaining the behavior change). The survey results suggested that the willingness to visit dentists improved, indicating a transition from the pre-contemplation stage to the contemplation stage. However, for patients to move to actual behavioral changes, a shift from the contemplation stage to the preparation and action stages is necessary, and building a trusting relationship is considered crucial for this transition . In the pharmacist's questionnaire, some respondents mentioned the challenge of "being unable to obtain non-verbal information, such as the patient's feelings and thoughts," highlighting the limitations of mechanical follow-ups. To build trust and promote behavioral change among patients, combining mechanical follow-ups with in-person consultations and guidance is necessary. Section title: Discussion Educational score: 2.6245639324188232 Domain: biomedical Document type: Study Language: en In addition, when asked "Have you ever consulted a pharmacist about your teeth or mouth?," the percentage of answering "Yes" significantly increased from 3.1% at baseline to 10.3% after six months. This suggests that follow-ups may lead to an increase in dental consultations at pharmacies. Given the current emphasis on the role of pharmacists in health support and dental-pharmaceutical collaboration , the indication that follow-up could promote pharmacists’ involvement in dental consultations is useful. However, the number of consultations remains insufficient. Thus, if further long-term follow-up is conducted, it may lead to a further increase in the number of consultations with pharmacists and an associated improvement in the rate of dental visits. Section title: Discussion Educational score: 3.894392728805542 Domain: biomedical Document type: Study Language: en In the patient questionnaire survey regarding usability, 52 (46.0%) answered that the messages were "Easy," while 12 (10.6%) answered that they were "Slightly easy.” These values were lower than those obtained in our study using diabetes follow-up data . This may be due to the high number of non-answers to this question (20.4 %). Given that a combined total of 7.1% of patients answered that it was "Slightly difficult" or "Difficult," the contents were adequately understood. Regarding the number of questions, 70.8% answered that it was "Appropriate,” and regarding the frequency of questions, 71.7% answered that it was "Appropriate,” indicating that the number was suitable. In this study, follow-ups were conducted only once every two months. In our previous report , follow-ups were planned for each consultation and conducted approximately once a month, with > 90% responding that this was appropriate. Although it is difficult to generalize the results because the participants and contents of follow-ups varied, the frequency of follow-up may not be an issue, depending on the patient. Therefore, pharmacists should consider medication and patient information and conduct frequent follow-ups. Section title: Discussion Educational score: 2.5703659057617188 Domain: biomedical Document type: Study Language: en Pharmacists often perform follow-ups on prescribed medications mainly to assess medication adherence, effectiveness, and side effects . Few studies report cases of pharmacies participating in dental care, which is not limited to follow-up care. Therefore, we believe that the results of this study, which confirmed the usefulness of dental follow-ups for patients with diabetes regardless of the prescribed medication, are significant when considering the scope of pharmacists' follow-up duties. Section title: Discussion Educational score: 3.1788806915283203 Domain: biomedical Document type: Study Language: en This study has two limitations. First, this study had a high rate of incomplete answers in the questionnaires. In the usability questionnaire, approximately 20% of the questions remained unanswered. Thus, improving the questionnaire method and questions is necessary for future surveys. Second, data, such as duration of diabetes, dental consultations, and participation in dental checkups, were obtained via questionnaire surveys administered to patients. Therefore, there is a possibility of recall bias and lack of accuracy. Further studies are needed to obtain accurate medical data and evaluate them. Section title: Conclusions Educational score: 3.5795421600341797 Domain: biomedical Document type: Study Language: en In the six-month follow-up using the periodontal contents of the "FollowNavi ® " platform, which leverages LINE ® for the follow-up system, there was no significant improvement in the dental consultation, checkup rates, or HbA1C levels in patients with diabetes. However, the awareness and understanding of oral care improved, and there was an increase in dental consultations at pharmacies. The results of this study, which confirmed the usefulness of dental follow-ups for patients with diabetes regardless of the prescribed medication, are useful when considering the expansion of follow-ups in the future and ways of involvement in the dental field. Section title: Conclusions Educational score: 1.9291526079177856 Domain: biomedical Document type: Other Language: en The findings show that pharmacists may be able to play a role in improving dental treatment for patients with diabetes through follow-up. This will be of great help in considering how community pharmacists can expand follow-up and become involved in the dental field. | Other | other | en | 0.999998 |
PMC11699589 | Section title: Introduction and background Educational score: 3.951324224472046 Domain: biomedical Document type: Review Language: en Joint degeneration characterized by cartilage deterioration and bone wear is the hallmark of osteoarthritis (OA), a condition that worsens over time . This debilitating disorder often manifests as persistent discomfort, potentially leading to impaired mobility and function. The impact of OA extends beyond the physical changes in joint structures, as the chronic pain associated with this condition can significantly compromise an individual's ability to perform daily activities . Section title: Introduction and background Educational score: 3.7810065746307373 Domain: biomedical Document type: Review Language: en Epidemiological data indicate that the Americas bear the highest overall OA burden globally, while Asia demonstrates a disproportionately high prevalence of knee-specific OA. To mitigate this substantial health challenge , it is needed to implement comprehensive prevention strategies and targeted interventions aimed at addressing modifiable risk factors associated with OA in order to reduce the increase in cases annually . One of the main reasons for the increase in the number of patients suffering from OA is the increase in the number of aged populations with other risk factors, such as obesity and not performing physical activities . Section title: Introduction and background Educational score: 3.997239828109741 Domain: biomedical Document type: Review Language: en Total knee arthroplasty (TKA), typically performed in advanced osteoarthritis cases, offers modest improvements in proprioception with higher safety than other interventions . However, some sensory deficits may persist post-surgery . Additionally, patients often experience significant quadriceps weakness on the operated side, with strength potentially reduced by up to 30% compared to the unaffected limb . These strength and balance impairments can lead to various functional issues, including uneven weight distribution between legs, compromised balance, modified movement patterns, and overall reduced physical functionality . TKA can be performed using cemented, cementless, or hybrid fixation methods. Previous meta-analyses have indicated no significant differences in clinical outcomes among these fixation techniques, with the availability of these techniques being performed in a conventional way or robotic-assisted . Section title: Introduction and background Educational score: 4.128383636474609 Domain: biomedical Document type: Study Language: en Most of the studies use low-intensity training as a therapeutic procedure, although recent research indicates that higher-intensity training protocols may yield superior outcomes for knee rehabilitation . These more vigorous exercise regimens are associated with improved functional strengthening of the knee and enhanced overall knee function . Importantly, these intensive training approaches have been found to maintain safety standards, suggesting they can be effectively implemented without increasing risk to patients . This study's objective is to evaluate and contrast the safety and efficacy of intensive therapy training (ITT) versus conventional therapy training (CTT) programs implemented preoperatively for TKA patients. We seek to provide clear guidance for clinical professionals on optimizing preoperative physical therapy to enhance recovery, improve functional outcomes, and ensure patient safety. Section title: Review Educational score: 1.3877626657485962 Domain: biomedical Document type: Other Language: cy Methods Section title: Review Educational score: 3.755347490310669 Domain: biomedical Document type: Study Language: en We performed our meta-analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist and the guidelines that were mentioned in the Cochrane Handbook for Systematic Reviews and Meta-Analysis . Section title: Review Educational score: 1.3406771421432495 Domain: biomedical Document type: Other Language: en Searching Databases and Keywords Section title: Review Educational score: 2.51997447013855 Domain: biomedical Document type: Study Language: en We did our search through five databases (PubMed, Cochrane Library, Web of Science, Scopus, and Embase), and it was completed in July 2024. The terms that we used were: (("heavy" OR "progressive" OR "maximal" OR "explosive" OR "resistance" OR "high-intensity" OR "intensified" OR "strengthening" OR "weight lifting" OR "weight bearing" OR "concentric" OR "eccentric" OR "endurance" OR "elastic tube" OR "pulleys") AND ("arthroplasty" OR "replacement" OR "surgery" OR "operation") AND (knee) AND ("training" OR "exercise" OR "rehabilitation") AND ("randomized" OR "randomly" OR "randomised" OR "random")). Section title: Review Educational score: 1.992926001548767 Domain: biomedical Document type: Study Language: en Eligibility Criteria and Study Selection Section title: Review Educational score: 4.078231334686279 Domain: biomedical Document type: Study Language: en The PICOS (Population, Intervention, Comparison, Outcome, and Study Design) framework was used to guide the development of search terms, strategy, and inclusion criteria for the study . The framework components were defined as follows: the population included individuals who recently underwent knee arthroplasty and performed preoperative exercise; the intervention was pre-operative intensive physical therapy; the comparison was standard pre-operative physical therapy; the outcomes included clinical measures and questionnaire results; and the study designs considered were randomized and non-randomized controlled trials. Only English articles with full texts were included Section title: Review Educational score: 2.6108505725860596 Domain: biomedical Document type: Study Language: en The study excluded observational research (including cohort studies, case series, and case reports), literature reviews, animal studies, qualitative investigations, and single-arm trials. Section title: Review Educational score: 1.128065586090088 Domain: biomedical Document type: Other Language: en Quality Assessment Section title: Review Educational score: 4.047975540161133 Domain: biomedical Document type: Study Language: en To maintain data integrity, two independent researchers extracted information from the selected trials. In cases of disagreement, additional authors provided oversight and facilitated consensus. We used two different tools according to the study type: for randomized controlled trials (RCTs), the Cochrane Risk of Bias (RoB) 2 (Cochrane Methods, London, UK) tool was utilized, evaluating five critical domains . Non-randomized studies were assessed using the Risk of Bias in Non-randomized Studies of Interventions (ROBINS-I) tool (Cochrane Methods, London, UK) . Section title: Review Educational score: 1.6088417768478394 Domain: biomedical Document type: Other Language: ca Data Extraction Section title: Review Educational score: 4.1434454917907715 Domain: biomedical Document type: Study Language: en Data extraction was performed independently by two authors using Excel spreadsheets (Microsoft Corporation, Redmond, USA), encompassing three main categories of information. The first category, summary data, included study timing and locations, design, protocol number, total patient count, ITT and CTT details, follow-up duration, and primary outcome. The second category covered baseline patient characteristics such as study groups, age, gender, BMI, height, and knee arthroplasty laterality and location. The third category focused on clinical outcomes, further divided into physical measures (including 6 or 10-minute walk test, quadriceps strength, range of motion for extension and flexion, isometric knee extension and flexion stair test, and Timed Up and Go test) and questionnaires (comprising the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Visual Analog Scale (VAS) . Section title: Review Educational score: 0.9184456467628479 Domain: biomedical Document type: Other Language: tl Data Analysis Section title: Review Educational score: 4.048376560211182 Domain: biomedical Document type: Study Language: en The analytical component of the study utilized Review Manager version 5.4 software (Cochrane Methods, London, UK). For continuous outcomes, results were presented as mean differences (MD) with 95% confidence intervals . Statistical significance was established at a p-value threshold of 0.05. Heterogeneity assessment employed χ2 and I-square (I2) tests, with heterogeneity considered present when the χ2 p-value was below 0.1 or the I2 value exceeded 50%. In cases where heterogeneity was detected, a random-effects model was applied; otherwise, a fixed-effects model was used. When heterogeneity persisted despite the use of a random-effects model, a leave-one-out sensitivity analysis was conducted. Section title: Review Educational score: 1.065622329711914 Domain: biomedical Document type: Other Language: et Results Section title: Review Educational score: 1.884278416633606 Domain: biomedical Document type: Study Language: en Literature Search and Study Selection Section title: Review Educational score: 4.025205612182617 Domain: biomedical Document type: Study Language: en After applying our search strategy, we found a total of 917 articles, and after the removal of the duplicates, a total of 605 studies proceeded for the title and abstract screening. We performed a title and abstract screening that led to the elimination of 561 articles. Finally, after full-text screening, a total of seven studies were included. The full, detailed PRISMA flowchart (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) is shown in Figure 1 . Details of excluded studies after full-text screening are in the Appendices. Section title: Review Educational score: 1.628010630607605 Domain: biomedical Document type: Study Language: en Study Characteristics Section title: Review Educational score: 3.5996925830841064 Domain: biomedical Document type: Study Language: en Seven studies were included, with a total of 490 participants. The locations of the studies were Japan, China, Switzerland, and Greece. Various rehabilitation programs were used, whether for intensive therapy or the conventional ways. There are some differences regarding the type of exercise, duration, and number of sessions. The follow-up duration ranged from three months to 13 months. Full details about the summary characteristics are shown in Table 1 . The majority of the included participants were females and elderly people more than 60 years of age. The BMI of the included population ranged from 29 to 35. The majority of the patients had unilateral knee arthroplasty. Full details about the baseline characteristics are shown in Table 2 Section title: Review Educational score: 1.698256492614746 Domain: biomedical Document type: Study Language: en Quality of the Included Studies Section title: Review Educational score: 4.090567111968994 Domain: biomedical Document type: Study Language: en The risk of bias assessment using the RoB 2 tool revealed varying levels of potential bias across the included studies. No studies demonstrated a high risk of bias. However, three studies were associated with some concerns due to randomization domain or others as deviation from the intended intervention or missing outcome domain. The remaining two RCTs were associated with a low risk of bias. A comprehensive visual representation of these assessments is provided in Figure 2 . On the other hand, regarding the ROBINS-I, one study showed a low risk of bias, and the other showed a serious risk due to bias in the measurement of the outcomes domain . Section title: Review Educational score: 1.5020114183425903 Domain: biomedical Document type: Other Language: fr Outcomes Section title: Review Educational score: 1.7866694927215576 Domain: biomedical Document type: Other Language: en Physical Measurements Section title: Review Educational score: 4.1487321853637695 Domain: biomedical Document type: Study Language: en Six- or 10-minute walk test (meters): In the follow-up of one to two months, two studies were included, and the result showed a significant increase in the distance that patients were able to walk in the ITT when compared to CTT with results of (MD = 47.71, 95% CI). Moreover, in the follow-up period of three to six months, there were significant differences between the two comparators (MD = 42.62, 95% CI (23.15 to 62.10), p-value < 0.0001). The overall results demonstrated a significant difference favoring ITT, and pooled results were (MD = 45.07, 95% CI (31.04 to 59.1), P-value < 0.000001). The overall results of the study were homogenous as p-value = 0.34 and I2 =11% . Section title: Review Educational score: 4.147109031677246 Domain: biomedical Document type: Study Language: en Isometric knee extension (Kg): In the follow-up period ranging from one to two months, there was no significant difference in the isometric knee extension (MD = 1.06, 95% CI (-2.09 to 4.20), P-value = 0.51). Additionally, the follow-up period ranging from three to six months showed a significance favoring CTT over ITT (MD = 7.83, 95% CI (4.11 to 11.55), p-value < 0.0001). Regarding the long-term follow-up (12 months), only one study was included, with no significant difference between the comparators. The overall results favored the CTT over ITT (MD = 3.45, 95% CI (0.49 to 6.41), p-value = 0.02) . Section title: Review Educational score: 4.139915466308594 Domain: biomedical Document type: Study Language: en Isometric knee flexion (Kg): In the follow-up period ranging from one to two months, there was no significant difference in the isometric knee flexion. Additionally, the follow-up period ranging from three to six months showed no significant difference between CTT and ITT (MD = 2.97, 95% CI (-0.21 to 6.16), p-value =0.07). Regarding the long-term follow-up (12 months), only one study was included with no significant difference between the comparators. The overall results favored the ITT significantly over CTT (MD = 2.32, 95% CI (0.07 to 4.56), p-value = 0.04). The results were heterogeneous and I2=90% . Section title: Review Educational score: 4.139968395233154 Domain: biomedical Document type: Study Language: en Quadriceps strength (Kg): In the follow-up period ranging from one to two months, there was a significant difference between ITT and CTT regarding the strength (MD = 0.06, 95% CI (0.02 to 0.10), P-value = 0.0003). The follow-up period ranging from three to six months showed another significant difference favoring ITT (MD = 0.08, 95% CI (0.02 to 0.13), p-value =0.005). The overall results favored the ITT over CTT (MD = 0.07, 95% CI (0.07 to 0.10), p-value <0.0001). The results were heterogeneous and I2=90% . Section title: Review Educational score: 3.0164151191711426 Domain: biomedical Document type: Study Language: en ROM extension: In the follow-up period ranging from one to two months, there was no significant difference between ITT and CTT regarding the extension. The follow-up period ranging from three to six months showed another insignificant difference between the two comparator groups. The results were heterogeneous and I2=83% . Section title: Review Educational score: 4.1485395431518555 Domain: biomedical Document type: Study Language: en ROM flexion: In the follow-up period ranging from one to two months, there was no significant difference between ITT and CTT regarding the flexion. The follow-up period ranging from three to six months showed another insignificant difference between the two comparator groups. Finally, the overall result showed that ITT was significantly improved in comparison to CTT (MD = 4.29, 95% CI (0.35 to 8.22), p-value = 0.03). The results were homogenous (p-value= 0.77) and I2=0% . Section title: Review Educational score: 4.128253936767578 Domain: biomedical Document type: Study Language: en SF-36 physical component: In the follow-up period ranging from one to two months, two studies were included showing a significant difference in the SF-36 (MD = 0.96, 95% CI (0.43 to 1.48), p-value = 0.0003). Additionally, the follow-up period ranging from three to six months showed a significant difference between CTT and ITT (MD = 1.00, 95% CI (0.09 to 1.91), p-value =0.03). Regarding the long-term follow-up (12 months), only one study was included with a significant difference between the comparators. The overall results favored the ITT over CTT (MD = 2.32, 95% CI (0.07 to 4.56), p-value = 0.04). The results were heterogeneous and I2=90% . Section title: Review Educational score: 4.137465476989746 Domain: biomedical Document type: Study Language: en Stair test (seconds): In the follow-up period ranging from one to two months, two studies were included showing a significant difference in the stair test (MD = -3.21, 95% CI (-4.76 to -1.67), p-value < 0.0001). Additionally, the follow-up period ranging from three to six months showed a non-significant difference between CTT and ITT (MD = -2.30, 95% CI (-5.54 to 0.93), p-value =0.16). Regarding the long-term follow-up (12 months), only one study was included with a non-significant difference between the comparators. The overall results favored the ITT over CTT (MD = -2.01, 95% CI (-3.61 to -0.41), p-value = 0.01). The results were heterogeneous and I2=89% . Section title: Review Educational score: 4.137491226196289 Domain: biomedical Document type: Study Language: en Timed up and go: In the follow-up period ranging from one to two months, two studies were included showing a non-significant difference in the time up and go (MD = -0.21, 95% CI (-4.76 to -4.35), p-value =0.93). Additionally, the follow-up period ranging from three to six months showed a significant reduction in time favoring ITT (MD = -1.26, 95% CI (-2.43 to -0.09), p-value =0.04). Regarding the long-term follow-up (12 months), only one study was included with a non-significant difference between the comparators. The overall results favored the ITT over CTT (MD = -1.12, 95% CI (-2.04 to -0.20), p-value = 0.02). The results were heterogeneous (p-value= 0.003) and I2=75% . Section title: Review Educational score: 1.087002158164978 Domain: biomedical Document type: Other Language: fr Questionnaires Section title: Review Educational score: 4.034056663513184 Domain: biomedical Document type: Study Language: en VAS score: No results were found significant according to the included studies at the VAS score results. In the follow-up period, which ranged from one month to two months, three studies were included, and the result showed a non-significant decrease in pain in the ITT on VAS when compared to CTT. Additionally, in the follow-up ranging from three to six months, there were no significant differences between the two comparators with three included papers. The long-term follow-up of 12 months was only mentioned in one study without any significant difference. The results of the outcome were heterogeneous as p-value <0.0001 and I2 = 85% . Section title: Review Educational score: 4.138213157653809 Domain: biomedical Document type: Study Language: en WOMAC score: Results showed a significant difference according to the included studies on WOMAC score. In the follow-up period that ranged from one to two months, two studies were included, and the result showed a significant difference in the ITT on WOMAC score when compared to CTT (MD = -10.87, 95% CI (-19.23 to -2.51), p-value = 0.01). Additionally, in the follow-up ranging from three to six months, there was a significant difference between the two comparators with two included papers (MD = -6.22, 95% CI (-8.70 to -3.74), p-value <0.00001). The overall results showed a significant difference in favoring ITT over CTT (MD = -8.43, 95% CI (-13.63 to -3.22), p-value = 0.28). The results of the outcome were heterogeneous as P-value < 0.0001 and I2 = 87% . Section title: Review Educational score: 1.083368182182312 Domain: other Document type: Other Language: de Discussion Section title: Review Educational score: 4.030045032501221 Domain: biomedical Document type: Review Language: en This systematic review and meta-analysis analyzed seven studies comparing ITT to CTT for knee arthroplasty patients. The study populations were predominantly female and elderly, with no weight specification. The results demonstrated that ITT generally outperformed CTT across several outcomes. Significant improvements were observed in walking tests, quadriceps strength, range of motion flexion, physical component scores, stair tests, timed up and go tests, and WOMAC scores. These benefits were noted across various follow-up periods, ranging from short-term to medium-term and long-term post-intervention. However, the results showed CTT showed better outcomes in isometric knee extension, particularly in the medium-term follow-up period Section title: Review Educational score: 3.9901039600372314 Domain: biomedical Document type: Study Language: en The selection of appropriate training intensity and duration is critical for optimizing muscle strength gains. While many previous studies focused on low to moderate-intensity regimens , recent research has highlighted the potential benefits of higher-intensity approaches . Notably, a randomized controlled trial demonstrated that preoperative high-intensity strength training can yield significant improvements. This intensive training was found to reduce pain levels, significantly increase lower limb muscle strength, and enhance performance in functional tasks, suggesting that higher-intensity protocols may offer superior outcomes for patients preparing for knee surgery . Rooks et al. found that preoperative progressive resistance training improved postoperative functional performance and muscle strength but did not significantly affect patient-reported outcomes . This suggests a potential disconnect between objective physical improvements and patients' subjective experiences. Section title: Review Educational score: 3.911039352416992 Domain: biomedical Document type: Other Language: en A comprehensive approach to TKA rehabilitation should integrate both preoperative and postoperative physiotherapy programs . Preoperative training for patients with end-stage osteoarthritis has been shown to enhance initial outcomes and improve patient satisfaction following TKA . Conversely, postoperative exercise regimens primarily focus on pain management, edema reduction, and improvement of knee range of motion. This dual-phase approach aims to optimize overall patient outcomes by addressing both preoperative conditioning and postoperative recovery needs . Section title: Review Educational score: 4.090570449829102 Domain: biomedical Document type: Study Language: en Our study has several strengths. By including only RCTs and non-randomized controlled trials, we ensured the inclusion of high-quality evidence, excluding observational studies to minimize potential biases. Furthermore, our study is the first meta-analysis to focus specifically on the preoperative differentiation between ITT and CTT, addressing a critical gap in the literature. The key guidance from our findings highlights that preoperative physical therapy tailored to intensity levels has significant implications for optimizing TKA outcomes. Specifically, the results suggest that ITT protocols may be more effective in improving functional recovery and postoperative strength, while CTT approaches might still hold value in specific patient populations or settings where ITT is less feasible. Section title: Review Educational score: 4.0824995040893555 Domain: biomedical Document type: Study Language: en Our study also had some limitations, as long-term follow-up (12 months) was limited, with only two studies reporting outcomes at this time point . Moreover, various training programs were introduced with different intervention settings which could add possible heterogeneity and make the results affected. The included studies exhibited considerable variation in exercise training protocols which were observed in the duration of training periods, frequency of sessions, and intensity of exercises. This diversity in intervention approaches may have contributed to heterogeneity among the study populations, potentially influencing the comparability and generalizability of results across studies. It's important to note that although ITT generally appears more effective, individual responses to therapy may vary as there is a high heterogenicity among the included studies. Additionally, taking into consideration that some outcomes had no significant difference in the specified follow-up durations and gave overall significance according to the analysis. Previous meta-analyses have suggested that robotic-assisted TKA may offer certain advantages over conventional surgical techniques, particularly in terms of knee alignment and functional outcomes which may add another factor to be considered for investigation and increase the requirement of having a homogenous population . Section title: Review Educational score: 4.0412211418151855 Domain: biomedical Document type: Review Language: en We recommend more long-term follow-up studies beyond 12 months, which are crucial to assess the sustained benefits of intensive therapy compared to conventional approaches. Given the observed heterogeneity in outcomes, future studies should aim to identify patient subgroups that benefit most from intensive therapy, enabling more personalized rehabilitation approaches. Additionally, economic analyses should evaluate the cost-effectiveness of intensive therapy, considering both short-term resource utilization and long-term functional outcomes. Clinical guidelines for post-operative care in TKA should be reviewed and potentially updated to reflect these benefits. Finally, patient education materials should be developed to inform individuals about the potential advantages of intensive therapy and encourage adherence to more rigorous rehabilitation protocols. Special training programs should be made to fit every participant, considering their health and condition. Section title: Conclusions Educational score: 4.001725673675537 Domain: biomedical Document type: Study Language: en We conclude that ITT generally demonstrates overall superior outcomes compared to CTT for patients undergoing knee arthroplasty when done preoperatively. ITT showed significant improvements across a range of functional and patient-reported outcomes, including walking capacity, quadriceps strength, range of motion, and quality of life measures. On the other hand, the isometric knee extension showed superiority for the CTT group. We recommend that healthcare providers should consider implementing preoperative intensive therapy protocols for TKA patients due to their superior outcomes across multiple recovery domains. However, further research is needed to optimize these protocols by focusing on specific exercises, frequency, and duration for the best results. | Review | biomedical | en | 0.999998 |
PMC11699598 | Section title: Introduction Educational score: 4.166028022766113 Domain: clinical Document type: Clinical case Language: en Out-of-hospital-cardiac arrest (OHCA) is a significant health concern with approximately 275,000 annual cases in Europe and 420,000 in the United States. 1 Immediate recognition of cardiac arrest, initiation of cardiopulmonary resuscitation (CPR) and early defibrillation are key factors to improve survival. 2 However, bystander CPR are only reported on average in 58 % (range 13 % to 83 %) of episodes. 2 In addition, there is considerable variation in the quality of bystander CPR, with only a minority demonstrating high quality CPR. 3 Simulation studies have shown that video assisted CPR (V-CPR) may improve cardiac arrest recognition, improve the quality of basic CPR and that it can be performed in a real-life setting. 4 , 5 In OHCA, clinical studies show promising results of V-CPR on CPR quality, return-of-spontaneous-circulation (ROSC) rates and survival to hospital discharge. 6 , 7 , 8 , 9 However, there are considerable knowledge gaps regarding possible benefits of applying this technology in patients with cardiac arrest. 10 We present a case with witnessed OHCA, where dispatcher V-CPR was initiated early and considered essential to provide a tailored approach to a complex OHCA. Section title: Case Description Educational score: 3.772897481918335 Domain: clinical Document type: Clinical case Language: en A 58-year-old male with known paroxysmal atrial fibrillation (AF) sustained an OHCA at 9:47 a.m. while changing tires on his jacked-up car together with his son. An emergency call was received at 9:48 a.m. The patient was reported unconscious with agonal respiration and no CPR was performed. Guided by the Norwegian Index for Medical Emergency Assistance, a stepwise tool for decision-making and to guide callers in emergency medical situations by the Emergency Medical Coordination Centre (EMCC), the patient was identified as unresponsive and not breathing normally. 11 The medical dispatcher immediately instructed the two laypersons on-scene to perform CPR, which they did without hesitation. None had previous CPR experience. Emergency medical services (EMS) and the on-call general practitioner were also immediately dispatched. No other emergency resources (e.g. volunteer first responders) were available during the incident. Since there were two persons on-scene, the medical dispatcher decided to initiate a video call (VC) to better guide the ongoing layperson CPR. The video transmission was initiated two minutes after the emergency call and showed high-quality chest compressions, and a patient with apparent spontaneous respiration and limb movements. After three minutes, they paused compressions according to Norwegian CPR guidelines. 12 The medical dispatcher then observed that the patient‘s breathing pattern changed into agonal respiration. When compressions were continued, the respiration was again considered normal. Because the breathing pattern was deemed normal due to well performed chest compressions, the medical dispatcher decided not to instruct in mouth-to-mouth ventilations according to standard CPR guidelines. The patient therefore received compression-only CPR until arrival of EMS at 10.21 am, 33 min after start of bystander CPR, at which point V-CPR was discontinued. The two laypersons alternated in performing CPR during the whole incident prior to EMS arrival. Section title: Case Description Educational score: 3.6846113204956055 Domain: clinical Document type: Clinical case Language: en On EMS arrival, the patient was still in cardiac arrest and presented with ventricular fibrillation (VF). A direct current (DC) defibrillation with 200 Joule was delivered by EMS personnel before they continued CPR. The patient obtained ROSC at 10:23 am. However, the ROSC only lasted for 30–40 s, and the patient resumed to VF. Another two DC 200 J defibrillations were delivered, but the patient remained with VF. A physician-staffed air ambulance helicopter arrived on-scene at 10:30 am. With ongoing CPR with the patient in VF, the patient showed signs of life with head and limb movements, verbal sounds and a normal respiration pattern with a respiration rate of 12 breaths per minute during chest-compressions. A fourth DC 200 Joule defibrillation was delivered which resulted in sustained ROSC with AF rhythm at 10:33, 46 min after the time of cardiac arrest. Before transportation a rapid sequence induction with intravenous (iv) analgesia (fentanyl 0.2 mg iv), sedation (ketamine 75 mg iv), muscle relaxation (rocuronium 50 mg iv) and endotracheal intubation was performed without any complications. An arterial line was inserted and an intravenous ketamine infusion (1.5 mg/kg/hour) was started for maintenance of sedation during transport to hospital. The patient was hemodynamically stable in-flight with a blood pressure of 150/80 and a pulse rate of 70–90/min, except a brief period of ventricular tachycardia during take-off from the scene. A dose of intravenous amiodarone 100 mg was administered, and the patient converted to AF. The measured oxygen saturation level was 98 % and the patient was normothermic. Transport to the nearest university hospital was uneventful and he was admitted to hospital at 11:59 a.m. Section title: Case Description Educational score: 3.753120183944702 Domain: clinical Document type: Clinical case Language: en The first arterial blood gas showed a metabolic acidosis with pH 7.20, pCO2 5.7 kPa, pO2 12.2 kPa, base excess −11.2 mmol/L, lactate 5.5 mmol/L, bicarbonate 17 mmol/L and glucose 13.6 mmol/L. Other values were normal. A percutaneous coronary intervention was performed with a successful revascularisation and stent implementation of partly occluded left anterior descending and circumflex arteries. A one-chamber cardioverter-defibrillator was implanted and he was discharged to a local hospital for follow-up on day 11, with full neurological recovery. Section title: Discussion Educational score: 4.196712017059326 Domain: biomedical Document type: Study Language: en Access to timely EMS response in time-critical situations (e.g. cardiac arrest) is a vital factor to improve patient outcome. 13 The EMS response time in this case was 33 min, which is often the case in sparsely populated areas, such as rural Norway. 14 However, the massive development of smartphone technology during the last two decades has opened new telemedicine possibilities in modern healthcare. 15 The use of VC to assist callers and EMS has the potential to compensate for longer response and travel times. However, despite considerable interest in this technological development, the evidence of effect on patient survival and outcome in real-life OHCA is limited as the specific effect of V-CPR has been difficult to isolate. Other promising technological developments, such as the use of drones carrying defibrillators in OHCA, have shown the potential for increased access to advanced devices and additional decision-making, especially in rural areas. 16 , 17 However, like V-CPR, there is a need for further testing in real-life situations to evaluate efficiency and how these interventions impact final outcome. 16 , 17 A recent meta -analysis found that V-CPR can improve bystander CPR during simulated cases, but that the process is substantially affected by poor video signals and lack of guidance procedures. 18 Several studies have found improvements in specific items of the CPR process, with better compression rates and hand placement. 10 , 5 , 6 , 7 In our opinion, the main effect of V-CPR in this case was a more tailored approach and thorough guidance of laypersons without previous CPR experience in a complex setting with long-lasting CPR without back-up of present EMS, resulting in high-quality CPR, CPR induced consciousness (CPRIC) and a good neurological outcome. Section title: Discussion Educational score: 3.5983288288116455 Domain: biomedical Document type: Other Language: en VCs were introduced in 2020 in Norwegian EMCCs for the use at the medical dispatcher’s discretion, in order to increase dispatcher situational awareness and improve the quality of care given by lay persons. 19 Currently, no specific guidelines exist on how VC can be used in the most appropriate way. The solution used in this case was developed by the Norwegian Air Ambulance Foundation and implemented in EMCC nationally in collaboration with the Norwegian Directorate of Health in 2020. 20 The EMCC sends a link to the caller’s mobile phone after approval by the caller, and the video link allows the medical dispatcher to use the available mobile phone camera. 20 , 21 Preliminary evaluations pertaining to general use have described high user-friendliness and that the medical dispatcher’s perception of patient’s acuity was affected in about half of the cases. 19 We strongly believe that video assistance by trained medical personnel has future unsolved potential. However, it is important to recognize the need for 1) clear guidance protocols 2) training in video-assisted dispatch and 3) training in the ability to instruct lay rescuers in psychological distress. It is also important to keep in mind, that the availability of live video transmission from scenes may expose the EMCC dispatchers to unpleasant visual impressions. 22 . Section title: Discussion Educational score: 4.200608253479004 Domain: biomedical Document type: Study Language: en Apparent signs of life (i.e. breathing and head/limb movements) were observed in this case during compressions by the laypersons and after arrival of EMS personnel. This situation added extra complexity, which potentially could have affected the decision to continue or stop ongoing CPR. In case reports and a systematic review describing the phenomena, the observation of breathing efforts has been inconsistently reported. 23 , 24 Agonal gasps or breathing efforts also may be present in patients with cardiac arrest not receiving CPR, so this sign may be less sensitive on CPRIC than movements. Increasing number of CPRIC events are reported, most likely due to a combined effect of community CPR responder programs, improved CPR quality and increased focus on the chain of survival. 25 A noteworthy point is that in a prospective study focusing on cognitive experiences in cardiac arrest survivors, only 2 % of patients retrieved visual or auditory awareness following their incident and no patients remembered any experience of pain. 26 In the described case, the patient could not recall any memory of the incident. Section title: Conclusion Educational score: 2.030858039855957 Domain: clinical Document type: Clinical case Language: en The case represents a complex cardiac arrest with long-lasting CPR, signs of life and no previous CPR experience in lay persons, where V-CPR was instrumental in providing on-scene guidance and decision-making. Section title: CRediT authorship contribution statement Educational score: 0.964204728603363 Domain: other Document type: Other Language: en Steinar Einvik: Writing – review & editing, Writing – original draft, Investigation, Data curation, Conceptualization. Ole Erik Ulvin: Writing – review & editing. Trond Nordseth: Writing – review & editing. Oddvar Uleberg: Writing – review & editing, Writing – original draft, Supervision, Investigation, Data curation, Conceptualization. Section title: Declaration of competing interest Educational score: 0.9818206429481506 Domain: other Document type: Other Language: en The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Clinical case | clinical | en | 0.999997 |
PMC11699613 | Section title: Introduction Educational score: 3.7201437950134277 Domain: biomedical Document type: Review Language: en According to the International Agency for Research on Cancer, female breast cancer (BC) had 2.3 million new cases in 2020, making it the most prevalent cancer worldwide . About 7 % of patients die, mainly due to metastasis. Optimizing diagnostics and discovering new prognostic factors are crucial, alongside well-accepted clinico-pathological factors like biomarker status, patient age, comorbidity, tumor size, grade, and lymph node involvement . Section title: Introduction Educational score: 4.08351993560791 Domain: biomedical Document type: Study Language: en Recently, the histological features of the acellular tumor microenvironment (TME) have gained attention. High breast tissue density, due to increased collagen, is a significant risk factor for BC. Tumor-associated collagen signatures (TACS) may serve as prognostic markers , correlating with tumor grade and prognosis [ , , ]. Notably, TACS5 (aligned collagen fibers) and TACS6 (randomly aligned fibers) at the tumor's invasive front strongly correlate with poor prognosis . Section title: Introduction Educational score: 4.158000469207764 Domain: biomedical Document type: Study Language: en These collagen signatures might act as “migration highways” , facilitating cancer cell dissemination. Cancer cells respond to biomechanical cues from the TME, which influence migration and gene expression [ , , ]. For instance, cells prefer migrating parallel to topographical features , impacting cell morphology and migration efficiency . Matrix alignment, more than stiffness, enhances migration for BC cells by promoting directional persistence . Section title: Introduction Educational score: 4.105955600738525 Domain: biomedical Document type: Study Language: en Topography-based contact guidance depends on cell-cell interactions. Epithelial clusters respond less to topographical alignment than single cells . This underscores the role of EMT, where cancer cells gain mesenchymal traits, enhancing interaction with TME cues. EMT is linked to increased BC malignancy [ , , ], suggesting a synergistic relationship with TACS in metastasis. Section title: Introduction Educational score: 3.84895396232605 Domain: biomedical Document type: Other Language: en Bioengineering applications aim to understand the relationship between tissue-relevant topographies and EMT, which could advance BC diagnostics and treatment targets. Electrospinning, a technique producing fiber mats mimicking native ECM, is valuable in biomedicine . Electrospun scaffolds serve as drug delivery systems, cancer biosensors, and 3D in vitro models . Section title: Introduction Educational score: 4.122466087341309 Domain: biomedical Document type: Study Language: en This study established an electrospun 3D in vitro BC model using poly (ε-caprolactone) (PCL) fibers to mimic TACS. By culturing BC cell lines on this model, the study addressed how TACS-mediated contact guidance and EMT phenotype interrelate, how EMT/MET-like changes influence contact guidance, and how phenotypic changes affect cellular mechanics. This interdisciplinary approach aimed to elucidate the reciprocal dependence of cancer cells and the TME in tumor progression. Section title: Materials and cell culture Educational score: 1.5938268899917603 Domain: biomedical Document type: Other Language: en Polycaprolacton (M N 80.000), 3-(Trimethoxysilyl)propyl methacrylate 98 %, Formaldehyde solution (≥36 %), Dichloromethane (DCM) anhydrous, N,N -Dimethylformamide (DMF) anhydrous, 4′,6–diamidino–2-phenylindole dihydrochloride (DAPI), FluorSave reagent, DNase I (recombinant, RNase-free), cOmplete™, EDTA-free Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail 2, RIPA buffer, Tris buffered saline powder, Ponceau S Stain, Tween 20, Amersham™ Protran® Western-Blotting-Membrane (nitrocellulose), Eagle's Minimum Essential Medium (EMEM), RPMI-1640 Medium, Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS), Penicillin-Streptomycin (Pen/Strep) solution, Dulbecco's Phosphate Buffered Saline (PBS), trypsin-EDTA solution 0.05 % and 0.25 %, 200 mM of L-glutamine solution, doxycycline hydrochloride (DOX) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Taufkirchen, Germany). GAPDH Monoclonal Antibody (ZG003), LIVE/DEAD™ Cell Vitality Assay Kit (C12 Resazurin/SYTOX™), Green Pierce™ BCA Protein Assay Kit, Novex™ 10 % Tris-Glycine Mini Gels (WedgeWell™ format, 15-well), Novex™ Value™ 4–20 % Tris-Glycine Mini Gels (1.0 mm, 10-well), Page Ruler™ Plus Prestained Protein Ladder 10–250 kDa, Tris Glycin transfer buffer, SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Hoechst 33342, Rhodamine Phalloidin, High capacity cDNA synthesis kit, Power SYBR™ Green PCR Master Mix, PureLink™ RNA Mini Kit and Leibovitz's L-15 Medium and MEM Non-Essential Amino Acids Solution (100X) were purchased from Thermo Fisher Scientific (Darmstadt, Germany). Collagen I , Collagen IV , m-IgGκ BP-HRP , COL1A2 antibody (H-9), Anti-Lamin A/C Antibody (636), Anti-Lamin B1 Antibody (B-10), E-cadherin Antibody (G-10) and Vimentin Antibody (V9) were ordered from Santa Cruz Biotechnology (Dallas, TX, USA). Hs_CDH1_Primer Assay , Hs_SNAI1_Primer Assay , Hs_VIM_Primer Assay , Hs_GAPDH_1_SG QuantiTect Primer Assay and Hs_XBP1_1_SG QuantiTect Primer Assay were purchased from Qiagen (Hilden, Germany). Rotiphorese 10× SDS Page, Rotilabo®-Blotting Papers and Methanol (blotting grade) were purchased from Carl Roth (Karlsruhe, Germany). rh-TGF-β 1 (Transforming Growth Factor beta 1) and rh-EGF (Epidermal Growth Factor) were acquired from ImmunoTools (Friesoythe, Germany). Laemmli loading buffer (4×) and round glass coverslips (Ø 13 mm) were purchased from VWR (Ismaning, Germany). HyClone trypan blue solution 0.4 % in phosphate-buffered saline was obtained from FisherScientific (Darmstadt, Germany). Injection Luer Lock needle, blunt end (Ø 0.7 × 30 mm) was purchased from Unimed S.A (Lausanne, Switzerland). Human CD44s Pan Specific Antibody was purchased from R&D Systems (Minneapolis, MN, USA). Braun Omnifix® Syringes (20 mL, Luer) were purchased from Neolab (Heidelberg, Germany). Section title: Materials and cell culture Educational score: 4.206264495849609 Domain: biomedical Document type: Study Language: en MCF7 (and MCF7 miR200c_KO) cells, a Luminal A breast cancer cell line, were cultured in EMEM supplemented with 10 % FBS, 1× Pen/Strep, 1× MEM Non-Essential Amino Acids Solution and 2 mM L-glutamine. The HER2-positive breast cancer cell line HCC1954 was grown in RPMI-1640 Medium supplemented with 10 % FBS and 1× Pen/Strep. MDA-MB-231 (and MDA-MB-231 i-miR200c) cells, a triple negative breast cancer (TNBC) cell line, were cultured in high glucose DMEM, and 10 % FBS, 1× Pen/Strep and 2 mM glutamine were added to the medium. For miRNA induction in MDA-MB-231 i-miR200c cells, the medium was equipped with 5 μg/mL doxycycline hydrochloride which was replenished every 48 h. The latter three cell lines were cultured in a humidified atmosphere with 5 % CO 2 at 37 °C. The second TNBC cell line MDA-MB-468 (DSMZ, Braunschweig, Germany) was grown in L-15 Medium supplemented with 20 % FBS and 1× Pen/Strep. These cells were grown in a humidified incubator with 0 % CO 2 at 37 °C. Section title: Electrospinning of TACS-like structures: set-up and processing of cell culture inserts Educational score: 4.138435363769531 Domain: biomedical Document type: Study Language: en The used electrospinning system was custom made by the Laboratory for Tissue Engineering and Cardiovascular Medical Technology of the Clinic of Cardiac Surgery at the Ludwig-Maximilian-University in Munich. Briefly, the test chamber included air conditioning for organic solvents to evaporate and contained molecular sieves to reduce humidity within the chamber. Either a static aluminum plate or a dynamic rotating cylinder was used as collectors to produce unaligned or aligned fibers, respectively. The cylinder (Ø 150 mm) itself was assembled out of 3D-printed constructs using the Keyence Agilista 3200W printer. The use of non-conductive AR – M2 (Keyence Corp, Osaka JP) acrylic polymer necessitated an additional wrapping step with aluminum foil to enable electrostatic attraction. A NEMA 17 motor (Nanotec Electronic GmbH, Feldkirchen, Germany) connected to a computer via an associated control card was used to drive the cylinder. The speed and direction of rotation were set using the “Plug & Drive Studio” (Nanotec) software. A high-voltage power supply from iseg GmbH (Radeberg, Germany) with a voltage range of 0–30 kV (positive and negative) was used to generate the electric field. The voltage supply was coupled to the needle and the collector by means of a high-voltage cable. The flow rate of the polymeric solution was controlled by a laboratory syringe pump (Fusion 100, Chemyx Inc., Stafford, TX, USA). Section title: Electrospinning of TACS-like structures: set-up and processing of cell culture inserts Educational score: 4.302924156188965 Domain: biomedical Document type: Study Language: en PCL (20 g) was dissolved in 100 mL of DCM/DMF (40/60) 24 h prior to the electrospinning process. The solution was stirred over night at 400 rpm covered with aluminum foil to avoid evaporation of the organic solvents and degradation of PCL. To increase adhesion of fibers to glass coverslips, coverslips were incubated for 5 min in 4 % ( v/v ) of 3-(Trimethoxysilyl)propyl methacrylate in absolute ethanol. The solution was removed and rinsed 3 times with pure ethanol. Coverslips were subsequently baked at 100 °C for 40 min. To produce cell culture inserts, optimized (methacrylated) coverslips were mounted either on the plate collector or rotating cylinder using double-sided adhesive tape as can be seen in Fig. S1a . The rotation speed was set to 1800 rpm. A 20 mL syringe was loaded with the polymer (PCL) solution and connected to the syringe pump. The flow rate was set to 1.0 mL/h. The high-voltage supply (+7–10 kV) was connected to a needle (Ø 0.7 × 30 mm) placed 21–23 cm away from the collector. Opposite, constant charge was applied to the collector (-1 kV). In case of the rotating cylinder, a sliding contact was used. To assure constant fiber deposition during the 10 min of the electrospinning process, Taylor cone formation was monitored at the tip of the nozzle. As shown in Fig. 1 , fiber-coated coverslips (FCCs) were consequently either subjected to SEM analysis or further processed to fulfill cell culture requirements. To remove excess amounts of organic solvents, FCCs were placed in a chemical hood for 48 h. Afterwards FCCs were sterilized using UV-light for 1 h and thereafter placed into a 24-well plate under sterile conditions where they were exposed to UV-light for another hour. Subsequently, for cell culture work, FCCs were coated with collagen (I/IV) as described elsewhere . Methacrylated glass coverslips coated with collagen were used as reference samples (referred to as blank or conventional) for all methods, if not stated otherwise. For AFM and live-cell imaging purposes, collagen-coated FCCs were fixed onto the surfaces of culture dishes. Therefore, 3 μL of silicon-based glue were placed in the center of the bottom side of the FCCs and let dry under sterile conditions for 1 h prior to cell seeding. For the seeding process, 25.000–50.000 cells were suspended in 200 μL of the respective medium and then slowly pipetted on the scaffold starting at the center and spirally approaching the margin of the FCC with a blob emerging and covering the entire FCC. After 15 min of incubation, allowing cells to sediment onto the scaffold, each well was filled up with pre-warmed medium to 1 mL and subjected to various analyses. Fig. 1 (a, b) SEM image analysis: Upper panel shows representative SEM images of (a) TACS6- and (b) TACS5-like structures (10.000× magnification); mid panel highlights single fiber orientations with same colors representing same fiber orientation; lower panel depicts distribution intensity of fiber orientation (−90°–90°). (c) Fiber diameters and (d) inter-fiber distances of TACS5- and TACS6-like structures analyzed with Fiji software. (e) Residual amount of organic solvents (DCM and DMF) in ng/mg fiber were analyzed with HS-GC-MS. Errors indicate standard error of mean (SEM). Fig. 1 Section title: Scanning electron microscopy (SEM) Educational score: 4.132721900939941 Domain: biomedical Document type: Study Language: en The topography of TACS5- and TACS6-like scaffolds (FCCs) were examined by scanning electron microscopy (SEM) using a JSM-6510LVLGS, 25 kV (JEOL, Peabody, MA, USA). For imaging, scaffolds were sprinkled on a stub covered with double-sided carbon tape and sputter-coated with gold (Ernest Fullan) under vacuum for 60 s. Topographic features including fiber orientation, diameter and density were analyzed from the SEM images by processing with the Fiji imaging software (Version 2.30/1.53q) . Fiber orientation and distribution of orientation were analyzed from representative samples of each collector type using the OrientationJ plug-in (written by Daniel Sage). Fibers represented with the same color indicate same directionality. Fiber diameters and inter-fiber distances were manually calculated from 10.000× images taken from samples of 2 independent electrospinning runs per collector type. Section title: GC analysis – residual solvents Educational score: 4.169246673583984 Domain: biomedical Document type: Study Language: en Residual DCM and DMF content was analyzed by static headspace-gas chromatography-mass spectrometry (HS-GC-MS). An Agilent Technologies 7890B gas chromatograph (Waldbronn, Germany), equipped with an Agilent J&W DB-624 UI ultra-inert capillary column (6 % cyanopropyl phenyl and 94 % polydimethylsiloxane) 30 m × 0.25 mm × 1.4 μm and an Agilent Technologies 7010B triple quadrupole detector with high efficiency source (HES) was used for analysis. Helium (99.999 %) was used as mobile phase. DCM (HPLC grade), 1,2-dichloroethane (DCE, purity 97 %), DMF (purity HPLC grade), DMF-d7 (purity 99.5 %), and DMSO (purity HPLC grade) were purchased from Sigma-Aldrich (Schnelldorf, Germany). As samples, 15 mg fiber mesh (obtained from three independent electrospinning runs, respectively) were filled into a 20 mL headspace vial, 10 μL of DMF-d7 and DCE (each 10 μg/mL in DMSO) as internal standards were added, and the vial was closed tightly. After sealing, the sample was analyzed by HS-GC-MS (see Table S1 ). The MS was operated in single ion monitoring mode (SIM; EI 70 eV). The retention times and characteristic ions of DCM (5.6 min, m / z 84.1), DCE (internal standard; 8.2 min, m / z 62.1), DMF (9.8 min, m / z 73.2), and DMF-d7 (internal standard; 9.9 min, m / z 80.2) were used for identification and quantification. For detailed information on HS-GC-MS conditions, see Table S1 in the supplements. Section title: Cell viability Educational score: 4.107102394104004 Domain: biomedical Document type: Study Language: en The cell viability of cells growing on TACS5- and TACS6-like (data not shown) structures was assessed by applying the LIVE/DEAD™ Cell Vitality Assay Kit (Invitrogen™) and subsequent fluorescence microscopy. For each sample, 35.000 MDA-MB-231 cells were seeded on collagen I – coated tissue plates, uncoated FCCs and coated FCCs. All samples were cultivated for 72 h and then stained according the manufacturer's protocol. Untreated cells on conventional culture dishes served as positive (live/red) control, whereas negative (dead/green) control cells were incubated for 30 min with ethanol 70 % ( v/v ) prior to the analysis. Fluorescence images were recorded with the Keyence BZ81000 Fluorescence microscope (Keyence, Osaka, Japan). Images of transmitted light, green and red channel were taken simultaneously with a 20× objective and further analyzed with Fiji software. Percentages of living/dead cells was calculated in duplicates (with each sample >100 cells), counting red/green fluorescence signals (cells) of the respective channels. Section title: Confocal scanning microscopy – morphological changes Educational score: 4.264739036560059 Domain: biomedical Document type: Study Language: en Confocal laser scanning microscopy was used to assess morphological changes across different cell lines growing on glass coverslips, TACS5- and TACS6-like structures. As performed with the FCCs, coverslips were coated with collagen I and IV. Thereinafter, 35.000–50.000 cells of the respective cell lines were seeded in 24-well plates following the protocol described above. In case of growth factor (GF) treatment, cells were allowed to attach within the first 4 h. Medium was then removed and replenished with GF-containing medium. Samples were either supplied with 10 ng/mL of TGF-β1 or 25 ng/mL of EGF (MDA-MB-468). DMEM medium used for miR200c induction in modified MDA-MB-231 cells contained 5 μg/mL of doxycycline hydrochloride (DOX). After 72 h of incubation, wells were washed 3 times with PBS before cells were fixed for 15 min with a 4 % formaldehyde solution. To stain the actin cytoskeleton, cells were incubated with 8.25 μM rhodamine-phalloidin solution for 40 min. Hereinafter, cells were washed another 3 times with PBS. Nuclei were stained with a 0.5 μg/mL DAPI solution for 10 min. Finally, after an additional washing step, samples were mounted on glass slides using FluorSave and stored at 4 °C until the next day. Fluorescence images were acquired using a confocal laser scanning microscope (Leica SP8 inverted, Software: LAS X, Leica microsystems GmbH, Wetzlar, Germany) equipped with a HC PL APO CS2 63×/1.40 oil immersion objective. Diode lasers (405 nm) and a semiconductor laser OPSL (552 nm) were chosen for excitation. Emission was detected in blue (PMT1: 410–520 nm) and yellow (PMT2: 560 nm–760 nm), respectively. Images were further processed with Fiji image analysis software. Nuclear circularity was calculated as C N = 4 π A P 2 , (n > 30; A: area, P: perimeter), and the cellular aspect ratio as A R = d min d max , (n > 15). The axes (d min and d max ) were drawn manually. Results are given as Whiskers Plots with error bars indicating minimal and maximal values. One-way ANOVA with Bonferroni multiple comparison test was performed in GraphPad Prism software version 5.00 (Graph Pad Software, La Jolla, CA, USA) to calculate P-values at a 95 % confidence interval. Section title: Live cell imaging – motility and contact guidance Educational score: 4.191780090332031 Domain: biomedical Document type: Study Language: en Live cell imaging was performed using the Eclipse Ti inverted microscope (Nikon, Düsseldorf, Germany) with a 4/10 phase contrast objective and a charge-coupled device camera (DS-Qi1Mc; Nikon). After 48–72 h of incubation, nuclei of cancer cells were stained with Hoechst 33342 dye (Invitrogen™) according to the manufacturer's protocol. Briefly, cells were washed once with PBS and subsequently incubated for 20 min in the cell culture incubator in 300 μL of 0.5 μg/mL dye in PBS. Afterwards cells were washed another 2 times, and 1 mL of the respective medium was replenished. The 24-well plates were inserted into a 37 °C heating and incubation system that was flushed with actively mixed 5 % CO2 (0 % in case of MDA-MB-468 cells) at a rate of 10 L/h, and the humidity was kept at 80 % to prevent dehydration of cells. The cells were imaged in bright-field, and the nuclei were detected at 405 nm using the integrated fluorescence LED. Time-lapse videos were taken with a time interval of 5 min between images over 24 h. Section title: Tracking of single-cell trajectories Educational score: 4.217254638671875 Domain: biomedical Document type: Study Language: en For single-cell trajectories, the position of nuclei was analyzed using the TrackMate plugin within the Fiji software . To extract nuclear circularity trajectory, the image series of the 405 nm channel was manually processed ( Enhance contrast (5.0 %) ≫ 2× Smooth ≫ Convert to mask ) and further analyzed with “Mask detector” function of the TrackMate plugin (see Table 1 ). Table 1 Description of variables calculated with the “Track analyzer” function of the TrackMate plugin (Fiji software) , which was used to evaluate differences in contact guidance amongst cell lines and treatments. Table 1 Track displacement Confinement Ratio Mean directional change Measure of the distance [μm] between first (0 h) and last spot (24 h) of a track. Indicative for how far a cell has migrated away from its starting point but not total distance it has traveled. = Linearity of forward progression = t r a c k d i s p l a c e m e n t [ μ m ] t o t a l d i s t a n c e [ μ m ] The confinement ratio indicates how efficient a cell displaced from its origin. Measures the angle between two succeeding links, which is then averaged over all links of a track. Low values indicate high consistency of directed migration/migratory persistence and vice versa . Section title: EMT-marker expression – RT-qPCR Educational score: 4.126680374145508 Domain: biomedical Document type: Study Language: en In 24-well plates, 40.000 cells of the respective cell line were seeded in triplicates. Importantly, to allow for the best possible comparison, all cells were seeded on collagen I coated coverslips or coated FCCs. GF and DOX supply was performed as described above. After 72 h incubation, cells were washed 3-times with PBS. Subsequently, 300 μL of ethanol-containing lysis buffer was added for 15 min. In order to maximize the amount of RNA for analysis, triplicates were pooled together prior to the RNA isolation step. Total RNA was isolated using the PureLink RNA mini kit according to the manufacturer's protocol with additional DNAse I digestion. Subsequently, 500 ng of RNA was used to synthesize cDNA using the High capacity cDNA synthesis kit. In the following, E-cadherin (CDH1)-, Snail (SNAI1)-, Vimentin (VIM)-, and XBP1-specific primers were used to amplify and quantify RNA using Power SYBR™ Green PCR Master Mix and the qTOWER real-time PCR thermal cycler (Analytik Jena, Jena, Germany). C t values were normalized to GAPDH RNA expression, and delta C t values were calculated for the comparison. One-way ANOVA with Bonferroni multiple comparison test was performed in GraphPad Prism software (Graph Pad Software, La Jolla, CA, USA) to calculate P-values at a 95 % confidence interval (n = 4). Section title: EMT-marker protein levels – Western blotting Educational score: 4.177305221557617 Domain: biomedical Document type: Study Language: en In 24-well plates, 40.000 cells of the respective cell line were seeded in triplicates. Importantly, to have the best comparison possible, cells were seeded on collagen I coated coverslips or coated FCCs (here: TACS5). DOX was supplied as described above. After 72 h incubation, cells were washed 3 times with PBS prior to cell lyses. Subsequently, 60 μL of proteinase- and phosphatase-inhibitor containing RIPA buffer was added to one well (of a triplicate) and cells were kept on ice for 20 min. Thereinafter, wells were thoroughly scraped and the extract was transferred to the next well of a triplicate. This step was repeated again for the last well of a triplicate (including 20 min of incubation). This step was necessary to maximize the protein yield. Finally, pooled triplicates were transferred into 1.5 mL microcentrifuge tubes. After a 10 min centrifugation step at 4 °C, total protein concentration was assessed according to the manufacturer's protocol (Pierce™ BCA Protein Assay Kit). Gels were loaded with 30 μg protein per sample, and electrophoresis was run for 90 min at 120 mV. Subsequent to 1 h of protein transfer at 100 mV, blots were washed, blocked and incubated overnight using CD44s-, CDH1-, VIM- and GAPDH-specific antibodies. HRP-bound secondary antibody was then added for 1 h under exclusion of light before blots were developed. Section title: Atomic force microscopy (AFM) Educational score: 4.13559627532959 Domain: biomedical Document type: Study Language: en All stiffness measurements were performed on a JPK Nano Wizard 4 in cell culture media using the AFM's contact mode (force spectroscopy mode). Tipless MLCT-D cantilevers (silicon nitride, resonance frequency 15 kHz, spring constant 0.03 N/m) glued to a glass bead (diameter microscopically determined) were used to indent the cells. These were calibrated with the contact-free method in liquid. The following parameters were used: set point 1 nN, z-length 10 μm and velocity 5 μm/s. The Petri dish was mounted onto the AFM stage and the cells were kept in cell culture media. A digital petri dish heater attached to the AFM stage was used to maintain the physiological temperature of 37 °C during the indentation. For every sample, 3–5 cells were indented overall, and force curves were collected. For measuring nuclear stiffness, the area of the nucleus was selected for the acquisition of force curves. One-way ANOVA with Bonferroni multiple comparison test was performed to calculate P-values at a 95 % confidence interval . Section title: Atomic force microscopy (AFM) Educational score: 4.16578483581543 Domain: biomedical Document type: Study Language: en For imaging the enhanced imaging QI mode of the AFM and pyramidal tip MLCT-D silicon nitride cantilevers were used (spring constant 0.03 N/m and resonance frequency 15 kHz), and the following parameters were taken: set point 1 nN, z-length 2500 nm, pixel time 40 ms. The acquiring area of the grid was set according to the cell size, and the pixel ratio was 256 ✕ 256. The data were analyzed using JPK data processing software. The stiffness/elastic modulus of the cells (Young's modulus) was calculated with the Hertzian contact model according to Ref. : E = 3 4 ∙ F ∙ ( 1 − υ 2 ) δ 3 ∙ R Section title: Optimized electrospinning set-up enables mimicry of TACS5 and TACS6 architecture Educational score: 3.5475761890411377 Domain: biomedical Document type: Other Language: en During electrospinning, the polymer jet from the nozzle experiences physico-chemical instabilities , causing random fiber deposition on the collector ( e.g. plate collector). These instabilities can be mitigated by using a rotating cylinder instead of a static collector, resulting in straight, parallel fibers. Section title: Optimized electrospinning set-up enables mimicry of TACS5 and TACS6 architecture Educational score: 4.175631046295166 Domain: biomedical Document type: Study Language: en For cell culture inserts, glass coverslips were mounted on the collectors and coated with fibers during spinning . Scanning electron microscopy (SEM) images of fiber-coated coverslips (FCCs) revealed strong fiber alignment and straightening (“TACS5-like structures”) for samples spun with the rotating cylinder compared to random deposited fibers (“TACS6-like structures”) obtained from the plate collector . Image analysis confirmed efficient alignment (equally colored fibers) of TACS5-like structures with a narrow peak angle distribution of 30° and an intensity of 80,000, versus the chaotic deposition of TACS6-like structures ( Fig. 1 a, (baseline: 7,000; maximum: 15,000). Both setups had similar fiber diameters and inter-fiber distances , with unaligned fibers slightly larger at 0.760 μm and 2.405 μm compared to 0.664 μm and 2.022 μm for aligned fibers, respectively. Thus, fiber orientation was adjusted without significantly affecting individual fiber characteristics. Section title: Collagen coating of fibers improves biocompatibility and cellular spreading on TACS-mimics Educational score: 4.120977401733398 Domain: biomedical Document type: Study Language: en The possible toxic effects of residual solvents or incompatibilities on cell growth were evaluated. GC analysis showed complete evaporation of DCM (below 10 ng) and negligible DMF residues (1.65 ng/mg), making it unlikely for residual solvents to affect cell viability . However, PCL is highly hydrophobic and unsuitable for cell attachment. To improve tissue mimicry and enhance cellular attachment, collagen coatings were applied. Confocal microscopy confirmed effective collagen deposition on fibers . Section title: Collagen coating of fibers improves biocompatibility and cellular spreading on TACS-mimics Educational score: 4.127491474151611 Domain: biomedical Document type: Study Language: en Biological compatibility of the in vitro model was assessed using a fluorescence-based cell viability assay. Fig. S1c shows that cell viability on coated fibers remained high (4.6 % dead cells), with collagen coating enabling widespread cellular infiltration. Without coating, toxicity tripled to 15.1 %, with cells clustering and indicating poor matrix infiltration. Thus, the hydrophobic nature of the PCL fiber surface, not solvent residues, threatened cell viability. Due to the improved compatibility and bio-similarity of coated fibers, collagen coatings were used in subsequent evaluations. Section title: TACS5-mimicking topographies induce EMT-like changes Educational score: 4.11787223815918 Domain: biomedical Document type: Study Language: en In a previous in vitro breast cancer study, our group established an EMT phenotyping system based on EMT-marker expression, morphological changes, and cellular motility. MCF7 and HCC1954 cell lines were designated EMT-negative, MDA-MB-468 as E/M-hybrid, and MDA-MB-231 as EMT-positive. Significant changes in cellular and nuclear morphologies, such as aspect ratio ( A R ) and nuclear circularity ( C N ), predicted EMT-like changes on the protein level during growth factor-dependent EMT . Section title: TACS5-mimicking topographies induce EMT-like changes Educational score: 4.235651969909668 Domain: biomedical Document type: Study Language: en Morphologically, the three studied cell lines remained unaffected when grown on TACS6-like structures as revealed by confocal imaging . Similarly, growth on TACS5-mimicking scaffold did not induce prominent changes in A R and C N , neither in MCF7 nor in HCC1954 cells . However, TACS5-like fibers caused a significant decrease of A R and C N in MDA-MB-468 cells, indicating pronounced cellular alignment on the scaffold. Cancer cells dispersed within the scaffold in contrast to conventional 2D-cell culture and TACS6-like scaffolds where cells formed sheet-like aggregates. On TACS5-like scaffolds, however, cells lost their cobblestone-like morphology and stretched into spindle-like shape . Similarly, EMT-positive MDA-MB-231 cells aligned with fiber orientation, implying a correlation between EMT-status and cellular alignment . Significant C N alterations were only observed in MDA-MB-468 and MDA-MB-231 cells. Fig. 2 Confocal images (63×) and morphological analysis of (a) MCF7, (b) HCC1954 and (c) MDA-MB-468 cells growing on conventional cell culture dishes and TACS-like scaffolds. The blue fluorescence (DAPI) represents the nucleus and the red fluorescence (Rhodamine-Phalloidin) depicts the actin cytoskeleton. Yellow arrows indicated mesenchymal-like morphologies. The graphs below demonstrate the morphological features, nuclear circularity C N (left) and aspect ratio A R (right), which were analyzed and calculated with the Fiji image software and plotted as Whiskers plot in GraphPad Prism 5. (d) Schematic representation of relative mRNA-expression of EMT-relevant markers (CDH1, VIM, SNAI1, XBP1) in MCF7, HCC1954 and MDA-MB-468 cells after 72 h incubation with growth factors or growth on TACS5-mimics. Colors define changes in relative mRNA expression as shown in the right box (green: down; grey: unchanged; orange: up). (e) Mean migration speed of the latter three cell lines was recorded either on conventional coverslips or on TACS5-mimics. Means were calculated based on three individual videos with n (cells) > 50. Errors indicate standard deviation (SD). (f) Western blot of MDA-MB-468 cells stained for CDH1 and VIM after 72 h incubation on conventional coverslips or TACS5-mimics. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05). Stars in brackets (∗) indicate statistical significance after exclusion of 1 out of 4 biological replicates. Fig. 2 Section title: TACS5-mimicking topographies induce EMT-like changes Educational score: 4.153111934661865 Domain: biomedical Document type: Study Language: en To correlate morphological changes on TACS5-like fibers with EMT-related gene expression, qPCR analysis was performed, also after growth factor treatments known to induce EMT-like changes . Expression levels of E-cadherin ( CDH1 ) and Vimentin ( VIM ) and mRNA levels of the EMT transcription factors SNAI1 and XBP1 (X-box binding protein 1) were monitored. Snail is known to downregulate CDH1 and upregulate VIM , while XBP1 has been recently linked to EMT and metastasis, impacting cancer progression and therapy outcomes . Section title: TACS5-mimicking topographies induce EMT-like changes Educational score: 3.66654372215271 Domain: biomedical Document type: Study Language: en Fig. 2 d summarizes relative mRNA expression with green indicating downregulation and orange representing upregulation of gene expression. TACS5-like topographies decisively influenced gene expression depending on the cell line, with accessibility to contact guidance cues increasing with EMT-status . Section title: TACS5-mimicking topographies induce EMT-like changes Educational score: 4.194097995758057 Domain: biomedical Document type: Study Language: en Overall, CDH1 expression was equally downregulated across cell line and stimulatory cue. Fiber topography induced significant upregulation of XBP1 (P < 0.001) in MCF7, VIM (≈1.7 fold) in HCC1954, and XBP1 (≈2.3 fold, P < 0.01), SNAI1 (≈2.1 fold) and VIM (≈1.9 fold) expression in MDA-MB-468. Western blotting confirmed topography-induced downregulation of CDH1 protein levels in MDA-MB-468, while VIM protein levels remained unaffected . Although similar EMT-related genes were affected by contact guidance, growth factor-mediated mRNA expression changes were 2–3 magnitudes higher . Section title: TACS5-mimicking topographies induce EMT-like changes Educational score: 4.195612907409668 Domain: biomedical Document type: Study Language: en Another feature comprised in EMT-like changes is accelerated cellular motility. Live cell imaging estimated mean migration speed on conventional coverslips and TACS5-mimicking scaffolds. Fig. 2 e demonstrates that TACS5-like topographies strongly influenced random cell migration. Mean speeds of MCF7 and HCC1954 cells decreased by 28 % and 38 %, respectively, while velocity of MDA-MB-468 significantly increased by 22 % on the fibrous matrix. Differences in motility and contact guidance among all four cell lines on the TACS5 model were measured. Besides mean migration speed, three trajectory features indicative of directional persistence were analyzed (see Table 1 ). Directional persistence increases with higher track displacement and confinement ratio but decreases with mean directional change. MDA-MB-468 and MDA-MB-231 displaced significantly further (46 % and 144 %) than MCF7 and HCC1954 over 24 h . Fig. S2e shows that MDA-MB-468 cells moved fastest (4.76 nm/s), followed by MDA-MB-231 (3.32 nm/s), MCF7 (2.67 nm/s) and HCC1954 (2.12 nm/s). MDA-MB-231 cells migrated most efficiently along the fibers, with the highest confinement ratio and the lowest mean directional change . Section title: TGF-β stimulation enhances contact guidance on TACS5-mimicking scaffolds and topographical cues potentiate EMT in HCC1954 Educational score: 4.252986431121826 Domain: biomedical Document type: Study Language: en Cancer cells gain phenotypic plasticity during EMT-like changes, enhancing cell-substrate interactions within the TME . Efficient migration through a porous matrix requires nuclear deformation, as the nucleus is the greatest cell organelle and dictates cellular migration and invasion in cancer . Since MCF7 and HCC1954 cell migration slowed down on TACS5-mimicking scaffolds, without elongation of cellular and nuclear morphologies , EMT was induced using growth factors (EGF, TGF-β1) and ECM components (collagen I/IV), promoting cancer cell spreading and alignment within the scaffolds . Growth factor treatment reduced cell-cell contacts (green arrows) and increased membrane protrusion (yellow arrows) prominently, fostering a mesenchymal-like morphology (yellow arrows) on TACS5-like mimics, especially in TGF-β1-treated HCC1954 cells. This led to alignment with fiber orientation on the TACS5-mimicking scaffolds and significantly decreased C N values on TACS5-like but not TACS6-like structures indicating the importance of unidirectional fiber orientation and growth factor treatment. Similar results were observed in the MCF7 cell line , where growth factors enhanced cell-fiber interaction and promoted mesenchymal-like morphologies (yellow arrows) in comparison to untreated cells clustered in sheet-like aggregates (green circle). Fig. 3 Morphological alignment of nuclei of HCC1954 cells on (a) TACS5- and (b) TACS6-like scaffolds depending on growth factor stimulation (EGF, TGF-β1) and growth surfaces (collagen I/IV). C N -values were plotted as Whiskers plot. (a, b) The dotted line represents the mean C N -value of control cells growing on conventional culture dishes. (c) Relative mRNA expression of CDH1 and VIM in HCC1954 cells. ΔΔC t -values were calculated after 72 h of either growth on TACS5-mimics and/or treatment with 10 ng/mL TGF- β1 (n = 2). Error bars depict SD. (d-g) Comparison of trajectory analysis of untreated (control) HCC1954 cells and cells treated with TGF-β1 using Fiji software (TrackMate). Displacement, mean migration speed, confinement ration and mean directional change were plotted as whiskers plot (5–95 percentiles). Statistical significance was assessed with a t -test based on > 200 cells/condition. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05, ns = not significant). Fig. 3 Section title: TGF-β stimulation enhances contact guidance on TACS5-mimicking scaffolds and topographical cues potentiate EMT in HCC1954 Educational score: 4.135660171508789 Domain: biomedical Document type: Study Language: en To evaluate combinatorial cue effects, mRNA expression of CDH1 and VIM in HCC1954 was analyzed. Fig. 3 c displays equally downregulated CDH1 mRNA levels (18–20 %) among the three conditions. However, the combined treatment significantly increased VIM expression, showing a 15.2-fold increase compared to a 11.6-fold increase upon TGF-β1 treatment alone. These results demonstrated that TACS5-mimicking scaffolds alone can induce EMT-like changes in HCC1954 cells which is fostered by combinatorial action with GFs. Section title: TGF-β stimulation enhances contact guidance on TACS5-mimicking scaffolds and topographical cues potentiate EMT in HCC1954 Educational score: 4.1576337814331055 Domain: biomedical Document type: Study Language: en Trajectory analysis revealed that TGF-β1-induced EMT significantly affected HCC1954 migration on TACS5-scaffolds. Cells migrated 70.5 % further and 39.3 % faster than control cells. With a 23 % increase in confinement ration and 12 % decrease in mean directional change, cells migrated more efficiently on the fibrous matrix, indicating reinforced contact guidance. Section title: Mesenchymal-to-epithelial-transition (MET)-like changes impair motility and contact guidance on TACS5-mimicking scaffolds Educational score: 4.114572048187256 Domain: biomedical Document type: Study Language: en The data indicated a reciprocal dependence between topographical cue and cancer phenotype. Growth factor-induced EMT-like changes enhanced cancer cells’ ability to use topographical input for cellular migration. To further confirm this observation and explore if MET-like changes reverse these effects, two genetically modified breast cancer cell lines were studied: miR200c knock-out MCF7 cells (EMT cell line), and miR200c-inducible expression in MDA-MB-231 cells (MET cell line) under doxycycline (DOX) exposure . Section title: Mesenchymal-to-epithelial-transition (MET)-like changes impair motility and contact guidance on TACS5-mimicking scaffolds Educational score: 4.201364517211914 Domain: biomedical Document type: Study Language: en First, CDH1 and VIM mRNA levels were measured in unmodified MCF7 cells (MCF7_WT), miR200c knock-out MCF7 (MCF7_KO), and inducible MDA-MB-231 (MB231_i-miR200c) cells with and without DOX stimulation. MiR200c knock-out in MCF7 decreased CDH1 expression by 50 % and increased VIM mRNA levels 3.2-fold. DOX-induced miR200c expression in MDA-MB-231 cells increased CDH1 mRNA 3.0-fold and decreased VIM expression by 40 % . CDH1 and VIM protein levels changed similarly, and CD44s protein levels, overexpressed in cancer stem cells and during EMT , were partially depleted upon DOX stimulation indicating MET . Fig. 4 (a) Relative expression of CDH1 and VIM mRNA in MCF7_miR200c_KO and DOX-treated MDA-MB-231_i-miR200c (MB231_i-miR200c) cells normalized to wild type (unmodified MCF7 or untreated MB231_i-miR220c, respectively). (b) Western blot of MB231_i-miR200c cells with and without doxycycline-induction stained for EMT-relevant marker CDH1, VIM and CD44s. (c) Whiskers plot showing alterations in C N -values (from 40× images) in MB231_i-miR200c cells with and without doxycycline-induction for growth on TACS5- and TACS6-like structures. One-way ANOVA with Bonferroni multiple comparison test was performed to calculate P-values at a 95 % confidence interval (n (cells) > 50). (d) Corresponding confocal images of (c) at 10× magnification. Cells were stained for nuclei (blue) and the cytoskeleton (red). Comparison of trajectory analysis of (e) WT cells with MCF7_KO or (f) MB231_i-miR200c, respectively, performed with Fiji software (TrackMate). Displacement, mean migration speed, confinement ration and mean directional change were plotted as whiskers plot (5–95 percentiles). Statistical significance was assessed with a t -test based on > 150 cells/condition. Stars indicate statistical significance (∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05, ns = not significant). Fig. 4 Section title: Mesenchymal-to-epithelial-transition (MET)-like changes impair motility and contact guidance on TACS5-mimicking scaffolds Educational score: 4.088407039642334 Domain: biomedical Document type: Study Language: en Second, nuclear circularity and cell growth of MB231_i-miR200c cells in TACS5- and TACS6-mimicking scaffolds were monitored. After 48 h, untreated cells aligned with fiber orientation , while DOX-induced miR200c expression impeded morphological polarization on aligned fibers . C N analysis confirmed this with elevated values for DOX-treated cells and no significant differences between TACS5 and TACS6-like scaffolds . Section title: Mesenchymal-to-epithelial-transition (MET)-like changes impair motility and contact guidance on TACS5-mimicking scaffolds Educational score: 4.1665544509887695 Domain: biomedical Document type: Study Language: en Third, live cell imaging-based trajectory analysis evaluated the effect of EMT-like (MCF7_KO) or MET-like (MB231_i-miR200c) changes on cellular motility on TACS5 mimics. miR200c KO increased migration speed significantly but did not improve contact guidance; track displacement remained unchanged, confinement ratio decreased (P < 0.001), and mean directional change increased (P < 0.001) . Conversely, miR200c induction in MDA-MB-231 cells reduced mean speed (WT: 3.625 ± 0.068 nm/s vs. i_miR200c: 3.151 ± 0.064 nm/s) and impaired contact guidance, affecting track displacement, confinement ratio and mean directional change . Overall, MET-like changes restricted contact guidance by TACS-like structures. Section title: EMT-status inversely correlates with total cellular stiffness Educational score: 4.227295398712158 Domain: biomedical Document type: Study Language: en Total cellular stiffness and nuclear stiffness of single cells were estimated using atomic force microscopy (AFM) on either the whole cell or the nucleus. The Elastic Young's modulus [Pa], a good approximation of cellular stiffness , was quantified. Cell mechanics varied significantly among the four cell lines. As demonstrated in Fig. 5 a, total cellular stiffness decreased with EMT-status, from 769.5 ± 54.5 Pa (MCF7) to 372.1 ± 20.8 Pa (MDA-MB-231). This trend was not observed for nuclear stiffness. While MCF7 cells had the highest Young's modulus (590.0 ± 280.0 Pa), the nuclear stiffness of the other three cell lines did not differ significantly . However, in Fig. 5 c, nuclear stiffness of the most invasive MDA-MB-231 cells contributed to the overall stiffness (92 %), whereas this contribution was less important for the other three cell lines (55–68 %). This indicates that the nucleus of this mesenchymal cell line is the main physical restriction for invasion. Lamins (A/C, B), nuclear envelope proteins, are thought to determine nuclear physical properties. Contrary to the literature , high levels of Lamin B1, instead of Lamin A/C, correlated with increased nuclear stiffness in the studied breast cancer cell lines . Section title: Early EMT-like events entail cellular/cytoskeletal stiffening during biochemically and topographically induced EMT Educational score: 4.148027420043945 Domain: biomedical Document type: Study Language: en These findings imply that EMT induction would decrease total cellular stiffness and nuclear stiffness. To challenge this hypothesis, MDA-MB-468 cells were stimulated with EGF for 24 h or 48 h to undergo EMT, and their biomechanical properties were analyzed . After 24 h, epithelial cancer cells adopted a spindle-like morphology . Contrary to expectations, total cellular stiffness significantly increased upon EGF-treatment, with Young's moduli rising by 39.6 % and 28.9 %, respectively . Nuclear stiffness also increased by 17.6 % within the first 24 h. However, after 48 h, the Young's modulus decreased by 21.4 % below the values of control cells, indicating nuclear softening . Section title: Early EMT-like events entail cellular/cytoskeletal stiffening during biochemically and topographically induced EMT Educational score: 4.227489948272705 Domain: biomedical Document type: Study Language: en To validate these findings, EMT was also induced in HCC1954 cells. Confocal scanning microscopy was used to monitor changes in the actin cytoskeleton, which is key to cell elasticity. Total cellular stiffness significantly increased upon TGF-β1 stimulation , with the Young's modulus rising by 117.9 % , surpassing the EGF-induced increase in MDA-MB-468 cells. However, nuclear stiffness did not change significantly upon TGF-β1 treatment. Image analysis illustrated cytoskeletal rearrangements explaining the dramatic cell stiffening. Control cells had a crosslinked, randomly orientated actin network , while TGF-β1-induced EMT elongated HCC1954 cells and restructured actin bundles into stress fibers . Actin fibers became significantly more parallel compared to the network-like structure in control cells, indicated by increased anisotropy . Section title: Early EMT-like events entail cellular/cytoskeletal stiffening during biochemically and topographically induced EMT Educational score: 4.155570030212402 Domain: biomedical Document type: Study Language: en Next, the effect of cell growth on TACS5-topographies on biomechanical changes was assessed to correlate EMT-marker expression changes with cell mechanics. Cellular stiffness was compared between cells growing on collagen I-coated coverslip and coated fibers. The Young's modulus remained unaffected in MCF7 cells and slightly increased in MDA-MB-231 cells on TACS5-mimicking scaffold. Interestingly, a significant increase in cellular stiffness occurred in HCC1954 and MDA-MB-468 cells growing on the fibrous matrix, with Young's moduli increasing by 72.9 % and 95.2 %, respectively, similar to GF-mediated EMT induction. Section title: Discussion Educational score: 4.1468000411987305 Domain: biomedical Document type: Study Language: en The goal of this in vitro study was to create a reproducible 3D electrospun matrix that mimics the topography and dimension of tumor-associated collagen signatures found in diseased mammary gland tissue. Two electrospinning collector types, namely a rotating mandrel and a plate collector, were used to mimic TACS5 or TACS6 structures, respectively. SEM images revealed that optimized PCL fibers on coverslips had either a parallel (mandrel) or a random (plate) orientation. Fiber diameters ranged from 400 to 1700 nm with comparable mean inter-fiber distances. Naturally occurring collagen structures have widths from a few hundred nanometers to one micron and inter-fiber distances in breast cancer tissues range from 2 μm to 3 μm . The optimized electrospinning setup imitated the in vivo architecture of collagen fibers by producing scaffolds with TACS5 and TACS6 orientations and appropriate submicron scale and fiber density. With negligible cytotoxicity on collagen-coated fibers, the in vitro model was successfully established. Section title: Discussion Educational score: 4.182162761688232 Domain: biomedical Document type: Study Language: en Several earlier studies demonstrated that breast cancer cells acquired a more malignant phenotype with EMT-like changes on fibrous 3D scaffolds compared to conventional 2D cell culture [ , , , , ]. This response to topographical cues, known as contact guidance, includes cellular polarization, alignment, altered cell motility, and gene expression. The transition magnitude depends on the fiber matrix’ physical characteristics and biological aspects of the cells, including scale (nanometric vs. micrometric), topography orientation (unidirectional vs. multidirectional), cell size and type, and the relation between cell size and substrate dimension [ , , , , , , ]. Additionally, the epithelial/mesenchymal-state of a cell influences the degree of contact guidance . This study found that cells with low EMT-status had weak contact guidance on the TACS5-mimicking model, while high EMT status related to strong guidance. TACS6-like structures did not provoke significant morphological changes. Cells were also described to align and migrate directionally on TACS5-like substrates but orientate randomly and migrate on multidirectionally on TACS6-like patterns . Section title: Discussion Educational score: 4.213708877563477 Domain: biomedical Document type: Study Language: en In case of MDA-MB-468 cells on TACS5-mimics , cytoskeletal and nuclear morphology changes coincided with EMT-phenotypic gene expression pattern , highlighting the predictive importance of (sub-)cellular morphologies. In contrast, MCF7 cells, which did not significantly change morphology, retained their epithelial expression pattern . Migration on TACS5-like scaffolds was slower in case of MCF7 and HCC1954 cells, while mesenchymal MDA-MB-468 cells demonstrated increased migration speed . The EMT-status was crucial in determining cellular responses, with cells having a partial mesenchymal phenotype showing polarization, pro-EMT transcription, and increased motility. Section title: Discussion Educational score: 4.530376434326172 Domain: biomedical Document type: Study Language: en TGF-β1-mediated downregulation of CDH1 and upregulation of VIM in HCC1954 supported alignment and migration within the TACS5-like fiber matrix . This increased scaffold interaction fostered mesenchymal transcription . As shown in Fig. 2 and Fig. S3 epithelial phenotypes in the TACS5-micking matrix tend to establish cell-cell contacts, while mesenchymal phenotypes interact more with the matrix. Focal adhesion-mediated recognition of topographical patterns likely induced cell polarization and actomyosin contractility, modulating EMT-related gene transcription by affecting chromatin arrangement [ , , ]. Ravikrishnan et al. demonstrated that HGF-dependent EMT-induction was necessary for epithelial cells to disintegrate, interact with, and migrate along nanofibers. E-Cadherin-based adherens junctions may have restrained contact guidance by counteracting anisotropic cell-substrate interaction . Additionally, the absence of vimentin in fully epithelial cells may explain impaired contact guidance, as vimentin aligns with grooved patterns in meningeal cells and affects focal adhesion maturation . Since vimentin templates microtubule-mediated directional migration , its presence and upregulation enhances contact guidance. These findings align with observations that miR200c-dependent MET induction in MDA-MB-231 cells downregulates vimentin, significantly reducing contact guidance on TACS5-like topographies . MET induction restricted cellular alignment and directed migration. Conversely, miR200c KO in MCF7 (EMT-induction) only marginally elevated contact guidance, likely because CDH1 mRNA expression still dominated over VIM expression. Section title: Discussion Educational score: 4.2280120849609375 Domain: biomedical Document type: Study Language: en Biochemically induced EMT resulted in stronger overall EMT-like changes than topographical triggers within the 72 h , corroborating findings by Saha et al. regarding EMT-relevant genes affected by contact guidance. Moreover, to the best of our knowledge, the present study is the first report to link XBP1 to contact guidance, with significant upregulation in two of three cell lines after 72 h of cell growth on the fibers . XBP1, known to upregulate SNAI1 expression and induce EMT-like changes , was upregulated alongside SNAI1 in the MDA-MB-468 cell line. Future studies should clarify XPB1's role in contact guidance, potentially involving ER stress and unfolded protein response (UPR) due to spatial confinements, which could provide survival advantage in cancer cells . Fig. 5 (a) Total cellular stiffness of the 4 breast cancer cell lines shown as Whiskers plot (5–95 percentiles). (b) Nuclear stiffness of the 4 breast cancer cell lines shown as Whiskers plot (5–95 percentiles). (c) Nuclear stiffness stacked over total cellular stiffness. Numbers indicate contribution (in percent) of nuclear stiffness on total cellular stiffness. Impact of nuclear stiffness on total nuclear stiffness increases with EMT-status. Stars indicate statistical significance (∗∗∗P < 0.001; ∗P < 0.05; ns = not significant). Fig. 5 Fig. 6 (a) Transmitted light photography images of MDA-MB-468 cells measured in AFM contact mode. Black arrows indicate analyzed cell. (b) Total cellular stiffness of MDA-MB-468 cells treated with EGF for 24 and 48 h. (c) Nuclear stiffness of MDA-MB-468 cells treated with EGF for 24 and 48 h (c). Total cellular stiffness of HCC1954 cells treated with TGF-β1 for 48 h. (d) Nuclear stiffness of MDA-MB-468 cells treated with TGF-β1 for 48 h. (e) Anisotropy of actin fibers of control HCC1954 cells compared with TGF-β1 stimulated cells ( t -test; ∗∗P < 0.01). Confocal images of (f) untreated and (g) TGF-β1-treated HCC1954 cells showing nuclei (blue) and actin cytoskeleton (red) at 63× magnification. Right panel (zoom-in) highlights actin cytoskeleton architecture to either show a cross-linked network (f) or parallel stress fibers (g). Stars indicate statistical significance (∗∗∗P < 0.001; ns = not significant). Fig. 6 Fig. 7 AFM analysis of (a) MCF7, (b) HCC1954, (c) MDA-MB-468 and (d) MDA-MB-231 cell growth on TACS5-like scaffolds. Left panel shows surface topography. Right panel depicts total cellular stiffness of the respective cell lines growing either on conventional culture dishes or TACS5-mimics. Stars indicate statistical significance ( t -test: ∗∗∗P < 0.001; ∗P < 0.05; ns = not significant). Fig. 7 Section title: Discussion Educational score: 3.864269495010376 Domain: biomedical Document type: Other Language: en Cell mechanical properties are defined by the nucleus, the actin cytoskeleton, microtubules, intermediate filaments and cell membrane, and are altered by interactions with adjacent cells and the ECM . EMT-like changes reorganize the cytoskeleton, affecting cell mechanics. Section title: Discussion Educational score: 4.110544681549072 Domain: biomedical Document type: Study Language: en AFM indentation revealed that Young's moduli of total cellular stiffness decreased with increasing EMT-status , aligning with cancer cell malignancy [ , , , , , ]. However, probing nuclear stiffness revealed some inconsistencies. In line with earlier studies nuclei were less compliant in highly invasive MDA-MB-468 and MDA-MB-231 cells compared to poorly invasive MCF7 cells . Contrary to literature , nuclei were not stiffer than the surrounding cytoskeleton. Section title: Discussion Educational score: 4.1623382568359375 Domain: biomedical Document type: Study Language: en Changes in cell mechanics during EMT were also investigated. While some studies report cell stiffening during GF-mediated EMT , others observed softening [ , , ]. This study supports cell stiffening within 24–48 h of GF induction in two cell lines . Phalloidin staining of HCC1954 cells demonstrated strongly aligned actin bundles under TGF-β1 stimulation , correlating stress fibers with increased stiffness [ , , , ]. Moreover, Tavares et al. revealed pre-metastatic cell stiffening before converting into a malignant but softer phenotype. Nuclear stiffness changes in MDA-MB-468 cells also supported initial stiffening followed by softening, highlighting the importance of transition time in biomechanical EMT evaluations. Section title: Discussion Educational score: 4.218909740447998 Domain: biomedical Document type: Study Language: en Vimentin, proposed to maintain mechanical integrity during invasion, correlated with cell stiffening in lung and breast cancer cells . However, vimentin expression did not directly correlate with cell stiffness in the studied breast cancer cell lines . MDA-MB-231 cells, with the highest vimentin levels , exhibited the lowest Young's modulus. Further studies on vimentin network assembly and cellular stiffness are needed to better understand context-dependent cell mechanics Presumably, as proposed elsewhere, geometry and organization of the vimentin network, which changes upon EMT, as well as interactions with other cells dictate vimentin's contribution to overall cell mechanics, which seems to be ultimately context-dependent . Section title: Discussion Educational score: 4.169448375701904 Domain: biomedical Document type: Study Language: en Cellular mechanics on TACS5 scaffolds conformed to prior findings. MCF7 cells showed no stiffness change in comparison to 2D cell culture , while already polarized MDA-MB-231 cells slightly stiffened. HCC1954 and MDA-MB-468 cells, undergoing EMT-like changes on the TACS5-like scaffold, significantly increased stiffness , similar to biochemically induced EMT. This suggests transient stiffening of breast cancer cells as a new marker for EMT-like changes. In future experiments, longer time intervals of the biomechanical evaluation need to be assessed to further study the kinetics of cellular mechanics of the EMT process. Ideally, understanding and interrupting the mechanical adaption may prevent tumor cell dissemination . Section title: Discussion Educational score: 3.6635425090789795 Domain: biomedical Document type: Study Language: en Discrepancies with existing literature may stem from different biophysical techniques, culture dishes or probing parameters . Examining single-cell versus epithelial layer mechanics yields distinct outcomes due to cytoskeleton reorganization. AFM parameters likely caused observed deviations, as the cytoskeleton contribution to nuclear stiffness was negligible. Section title: Conclusion Educational score: 4.209428787231445 Domain: biomedical Document type: Study Language: en This in vitro study provided strong evidence that TACS5-like structures support and promote EMT-like changes in breast cancer cells. Contact guidance highly depended on the EMT-status, not necessarily requiring a full transition to a mesenchymal phenotype. Epithelial depletions or mesenchymal acquisitions creating E/M-hybrids improved single-cell contact guidance of single cells. In vivo, EMT-phenotypic changes and restructuring of the stromal compartment's acellular fraction coincide in breast cancer. This co-action likely creates a positive feedback loop that is sustains tumor progression, ultimately leading to metastasis. A plausible strategy to inhibit this loop is to target initial transition into a (partial) mesenchymal phenotype or disturb biomechanical adaption of cancer cells. While the diagnostic value of TACS is yet to be confirmed, early detection may allow therapeutic prevention of disease progression. Section title: Declaration of generative AI and AI-assisted technologies in the writing process Educational score: 0.9310556650161743 Domain: other Document type: Other Language: en During the preparation of this work the authors used ChatGPT in order to shorten parts of the original manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Section title: CRediT authorship contribution statement Educational score: 0.9843976497650146 Domain: other Document type: Other Language: en Lorenz Isert: Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Mehak Passi: Methodology, Investigation, Formal analysis. Benedikt Freystetter: Methodology, Investigation. Maximilian Grab: Resources, Methodology, Investigation. Andreas Roidl: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Conceptualization. Christoph Müller: Writing – review & editing, Resources, Methodology, Investigation, Formal analysis. Aditi Mehta: Writing – review & editing, Supervision, Methodology, Investigation, Conceptualization. Harini G. Sundararaghavan: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Conceptualization. Stefan Zahler: Writing – review & editing, Supervision, Resources, Methodology, Conceptualization. Olivia M. Merkel: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. Section title: Declaration of competing interest Educational score: 0.9490618705749512 Domain: other Document type: Other Language: en O.M.M. acknowledges the Munich Multiscale Biofabrication Network, funded by the German 10.13039/501100002347 Federal Ministry of Education and Research . A.R and S.Z were financially supported by the 10.13039/501100001659 Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 201269156 - SFB 1032 (Projects B04 and B08). M.P was supported by the DAAD (German Academic Exchange Service). O.M.M. is a co-founder of RNhale GmbH, an SAB member for AMW GmbH, Corden Pharma International GmbH, and Coriolis Pharma GmbH and a consultant for AbbVie Deutschland GmbH and PARI Pharma GmbH on unrelated projects. | Review | biomedical | en | 0.999997 |
PMC11699620 | Section title: Introduction to polylactic acid Educational score: 4.047855377197266 Domain: biomedical Document type: Review Language: en Polylactic acid (PLA) stands one of the most studied and applied biodegradable aliphatic polyesters in the biomedical field. Produced from fermentation of sustainable sources like corn syrup, potatoes, and sugar cane, PLA is then synthesized by polymerization of lactic acid. 1 At the end of product life, PLA is degraded hydrolytically into lactic acid, a common metabolic product, and thus considered as biocompatible and environmentally friendly. 2 , 3 , 4 PLA has demonstrated the significant potential, either as a substitute for traditional petrochemical-based polymers in industry or as a prominent biomaterial for various medical applications. 5 Section title: Synthesis of PLA Educational score: 3.6176013946533203 Domain: biomedical Document type: Other Language: en Lactic acid is the monomer of PLA, and due to its asymmetric carbon atoms, there are two spatial isomers: the dextrorotatory form called r or d -lactic acid and the levorotatory form called s or l -lactic acid. 6 Since only l -lactic acid exits in mammalian metabolism, d -lactic acid or a mixture of d - and l -lactic acid is generally not advisable in the food, drink, and pharmaceutical industries to avoid metabolic issues. 7 , 8 , 9 Section title: Synthesis of PLA Educational score: 4.267870903015137 Domain: biomedical Document type: Study Language: en The manufacturing process of PLA mainly consists of two steps: the preparation of high-purity lactic acid monomers and the synthesis of high molecular weight PLA. 10 There are two approaches for lactic acid production: chemical synthesis and microbial fermentation. 11 The primary method for chemical synthesis is through the addition reaction of acetic aldehyde and hydrogen cyanide, followed by hydrolysis to obtain lactic acid. The chemical synthesis approach has problems such as limited production capacity, high costs, and production of only racemic lactic acid. 12 Therefore, most of lactic acid worldwide is produced through microbial fermentation. 13 , 14 , 15 Lactic acid bacterial fermentation can be divided into homofermentative fermentation, where glucose is mainly converted by lactic acid bacteria into lactic acid, counting for more than 90% of the products, and heterofermentative fermentation, where glucose is converted by lactic acid bacteria into various products including lactic acid, ethanol, acetic acid, etc. 16 Currently, industrial companies commonly use engineered strains such as recombinant Escherichia coli to efficiently produce lactic acid monomers by adding neutralizing agents like calcium hydroxide during fermentation. 17 This method yields over 140 g per liter, which achieves high fermentation intensity and produces only l -lactic acid or d -lactic acid. 18 Additionally, acid-resistant yeast or Lactobacillus can produce lactic acid in non-neutralizing fermentation systems, though the lactic acid yield is relatively low. 19 , 20 Section title: Synthesis of PLA Educational score: 4.102699279785156 Domain: biomedical Document type: Study Language: en The downstream purification process includes the removal of bacterial cells, separation of by-products, and the refining and purification of lactic acid monomers. 21 Currently, the widely used approach for the separation and purification of lactic acid monomers is the calcium lactate crystallization-acid hydrolysis process, which provides high yield and purity. Due to the technical maturity, operational simplicity, and high efficiency, traditional neutralization precipitation methods are still used in industrial production. Meanwhile, the development of emulsion liquid membrane extraction, reactive distillation, and advanced membrane separation techniques have shown great potential for purifying lactic acid in an energy-efficient manner. 22 , 23 Section title: Synthesis of PLA Educational score: 4.4978485107421875 Domain: biomedical Document type: Study Language: en After extraction and purification, the lactic acid monomers are polymerized through a polycondensation reaction to form PLA. There are three primary methods for massive PLA production: direct lactic acid polycondensation, lactic acid azeotropic dehydrative condensation, and ring-opening polymerization. 24 , 25 The direct polycondensation involves the continuous dehydration of lactic acid to form oligomers and then PLA. 26 However, water is slowly diffused in the viscous polymer melt during processing. The residual water in the PLA melt affects the efficiency of the polycondensation reaction and limits the molecular weight and performance of PLA. Therefore, azeotropic dehydrative condensation is developed for high molecular weight PLA, using a high-boiling-point organic solvent to achieve dehydration. The ring-opening polymerization involves the dehydration and polycondensation of lactic acid to form oligomers under high-temperature vacuum conditions, eliminating water. 27 Subsequently, these oligomers undergo depolymerization to produce lactide (cyclic dimer of lactic acid) under reduced pressure. Finally, purified lactide, separated from residual water and lactic acid, undergoes ring-opening polymerization reaction to form PLA. By using various catalysts, the ring-opening polymerization method can achieve both high molecular weight and high optical purity and has been the most used method for large-scale production of PLA. 28 , 29 Section title: Synthesis of PLA Educational score: 4.397647857666016 Domain: biomedical Document type: Study Language: en Furthermore, the strategies for synthesizing PLA are constantly evolving. 30 , 31 , 32 , 33 , 34 In addition to optimizing the process for ring-opening polymerization and developing new catalysts, reports have indicated the successful construction of PLA metabolic pathways in chassis cells such as E. coli , 35 Yarrowia lipolytica , 36 and Saccharomyces cerevisiae , 37 using glucose as a substrate for the biosynthesis of PLA homopolymers. 38 In the biological polymerization process, the key enzyme propionyl-CoA transferase is responsible for converting lactic acid monomers into lactyl-CoA, which is further polymerized into PLA under the catalytic action of polyhydroxyalkanoate synthase. Compared to chemical processes, the biological polymerization of PLA faces challenges such as low polymer content within cells and low molecular weight of the polymers, directly impacting the production costs and material properties of PLA. 35 Currently at the forefront, synthetic biology has also proposed the utilization of cyanobacteria as host cells for synthesizing PLA directly from carbon dioxide. By employing a combination of metabolic engineering and high-density cultivation on a light-driven cyanobacteria platform, one-step biosynthesis of PLA using carbon dioxide achieved a PLA concentration as high as 108.0 mg/L. 39 This “negative carbon” production technology can not only address plastic pollution but also achieve carbon capture, offering multiple benefits on society, economy, and the environment. 40 Section title: Properties of PLA materials Educational score: 4.185979843139648 Domain: biomedical Document type: Study Language: en Properties of PLA depend on its component isomers, molecular weight, annealing time, and processing temperature. 41 Component isomers can affect the crystallinity, melting temperature, and glass transition temperature. 42 PLA with poly l -lactic acid content higher than 90% tends to be crystalline, while a lower proportion often leads to amorphous PLA with reduced melting temperature and glass transition temperature. 43 Molecular weight primarily affects the degradation, mechanical strength, and solubility of polymers. PLA materials with a high molecular weight take approximately 2–8 years for complete resorption. 44 The PLA used in implants, such as orthopedic fixation screws, has a higher molecular weight, providing mechanical strength for a period, allowing the bone fracture to heal properly. In contrast, PLA materials used in drug delivery systems often have lower molecular weights, allowing for relatively rapid degradation and reduced drug accumulation in tissues. 44 Section title: Modification of PLA materials Educational score: 4.71737003326416 Domain: biomedical Document type: Review Language: en Copolymerization of lactic acid and different monomers together may control the degradation time of PLA materials or enhance their hydrophilicity, flexibility, and ductility. 45 , 46 For example, copolymerization of lactic acid and glycolic acid forms poly(lactic-co-glycolic) acid (PLGA), which can shorten the degradation time of PLA from 6 months to 2 months. 47 Since the degradation time is negatively correlated with the content of polyglycolide, the biocompatible PLGA can be used in drug delivery systems with controlled medicine release. 46 , 48 , 49 , 50 , 51 In addition, hydrogels formed by copolymerization of lactide and polyethylene glycol (PEG) exhibit good biocompatibility, adjustable degradation rates, and enhanced flexibility. 48 , 52 These hydrogels can absorb large amounts of water, forming soft gel networks with excellent plasticity, and then be used in drug delivery systems and as cell carrier scaffolds for tissue regeneration engineering. 53 , 54 , 55 Moreover, the PLA-polycaprolactone copolymer is a semi-crystalline, biodegradable thermoplastic with good flexibility and a low melting point (∼60°C). 56 , 57 Compared with PLA, this copolymer demonstrates enhanced flexibility and ductility, reduced brittleness, and improved mechanical properties, making it an ideal material for biodegradable implants and three-dimensional (3D) printing. 58 , 59 Section title: Modification of PLA materials Educational score: 3.4481496810913086 Domain: biomedical Document type: Study Language: en Blending PLA with other polymers through melting or solution mixing can significantly change its properties, 60 , 61 offering a cost advantage over copolymerization. 61 , 62 Blending with heat-resistant polymers can be used for heat-resistant flame-retardant materials, while blending with flexible polymers can improve PLA’s toughness and elasticity for 3D printing applications. 63 However, the application of PLA blended materials in the medical field is relatively limited. 64 Section title: Modification of PLA materials Educational score: 3.9697623252868652 Domain: biomedical Document type: Study Language: en PLA materials can also be modified by adding fillers or plasticizers. 65 Since PLA is hydrophobic and lacks inherent bioactivity, appropriate fillers can promote cell migration, extracellular matrix deposition, and vascularization. 50 , 55 Calcium-based inorganic fillers, such as calcium phosphate and calcium silicate, have been shown to promote the migration and mineral deposition of mesenchymal stem cells, making them widely applied in PLA modification for bone tissue regeneration. 66 , 67 Section title: Advanced manufacturing for PLA Educational score: 3.617629051208496 Domain: biomedical Document type: Study Language: en Various processing techniques have been developed to meet the thermal and mechanical demands of PLA materials, which include but not limited to drying and extrusion, 68 injection molding, 69 injection stretch blow molding, 3 and casting (film and sheet). 70 PLA can also be transformed into films, fibers, particulates, and porous structures of various shapes and sizes through techniques like solvent blending or melt blending. 62 These processes have largely broadened the applications of PLA materials. 70 , 71 Section title: Advanced manufacturing for PLA Educational score: 4.003876686096191 Domain: biomedical Document type: Review Language: en PLA can be engineered into nanomaterials, unlocking exciting possibilities for various applications. 72 Advanced techniques like nanoparticle fabrication, electrospinning, and nanocomposite synthesis are employed in producing PLA nanomaterials. 73 These methods enable precise control over the size, shape, and composition of the nanoparticles or nanofibers. For example, PLA nanoparticles have been employed in drug delivery systems to enhance drug solubility and release profiles. 74 PLA nanofibers are utilized in applications such as tissue engineering scaffolds, wound dressings, and filtration membranes due to their high surface area-to-volume ratio and favorable mechanical properties. 75 Section title: Advanced manufacturing for PLA Educational score: 3.8905251026153564 Domain: biomedical Document type: Other Language: en 3D printing is an innovative and forward-looking technology that enables the fabrication of multiscale, biomimetic structures according to specific patient needs and clinical requirements. 76 In clinical applications, 3D printing technology can be utilized to manufacture personalized medical devices, orthopedic scaffolds, artificial tissues, and biomedical models. In the treatment of bone defects, 3D printing, guided by imaging technologies like magnetic resonance and CT scans, can easily reconstruct the required 3D models. This enables the precise manufacturing of bone grafts that match the defect area accurately. 77 Section title: Advantages of PLA materials in clinical treatments Educational score: 4.0911970138549805 Domain: biomedical Document type: Review Language: en Compared with other bio-polymers, PLA is characterized for its biocompatibility, biodegradability, and plasticity in medical applications. Firstly, PLA is not toxic or carcinogenic and it undergoes degradation through non-enzymatic hydrolysis, where its monomer lactic acid is a common metabolic product and eliminated through the tricarboxylic acid cycle. 78 , 79 Secondly, the degradation rate of PLA can be controlled by adjusting factors such as molecular weight, d/l isomer purity, and environment temperature. The degradation time can vary from several hours in drug delivery systems to several months in orthopedic fixation implants, which allows PLA to achieve controlled degradation suitable for various medical treatments and repairs. 44 Thirdly, PLA can be processed into various forms, including films, fibers, particles, and porous structures. 62 New manufacturing techniques such as 3D printing technology and nanomaterials further expand its applications in the medical field. Section title: Advantages of PLA materials in clinical treatments Educational score: 3.9115569591522217 Domain: biomedical Document type: Review Language: en Therefore, PLA materials find a wide range of applications in the clinical treatments. 80 PLA and PLA blends have been investigated for various drug delivery approaches, encompassing nanosystems, hydrogels, films, and fibrous matrices. 81 In orthopedic and dental applications, PLA-based materials have found widespread use as fixation devices such as pins and screws in reconstructive surgeries for various fractures. 82 In tissue regenerative engineering, PLA-based biomaterials bring new developments for bone, ligament and cartilage regeneration. 75 PLA can also be used in wound management, such as surgical sutures. 83 Section title: Improved drug delivery systems through PLA nanoparticles Educational score: 4.276129722595215 Domain: biomedical Document type: Review Language: en The introduction of controlled release drug delivery has brought a significant transformation in the pharmaceutical sector, which provides the advantages of low side effects and specific targeting abilities. 84 , 85 PLA and its copolymers have been extensively investigated in this field because of their remarkable biocompatibility, biodegradability, low immunogenicity, and desirable mechanical properties. 86 For instance, PLA and PLGA have been approved by FDA for human use in formulations for controlled release drug delivery and as carriers for vaccine antigens. 87 Carried by PLA nanoparticles, macromolecules such as nucleic acids, peptides, proteins, etc., now can be used in therapeutics, by overcoming the challenges of rapid clearance through kidney or liver, enzyme degradation, cell membrane permeability, barriers between blood and other organs. 88 , 89 Additionally, nanoparticle drug delivery system can enhance the performance of various medications beyond conventional formulations, by reducing the size of compounds and modifying the surface characters. 90 For instance, nanoparticle drug delivery system can be utilized to encapsulate drugs, leading to (1) improved solubility especially for hydrophobic drugs, (2) protection from degradation, (3) increased absorption through epithelial barriers and extended circulation in the bloodstream, (4) precise targeting of drugs to specific cells, tissues, or organs, and (5) enhanced cellular uptake. A few methods have been employed to produce PLA nanoparticles, such as single or multiple emulsion, 91 , 92 nano precipitation or salting out, 93 melting based direct compositing method, supercritical fluids technique, 94 and template/mold based technique. Section title: Targeted therapy in oncology Educational score: 4.192832946777344 Domain: biomedical Document type: Study Language: en Due to the presence of blood-tumor barrier, conventional drugs generally struggle to reach sufficient concentrations at tumor sites, especially for central nervous system tumors that are further blocked by blood-brain barrier. 88 , 95 , 96 In oncology, loading usually hydrophobic chemotherapy drugs onto PLA nanoparticles is a promising approach, which can enhance the targeting effect, increase drug concentration at the tumor site, reduce systemic toxicity, and particularly, facilitate penetration through barriers such as the blood-brain barrier. 97 Additionally, modifications can be made to PLA nanoparticles to change surface chemistry, such as surface charge, influencing the pathways of cellular uptake; or add macromolecules such as antibodies, proteins, nucleic acids, etc., enhancing targeted actions on tumors; or manipulate the chemical composition of nanoparticle themselves, controlling the degradation rate and drug release rate, enhancing drug metabolism kinetics. 98 , 99 , 100 For example, a study employed PEG-PLA nanoparticles to deliver peptides for effective tumor targeting and internalization. The F3 peptide, which specifically bound to the overexpressed nucleolin marker on glioma cells, was used to modify the PEG-PLA nanoparticles, resulting in greater accumulation in the glioma regions. 101 Section title: Delivering polynucleotides and peptides in gene therapy and vaccination Educational score: 4.142326354980469 Domain: biomedical Document type: Study Language: en Gene therapy operates on the fundamental idea that polynucleotides delivered into cells can change the expression of a specific protein, leading to therapeutic advantages. This process encompasses the delivery of polynucleotides like DNA, RNA, anti-sense oligonucleotides, and small interfering RNA, either at a specific location or throughout the entire system. 102 Compared to viral vectors, non-viral vectors are mostly non-immunogenic, cost-effective, safer, and capable of carrying more genetic material. Among them, PLA nanoparticles can serve as non-viral carriers to deliver functional nucleic acids. 103 , 104 It has been reported that biodegradable polymeric nanoparticles through the combination of PLA, PLGA, and polyethylenimine exhibited DNA binding capabilities and performed well in vitro. 105 Section title: Delivering polynucleotides and peptides in gene therapy and vaccination Educational score: 4.039490699768066 Domain: biomedical Document type: Study Language: en The therapeutic proteins and peptides have been approved by FDA to cure many diseases, including Alzheimer’s disease, diabetes and melanoma. 106 , 107 , 108 The nanoparticle-based systems have significantly enhanced the delivery of antigens for vaccine therapies, which can be custom-designed to resemble cellular components so that they can enter cells through endocytosis and pinocytosis. 109 Tetanus toxoid, serving as a representative antigen, was integrated into either PLA or PEG-PLA nanoparticles to facilitate nasal vaccine administration, and the properties of these formulations were examined both in laboratory settings and in living organisms. 110 Section title: The PLA materials in orthopedic fixation devices Educational score: 4.050809860229492 Domain: biomedical Document type: Review Language: en PLA-based materials have found extensive use as fixation devices in orthopedic and dental applications. 82 Current orthopedic surgical procedures primarily involve the use of autografts, allografts, and metal and plastic implants. 111 However, metal and plastic implants face various challenges, including low fatigue strength, creep, poor adhesion, and biocompatibility issues with native tissue. Compared to traditional metal fixation materials such as steel or titanium alloys, PLA polymers with modifications to improve mechanical properties and corrosion and creep resistance, can also withstand the loads inside the body. 112 , 113 Moreover, PLA materials have advantages in biodegradability, and the degradation rate can be adjusted by modifications to match the patient’s tissue healing needs. Biodegradability also allows PLA materials to release stress to the affected area over time, facilitating tissue healing. Another significant benefit is the avoidance of a secondary surgical procedure to remove unnecessary hardware. This not only lowers medical expenses but also enables the gradual restoration of tissue function. 82 , 114 Section title: The PLA materials in orthopedic fixation devices Educational score: 3.9792473316192627 Domain: biomedical Document type: Review Language: en PLA polymers have been utilized in orthopedics for the fabrication of biodegradable screws, fixation pins, plates, and suture anchors, 115 which have been increasingly used in fractures in knee, shoulder, ankle, and foot. 116 , 117 The ligaments in knee and ankle joints are susceptible to tears or injuries because they bear weight and are exposed to high stress. During the surgical for reconstructions, orthopedic screws made by biodegradable polymeric are employed either independently or in conjunction with grafts, depending on the nature of the ligament injury. 118 Synthetic bioabsorbable screws have been integrated with bioceramic osteoconductive materials such as calcium phosphates and other composites to create biocomposite-based interference screws. 119 Section title: The PLA based drug delivery system for osteoarthritis Educational score: 4.27626895904541 Domain: biomedical Document type: Review Language: en Osteoarthritis is characterized by progressive cartilage degeneration and inflammation, leading to severe joint pain. 120 Traditional intra-articular drug injection therapies often provide only short-term benefits due to the rapid clearance of drugs from the joint space. The transport of drugs into cartilage to reach cellular targets is also hindered by the dense and negatively charged extracellular matrix of cartilage. 121 , 122 Therefore, developing drug delivery systems capable of crossing the cartilage barrier and achieving long-lasting therapeutic responses has become a research focus. Among these, PLA nanoparticles have garnered significant attention due to their controllable degradation properties and modifiable surfaces, which enable prolonged retention of drugs within the joint and penetration of the extracellular matrix to deliver drugs specifically to diseased chondrocytes. Current research directions include the following aspects. (1) Development of synovial joint-targeted hybrid systems: this involves using a combination of hydrogels, liposomes, and nanoparticle carriers to target the synovial joint and alleviate pain and inflammation. 123 , 124 , 125 (2) Drug delivery systems targeting intra-cartilage components: these systems aim to penetrate cartilage by targeting components such as aggrecan, collagen II, and chondrocytes through surface protein or charge modifications, facilitating drug transport through the cartilage. 126 , 127 (3) Steroid-encapsulated polymer microparticles: these microparticles, which have been approved for clinical use, provide prolonged release of steroids to alleviate pain more effectively. 128 , 129 Section title: The PLA based drug delivery system for osteoarthritis Educational score: 4.53508186340332 Domain: biomedical Document type: Review Language: en A novel multi-arm cationic nanostructure based on PLA has recently been developed, which possesses 28 covalent drug conjugation sites, allowing it to penetrate the entire thickness of the cartilage at high concentrations. When conjugated with dexamethasone, it achieves prolonged intra-cartilage retention and sustained drug release for up to two weeks. 130 PLA nanoparticles can also be loaded with various regenerative drugs and combined with structures such as hydrogels and cartilage cell membranes to serve as stem cell expansion vectors. 122 , 131 , 132 It has been reported that a novel porous microsphere was made by PLGA and loaded with kartogenin, serving as a stem cell expansion vector. Its advantages include a high cell-carrying capacity (up to 1 × 10 4 cells/mm 3 ) and the ability to effectively protect stem cells, promoting their controlled release in the osteoarthritis microenvironment, and inducing the differentiation of mesenchymal stem cells into chondrocytes . 133 Figure 1 Advantages and strategies of PLA-based targeted drug in osteoarthritis PLA-based targeted drugs in osteoarthritis can persist within the joint cavity for extended periods and effectively translocate across the cartilage barrier due to their controlled degradation properties and modifiable surfaces. 134 This figure illustrates two common strategies for PLA-based drug delivery: hydrogels and microspheres. Upon injection into the joint cavity, both hydrogels and microspheres form repair zones at cartilage defect sites. Stem cells encapsulated within these delivery systems can differentiate into new chondrocytes under the influence of the drugs, facilitating cartilage repair in osteoarthritis. 133 Both delivery systems utilize PLA-based nanofibers as fundamental units, capable of loading therapeutic agents and being modified to target synovium or cartilage. Common drugs in these systems include dexamethasone 135 and kartogenin. 133 Section title: The PLA materials in orthopedic tissue regeneration engineering Educational score: 4.041873455047607 Domain: biomedical Document type: Review Language: en The bone formation process, known as osteogenesis, can occur through either endochondral ossification or intramembranous ossification pathways. 136 , 137 In addition to being used as fixation devices, PLA is also utilized as a repair and filler material in orthopedics, dentistry, and neurosurgery. 138 In these fields, PLA can be used for bone defect repairs, providing temporary support and promoting the formation and regeneration of new bone. 139 Common applications include cranial bone plates in cranioplasty, 140 bio-substitute materials for mandibular fractures, bioabsorbable bone plates for orbital floor fracture 141 and filler materials for large bone defects. 142 Repair and filler materials made by PLA can also carry drugs, controlling the release of drugs during slow degradation to maintain the local concentration. The common drugs loaded are antibiotics that inhibit infection and various growth factors that promote tissue regeneration . 48 Section title: The PLA materials in orthopedic tissue regeneration engineering Educational score: 4.396705627441406 Domain: biomedical Document type: Review Language: en Moreover, researchers have explored more complex scaffold designs and material modifications to better support bone formation and the healing process. The characteristics required for bone tissue regeneration scaffolds are summarized in Figure 2 . In orthopedics, the latest technologies involve the use of 3D printing and electrospinning to fabricate composite scaffolds comprising organic and inorganic phases. Polyhydroxyapatite/PLA 3D composite scaffolds have been investigated in bone repair and exhibited improved compatibility, bioactivity, and osteoinductivity, with a reduced likelihood of inflammation. 143 Furthermore, the incorporation of antibacterial silver nanoparticles, graphene oxide, and selenium nanoparticles made from PLA materials, on the surface or within bone regeneration scaffolds, has been proven to be highly effective for addressing the issues of infection and accelerating bone regeneration after bone tumor treatment. 144 , 145 Also, coordinated growth factors, such as bone morphogenetic proteins, vascular endothelial growth factors, and fibroblast growth factors can be added to the scaffolds for stable and controlled release within the core region of bone regeneration. 146 , 147 A recent study reported a porous bio-composite PLA scaffolds integrated with nuciferine-loaded chitosan hydrogel through 3D printing, which demonstrated uniform pore distribution, sustained nuciferine release, and favorable cytocompatibility with mouse mesenchymal stem cells, leading to enhanced new bone formation. 148 Figure 2 Bone structure and the characteristics required for biomaterial-based bone regeneration scaffolds Bones are essential components of the human skeletal system with a complex anatomical structure. Osteon is the fundamental structural unit of bone, comprising a central canal, concentric bone matrix layers, and osteocytes embedded within. 136 , 137 Osteocytes are responsible for the production of new bone tissue. The central canal contains blood vessels and nerves, ensuring nutrient supply to the bone. The ordered arrangement of osteons imparts strength and stability to the bone. Biomaterial-based scaffolds for bone regeneration are expected possess several beneficial characteristics. 48 , 139 Section title: The PLA materials in orthopedic tissue regeneration engineering Educational score: 4.097626686096191 Domain: biomedical Document type: Study Language: en During ligament injury, the anterior cruciate ligament that lacks vascular tissue is typically difficult to heal and thus biomaterial-based regeneration has been employed. 149 Multiphasic scaffolds, designed to mimic the process of ligament-cartilage-bone regeneration, have been investigated for osteochondral regeneration. 150 In a recent report, braided PLGA is used to build multiphasic scaffolds, where the upper phase includes PLGA microspheres to encourage the development of non-calcified cartilage, the middle phase includes a small amount of bioglass added to PLGA microspheres to aid in cartilage tissue calcification, and the lower phase comprises PLGA microspheres with a higher bioglass concentration, promoting bone regeneration. 151 Section title: The PLA materials in orthopedic tissue regeneration engineering Educational score: 4.507573127746582 Domain: biomedical Document type: Review Language: en In the mid-to-late stages of osteoarthritis, when irreversible cartilage defects occur, drug treatments become very limited. At this point, additional surgical interventions are required. The main clinical treatments currently include microfracture, osteochondral transplantation, and autologous chondrocyte implantation. A recent study reported a novel 3D nanocomposite scaffold made from poly-ε-caprolactone and PLA, incorporating transforming growth factor β 1 (TGF-β1)-loaded chitosan-dextran nanoparticles, and fabricated using the electrospinning method to create a bead-free, semi-aligned nanofiber structure. The scaffold’s advantages include its biomimetic properties, high hydrophilicity, high porosity, and the sustained release of TGF-β1, which collectively enhance the expression of aggrecan and collagen type Ι genes, crucial for cartilage tissue engineering . 152 Figure 3 From repair fillers to composite and multiphase tissue regenerative scaffolds (A) In orthopedics, PLA was initially used for manufacturing repair fillers for bone defects. 139 These fillers are dense in structure and possess high strength, gradually degrading over time within the bone defect area while releasing antibiotics and bone regeneration-promoting growth factors. 48 As the filler degrades, new bone tissue grows into the defect site. With advancements in stem cell technology and 3D printing, repair fillers have evolved into composite tissue regenerative scaffolds. These scaffolds feature larger pores and are composed of both inorganic and organic phases. 153 The inorganic phase includes fillers such as hydroxyapatite, which promotes bone mineral deposition. 154 The organic phase is loaded with stem cells, antibiotics, and growth factors. (B) According to clinical practice, the latest multiphase scaffolds are frequently used for complex defects spanning bone, ligament, and joint tissues. 50 These scaffolds provide more comprehensive tissue repair and regeneration by replicating the natural transitions between different types of tissues. A multiphase bone-ligament scaffold for treating anterior cruciate ligament injuries. The scaffold includes three regions: the ligament region directly connecting with the ligament repair material, the bone region containing anchor points for fixation and a porous scaffold to promote bone regeneration, and the bone-ligament interface region replicating the gradual transition from fibrocartilage to bone, inducing a mechanical fixation to long-term biological fixation between the implant and the host bone. 151 , 155 Section title: Tissue regenerative engineering Educational score: 4.061532974243164 Domain: biomedical Document type: Review Language: en The concept of tissue regenerative engineering has arisen from the fusion of tissue engineering, advanced materials science, stem cell research, and developmental biology with the aim of regenerating complex tissues that have been damaged. 156 Advanced materials science offers scaffolds featuring precise geometry and architecture, ensuring sufficient mechanical support and potentially regulating cellular activities by delivering chemical and biochemical materials in both spatial and temporal control. 156 Section title: Tissue regenerative engineering Educational score: 4.070333957672119 Domain: biomedical Document type: Study Language: en The PLA-based nanofibers in tissue engineering possess unique characteristics, such as a high surface area-to-volume ratio, high porosity, and excellent mechanical strength. 105 Additionally, PLA nanoparticles containing growth factors or antimicrobial agents can also be embedded in regenerative scaffolds. 157 PLA nanoparticles can also be coated on the surface of regenerative scaffolds to improve the compatibility of the scaffold’s surface with tissue materials. 158 Additionally, the polymer surface can be modified by adding chemically charged end groups (e.g., –OH – , –COO – , and –NH 3 + ) to increase protein adsorption, or incorporating peptide segments or synthetically engineered proteins to enhance cell receptor binding and guide cell migration. 159 Section title: The PLA materials in nervous tissue engineering Educational score: 4.616664886474609 Domain: biomedical Document type: Review Language: en The effective treatment of severe peripheral nerve injuries remains an unmet clinical challenge, and biomaterials provide a hopeful avenue for stimulating nerve regeneration. 160 , 161 , 162 Scaffolds used in neural tissue engineering are typically manufactured in hollow tube-like structures, characterized for biocompatibility and biodegradability. They are designed to be neuro-compatible to support cell growth and the release of neurotrophic factors, thereby promoting regeneration. 163 , 164 Recent study reported that a multi-channel scaffolds made from electrospun poly- l -lactic acid and poly-ε-caprolactone have been developed, in which a suspension of autologous adipose-derived stromal and stem cells was injected during implantation in order to bridge a 10 mm gap in the rat sciatic nerve. 143 However, while the scaffold was observed to support nerve regeneration during the 4 weeks recovery period, the implanted cells also induced an inflammatory response. 165 Further research has reported the enhancement of neural repair in rats following spinal cord injury, by integrating PLA nano scaffolds fabricated using electrospinning and hydrogel coatings on the scaffold surfaces. Additionally, regenerative factors like brain-derived neurotrophic factor and stromal cell-derived factor-1α were incorporated into both the scaffolds and coatings, which released over time and promoted neural repair . 164 Figure 4 Hollow tubular PLA scaffolds in tissue regeneration engineering In tissue engineering, PLA scaffolds with a hollow tubular scaffold play a significant role in nerve tissue regeneration and cardiovascular tissue regeneration. This scaffold can mimic the morphology of natural tissues, such as nerve conduits and blood vessels, providing an appropriate environment for cell growth, tissue repair, and drug delivery. (A) Nerve regenerative scaffolds are commonly used to repair and regenerate damaged nerves, particularly in spinal cord 164 and peripheral nerve injuries. 166 A typical structure consists of a hollow tubular form, composed of PLA nanofibers coated with a hydrogel layer on the surface as the basic unit. This scaffold can carry neurotrophic factors and tissue growth factors that are gradually released over time to promote nerve cell growth and repair. (B) Vascular regenerative scaffolds are primarily used for small-diameter blood vessels and myocardial repair, divided into two common structures. The first is a multilayer tubular electrospun scaffold, featuring a porous outer layer that supports cell growth and tissue repair, facilitating the adhesion of endothelial cells and fibroblasts, and promoting capillary infiltration and endothelial regeneration. 167 The inner layer is drug-loaded, incorporating stem cells, antibiotics, and tissue regeneration factors. 168 The second structure is a multilayer rolled structure made from layered PLA composites, loaded with vascular-related cells. After rolling, it forms an arrangement of three types of vascular-related cells (endothelial cells, smooth muscle cells, and fibroblasts) from the inner to outer layers, mimicking the structure of artificial blood vessels. The inner layer degrades slowly to maintain vascular stability, while the outer layer degrades to promote cell growth and angiogenesis. 169 , 170 Section title: The PLA materials in cardiovascular tissue engineering Educational score: 4.239331245422363 Domain: biomedical Document type: Study Language: en Cardiovascular disease is the most common cause of death globally, prompting a massive need of cardiovascular tissue engineering. 171 PLA nanofibrous have been used in vascular tissue engineering, especially in small-diameter blood vessels and cardiac muscle repairing. 172 , 173 The model of PLA fibrous vascular scaffolds, like nerve conduit scaffolds, is a tube with a hollow lumen. 73 Researchers have built a double-layered electrospun scaffold composed of a micro-scale PLA fibrous outer layer and an inner layer made by silk fibroin-gelatin nanofibrous. The outer layer was found to promote the growth and multiplication of mouse fibroblasts, while the inner layer facilitated the adhesion and growth of human umbilical vein endothelial cells. During treatment, there was less inflammatory response and more coinciding between a vascular network forming and the implant denigrating. 174 Additionally, a self-regulating multilayered polymeric rolled structure composed of polycaprolactone and PLGA has been reported for the fabrication of artificial blood vessels. This tubular structure is formed by the stress-induced rolling of composite polymer membranes and is loaded with three types of vascular cells (endothelial cells, smooth muscle cells, and fibroblasts). After rolling, the structure features an inner layer of polycaprolactone and an outer layer of PLGA, with the three cell types arranged in a manner similar to natural blood vessels . During degradation, the inner polycaprolactone layer expands outward while the outer PLGA layer contracts inward, effectively maintaining the structural stability of the artificial vessel and facilitating the growth of the loaded cells. 169 Section title: The PLA materials in skin tissue engineering Educational score: 4.057544231414795 Domain: biomedical Document type: Study Language: en Skin is the body’s primary defense against external factors, and a compromised skin barrier can lead to various problems such as infection, hydration issues, and temperature dysregulation. 175 Nanofibrous mats made by PLA are exceptionally appropriate for promoting skin healing. The mesh structures replicate the natural structure of the dermal bed, aiding in wound protection, moisture retention, protein preservation, and exudate removal. 176 The large surface area-to-volume ratio of nanofiber mesh structures further promotes cell adhesion and growth, thereby facilitating the healing of large-area wounds. 177 Also, PLA nanofibrous mats can incorporate drug delivery systems of antibiotics, 178 silver nanoparticles, 179 or growth factors 180 to prevent infection, facilitate wound healing and promote the maturation of skin tissues. Section title: The PLA materials in oral medicine Educational score: 4.43617057800293 Domain: biomedical Document type: Review Language: en In dental, oral, and craniofacial tissue regenerative engineering, including teeth, dental pulp, periodontal tissues, hard and soft tissues of the craniofacial complex, polymeric materials have a broad range of applications as tissue engineering scaffolds, carriers for cell-based therapies, and delivery devices for drugs and biologics. 54 In oral medicine, PLA materials have been widely applied in the reconstruction and repair of bone tissues, especially in the alveolar bone, sinuses, and temporomandibular joint. The utilization of 3D printing technology to manufacture personalized bi-phasic bone and cartilage scaffolds, followed by seeding the scaffolds with isolated cells and bio-active molecules to promote bone formation, has been demonstrated to achieve favorable clinical outcomes. 181 Research has shown that in periodontal surgery for the treatment of periodontal intrabony defects, the use of nanohydroxyapatite powder in combination with PLA/PLGA as bone replacement grafts has improved postoperative periodontal parameters in patients. 182 Additionally, PLGA is efficacious in promoting chondrogenesis by facilitating the colonization and proliferation of mesenchymal stem cells, and it interacts with chondrocytes and other cells in the temporomandibular joint disc. 183 In periodontal and peri-implant tissue repair and dental pulp regeneration, regenerative scaffolds are primarily used post-periodontal surgery to prevent oral epithelial and soft tissue infiltration into bone defects, and promote the regeneration of periodontal soft and hard tissues. 184 For more complex dental pulp tissue regeneration, PLA materials combined with stem cell therapy hold significant potential. Recently a study presented an innovative approach for dental pulp regeneration by injecting simvastatin-functionalized GelMA cryogel microspheres loaded with stem cells from human exfoliated deciduous teeth. The system enhances stem cell functions, promotes vascularized pulp-like tissue formation, and demonstrates significant potential for clinical use in endodontic regenerative dentistry. 185 Section title: The PLA materials in suturing and surgery Educational score: 4.0062408447265625 Domain: biomedical Document type: Study Language: en PLA and its copolymers have found extensive applications in surgical sutures and wound management. Because of the biocompatibility and biodegradability of PLA, PLA based suture is well-tolerated by the human body and cause less inflammation or foreign body reactions. Also, PLA breaks down naturally in the body, which is particularly beneficial in surgical sutures, as it eliminates the requirement for a second surgery to remove non-absorbable sutures. 54 PLA-based sutures can be engineered to have the necessary strength and flexibility for various surgical applications, so that surgeons can choose sutures with specific properties to suit the type of tissue being sutured. 186 Recent research has reported a new type of antibacterial PLA suture, which uses polyglycolide and polycaprolactone coatings as carriers to load the antibacterial drug ciprofloxacin onto PLA sutures. This drug-loaded antibacterial PLA suture has the dual advantages of being antibacterial and biodegradable. 187 Section title: The PLA materials in suturing and surgery Educational score: 4.215302467346191 Domain: biomedical Document type: Study Language: en The PLA materials can also serve as novel bandages and dressings for wound healing. Compared to traditional gauze and other biological dressings such as polyvinylpyrrolidone and sodium alginate fibers, PLA-based biomaterials exhibit more collagen deposition, angiogenesis, and cellular activity, resulting in accelerated wound healing. 188 , 189 This is attributed not only to PLA’s good biocompatibility and biodegradability but also to its moisture management properties, which mimic the extracellular matrix by absorbing and transferring moisture in the wound environment. 190 Additionally, when PLA materials are combined with antimicrobial agents such as antibiotics, silver, etc., they demonstrate enhanced ability to prevent wound infections. 191 , 192 Recent research reported a novel sandwich-structured PLA-based dressing for hard-to-heal diabetic wounds. 193 This dressing is composed of an electrospun three-layer structure of PLA-polyvinyl alcohol-PLA. The hydrophilic polyvinyl alcohol inner layer is loaded with metformin hydrochloride, which is slowly released during the wound healing process, promoting the healing of diabetic wounds. The hydrophobic PLA outer layers on both sides are loaded with erythromycin and puerarin, which possess antibacterial properties. This laminate film dressing demonstrated excellent mechanical properties, high water vapor permeability, and promoted wound healing in a diabetic animal model. 194 , 195 Section title: The PLA materials in suturing and surgery Educational score: 4.157267093658447 Domain: biomedical Document type: Study Language: en Surgical meshes are primarily used for tissue repair and reconstruction surgeries, such as hernia repair and soft tissue repair. The biocompatibility and biodegradability of PLA material make it an ideal choice for surgical meshes. 196 Abdominal wall hernias are conditions where visceral organs protrude due to weakened or lost continuity of the fascia or muscle, typically requiring surgical intervention. 197 In most hernia surgeries, mesh implants are commonly used for reinforcement. PLA materials are often blended with other polymers to enhance performance, improve thermal stability, and increase cell adhesion, as well as to add antimicrobial coatings. 198 , 199 PLA meshes provide a temporary support structure that helps repair abdominal wall defects while allowing new tissue to grow. During the healing process, the PLA mesh gradually degrades and is eventually replaced by newly formed tissue. 200 , 201 According to research, a novel 3D-printed PLA mesh combined with acellular dermal matrix composite material was used to repair abdominal wall defects in rats. This composite scaffold can effectively reduce surrounding inflammation and significantly promote the repair of abdominal wall defects. 202 Section title: Biodegradable polymeric stents and balloons Educational score: 4.277462959289551 Domain: biomedical Document type: Review Language: en Inserting cardiovascular stents is a crucial therapeutic approach for addressing coronary artery diseases. Compared with bare-metal stents, the development of bio-resorbable stents provides new potentials. 203 The degradation of bio-resorbable stents left no foreign objects in the blood vessel for an extended period, and thus the risks of late and very late stent thrombosis may be reduced or even eliminated. 204 The PLA polymer-based stents can effectively intermediate revascularization of coronary artery lesions and demonstrate relatively low rates of major adverse cardiac events in early follow-up. 173 , 205 The Igaki-Tamai stent (Kyoto Medical Planning Co., Ltd., Kyoto, Japan) made of PLA is the pioneering bio-resorbable stent employed in human patients, which undergoes self-expansion at body temperature until it reaches equilibrium with vessel wall dilation and resistance. 206 Biodegradable stents present an appealing alternative to self-expanding stents for managing biliary, coronary, and various duct-related ailments, 207 where other chemical components can be incorporated into PLA to tailor the characteristics, e.g., ranging from soft and elastic materials to rigid and high-strength materials. 208 There are also reports about the implantable, biodegradable and inflatable balloons made by polymers of PLA, 209 where the balloons were developed as a sub-acromial spacer in the treatment of extensive irreparable rotator cuff tears. 210 Section title: Antimicrobial materials and surgical instruments Educational score: 3.7505319118499756 Domain: biomedical Document type: Study Language: en Antimicrobial materials are crucial in the medical field, particularly in surgical environments, to prevent infections and promote patient recovery. 196 Several additives, including natural compounds, peptides, enzymes, metals, chelating agents, and antibiotics, have been incorporated into PLA polymer matrix to impart antimicrobial activity. 211 , 212 , 213 , 214 , 215 Section title: Antimicrobial materials and surgical instruments Educational score: 4.141873359680176 Domain: biomedical Document type: Study Language: en PLA silver ion nanoparticles are synthesized by introducing silver ions into the PLA matrix. These nanoparticles combine the biocompatibility of PLA with the broad-spectrum antimicrobial properties of silver ions, resulting in significant antimicrobial efficacy. 216 Silver ions can disrupt bacterial cell membranes, interfere with bacterial DNA replication, and inhibit protein synthesis, effectively hindering bacterial growth. As a carrier, PLA allows for the sustained release of silver ions, extending the antimicrobial action, and degrades gradually within the body, reducing the side effects in long term. 217 , 218 Recent studies have reported a new antimicrobial surgical retractor manufactured using 3D printing technology with PLA materials, and a thin layer of silver ion PLA nanoparticles is uniformly deposited and fixed on the surface via sonochemical thin-film deposition technology. This retractor exhibits excellent antimicrobial properties and costs only one-tenth of the stainless-steel retractor. 216 Section title: Antimicrobial materials and surgical instruments Educational score: 3.780965566635132 Domain: biomedical Document type: Other Language: en Overall, surgical tools such as scalpels, sutures, and needles can be coated or embedded with PLA-metal nanoparticles to create a durable antimicrobial layer that protects wounds from bacterial invasion. 219 Additionally, these nanoparticles can be used to produce antimicrobial dressings, catheters, and implants, providing extensive antimicrobial protection. 220 Section title: Challenges in characteristics of the PLA materials Educational score: 4.108839988708496 Domain: biomedical Document type: Study Language: en Controlled biodegradability is considered the core value of PLA materials, but matching the degradation rate precisely with the clinical treatment cycle is still a challenge. 221 The degradation of PLA scaffolds needs to establish a give-and-take relationship with the regeneration of human tissues, and the drugs released from PLA nanoparticles also need to meet the pharmacokinetic demand of targeted tissue. Therefore, research efforts have been made to investigate the degradation rate of PLA materials in different microenvironments of the human body, e.g., tumor microenvironments, skeletal muscle tissues, blood vessels, etc. 222 Additionally, it is worth considering the local inflammatory effects caused by the lactic acid produced during PLA degradation. PLA is generally considered nontoxic and biocompatible, but when it degrades rapidly and the local tissue circulation is restricted, the accumulation of lactic acid may cause a decrease in pH, possibly leading to inflammation and tissue irritation, although the syndrome is usually limited and manageable. 223 , 224 Therefore, PLA material design needs to consider controlling the degradation rate to prevent excessive lactic acid buildup. Section title: Challenges in characteristics of the PLA materials Educational score: 4.325181007385254 Domain: biomedical Document type: Review Language: en The tensile strength of PLA is moderate among bioplastics, sufficient for static load applications, and PLA has a high compressive strength, allowing it to withstand substantial compression loads. It also possesses relatively high stiffness and modulus, providing good hardness and stability. 10 , 225 However, PLA has limitations in terms of brittleness and ductility, making it more prone to fracture under impact or tensile stress. 226 Additionally, its heat resistance is insufficient, with a glass transition temperature typically ranging from 60°C to 65°C, which may lead to softening and deformation in vivo. 227 When applied in the human body, the mechanical strength of PLA can become unstable under prolonged exposure to body temperature and humidity. 228 In tissue regeneration scaffolds, the porous and loose structure of PLA is beneficial for cell adhesion and growth, but it also reduces mechanical strength, compromising the long-term stability of the scaffold. 229 Therefore, new modification strategies are needed to expand the application range of PLA materials. Enhancing the mechanical performance of PLA can be achieved through copolymerization, blending, the incorporation of additives or fillers, and optimization of processing techniques. 10 , 225 , 230 , 231 Section title: Challenges in characteristics of the PLA materials Educational score: 4.344043731689453 Domain: biomedical Document type: Study Language: en The hydrophobicity of PLA material is a disadvantage for medical applications by impairing cell adhesion. 232 Surface modification methods such as plasma treatment, 233 chemical coating 234 or UV light-induced free radical reactions can be used to increase the hydrophilicity of PLA, 235 and then enhance cell adhesion and extracellular mineralization. 236 Additionally, blending PLA with other hydrophilic materials can increase the hydrophilicity of the composite. 237 However, uneven dispersion of the hydrophilic materials within the PLA matrix may happen during blending, leading to phase separation. 238 For example, when blending natural fibers with PLA, insufficient interfacial adhesion between the fibers and the PLA matrix can lead to uneven fiber distribution and reduced mechanical properties. 167 Using chemical coupling agents or compatibilizers, along with optimizing the blending process, can improve the interface bonding between PLA and hydrophilic materials, reduce uneven dispersion, and thereby enhance the overall mechanical properties and uniformity of the composite. 239 Enhanced hydrophilicity of PLA materials has been shown to improve cell adhesion and proliferation, thereby promoting tissue regeneration and extracellular mineralization in bone repair, and also accelerating wound healing. 236 Section title: Promoted PLA clinical applications with advanced manufacturing Educational score: 4.04354190826416 Domain: biomedical Document type: Study Language: en Currently, 3D printing technology has been widely utilized in the processing of PLA materials, including bone defect implants based on the precise 3D modeling of imaging data, and porous, biologically labeled, multi-phase tissue regenerative scaffolds. 240 , 241 , 242 The newly proposed concept 4D printing refers to the additional feature that products printed in 3D can undergo shape and structural changes when stimulated externally. 243 The combination of PLA materials with 4D printing may find applications in bioresorbable coronary stents. 232 When placed inside blood vessels, a bioresorbable coronary stent changes its shape in response to temperature variations after coronary revascularization and degrades over time to reduce the occurrence of blood clots. Section title: Promoted PLA clinical applications with advanced manufacturing Educational score: 4.0202436447143555 Domain: biomedical Document type: Study Language: en Another concept to be mentioned is 3D bioprinting, which produces more complex structures to meet the requirements of multi-level and multi-space regeneration engineering. 244 Currently, PLA has been used in 3D printing to make a cartilage-bone biphasic scaffold for knee joint cartilage repair or a bone-ligament-muscle triphasic scaffold for ligament regeneration. 243 In the field of skin regeneration engineering, multiphase PLA scaffolds have been proposed to include epidermis, dermis, blood vessels, and nerves, along with PLA hydrogels or nanoparticles loaded with antimicrobial drugs and growth factors. Furthermore, 3D bioprinting may integrate stem cell materials into printing inks, which facilitates complete organ regeneration on PLA scaffolds. Section title: Conclusions Educational score: 3.7524447441101074 Domain: biomedical Document type: Review Language: en PLA has established itself as a remarkable and versatile material in the realm of medical applications. Its biocompatibility, biodegradability, controlled degradation, and versatile processing have made it indispensable in various medical fields, ranging from drug delivery systems, fixation devices and implants, tissue regenerative engineering to wound management and beyond. Nevertheless, the PLA material itself still has some drawbacks, which can be further improved in the future through material modification and advanced manufacturing. In summary, the PLA materials have shown a promising future in medical innovation and will contribute significantly to healthcare advancement and patient well-being. Section title: Acknowledgments Educational score: 1.0442250967025757 Domain: other Document type: Other Language: en This research is supported by the program CNSB (Carbon-Negative Synthetic Biology for Biomaterial Production from CO2) under the Campus for Research Excellence and Technological Enterprise (CREATE) of National Research Foundation , Prime Minister’s Office, Singapore. Section title: Author contributions Educational score: 1.0175353288650513 Domain: other Document type: Other Language: en Conceptualization, P.X. and S.S.; investigation, Z.Y.; writing – original draft, Z.Y.; writing – review and editing, S.S.; visualization, Z.Y.; supervision, P.X. and S.S.; project administration, G.Y. and P.X. Section title: Declaration of interests Educational score: 0.8786384463310242 Domain: other Document type: Other Language: en The authors declare no competing interests. | Review | biomedical | en | 0.999995 |
PMC11699633 | Section title: Background Educational score: 2.024301290512085 Domain: biomedical Document type: Other Language: en More than half of the adult population in Germany is overweight, about 20% obese . Corresponding figures for children are 15% and 6%, respectively . Improving diet quality is an important means of tackling this growing health problem. Nutrition policies such as the reformulation of packaged food as well as the use of front-of-pack nutrition labels (FoPLs) are seen as effective interventions to create a healthier food environment and thus improve population diets . Section title: Background Educational score: 2.999330520629883 Domain: biomedical Document type: Other Language: en The World Health Organization defines food reformulation as “altering the processing or composition of a food or beverage product, to improve its nutritional composition or to reduce its content of ingredients or nutrients of concern” . In 2016, the European Union urged Member States to implement corresponding reformulation strategies . In response, Germany initiated the “National Reduction and Innovation Strategy for Sugar, Fats, and Salt in Processed Food” (NRI), which is based on voluntary commitments by the industry and accompanied by an annual product monitoring . One voluntary commitment by the food sector concerns breakfast cereals and aims to reduce the sugar content of products targeted at children by at least 20% until the end of 2025 . Breakfast cereals is one of the food categories with a high customer reach, especially popular among children, and extensively marketed to them , they are a prioritised food group in reformulation strategies of various countries [e. g. 11 – 14 ]. Section title: Background Educational score: 2.572805166244507 Domain: biomedical Document type: Other Language: en Although reformulation of packaged food can help improve the nutritional quality of food and enable individuals to consume a healthier diet to a certain degree , nutritionally favourable options are often not easily recognisable for consumers. Here, the role of FoPLs as consumer guidance comes into play . Among FoPLs, the Nutri-Score as a colour-coded, evaluative FoPL is an efficient tool to facilitate comparisons between products within a food category, enabling consumers to make informed and potentially nutritionally favourable food choices [ 16 – 19 ]. Section title: Background Educational score: 3.2095744609832764 Domain: biomedical Document type: Other Language: en Developed and first introduced in France in 2017, the Nutri-Score has now been adopted as the official FoPL in seven European countries, including Germany in 2020. A proposal for a harmonised mandatory FoPL at EU level, as part of the Farm to Fork strategy, is currently pending, with the Nutri-Score being one of the potential labels discussed . Although the use of the Nutri-Score is so far voluntary, food manufacturers or retailers that choose to use the Nutri-Score are obliged in general to display the FoPL on all their products within 2 years . The Nutri-Score undergoes regular revisions, including the most recent one in 2023, with the consistent goal to improve the discrimination between products with high and low contents of unfavourable nutrients. Smaller increments in the point allocation scale for instance for sugar allow for a stricter and therefore more adequate Nutri-Score rating. As the discrimination of sugary products was considered a key focus in this context, breakfast cereals were included as a priority product group . In Germany, the 2023 algorithm was introduced in January 2024, with a 24-month transition period . Section title: Background Educational score: 1.235750436782837 Domain: other Document type: Study Language: en With regard to current national reformulation efforts within the NRI, this paper aims to answer the following research questions: Section title: Background Educational score: 1.1841310262680054 Domain: other Document type: Other Language: en Are there changes in the energy and nutrient content of breakfast cereals between 2019 and 2022 on the German market? Did producers reformulate existing products within this time frame? If products have been specifically reformulated, does this affect the Nutri-Score classification? Section title: Background Educational score: 2.8195745944976807 Domain: biomedical Document type: Study Language: en To this end, based on data from the German monitoring of packaged food , changes in energy and nutrient contents of breakfast cereals targeted at children (children’s breakfast cereals) and breakfast cereals not targeted at children (non-children’s breakfast cereals) on the German market between 2019 and 2022 were evaluated. To determine whether the nutrient composition of individual breakfast cereals had changed, a sample of paired products that were available at both time points of data collection was analysed. Based on this subsample, it was assessed which of the observed changes in the nutrient composition affected the Nutri-Score classification. Section title: Data collection and management Educational score: 3.239971876144409 Domain: biomedical Document type: Study Language: en Data on breakfast cereals were gathered from August to December in 2019 and 2022, respectively, as part of the German monitoring of packaged food. Data collection comprised the energy and nutrient contents as stated in the mandatory nutrition declaration (fat, saturated fat, carbohydrate, sugar, protein, and salt) . Data on fibre was recorded when provided. Where available, additional labelling information such as ingredient lists, label on organic production or Global Trade Item Number (GTIN) was collected. In order to cover the market as broadly as possible, data was predominantly collected online via the manufacturers’ websites. To fill data gaps, the online research was complemented by enquiries with manufacturers and visits to grocery stores. The data were managed with a customised branded food module within the FoodCASE software, version 7.9.1 (Premotec GmbH, Winterthur, Switzerland). In the case of implausible values or missing data concerning energy or nutrient content, manufacturers were contacted to verify, correct, or complete the respective information. In case of no answer, these products were excluded from analysis. Detailed information on the design and methods of the German monitoring of packaged food is published elsewhere . Section title: Inclusion criteria and definition of food categories Educational score: 2.260711431503296 Domain: biomedical Document type: Study Language: en Breakfast cereals for which the nutritional information was available for the unprepared product, i.e. without the addition of milk or other components, were considered. Plain rolled grains without any other ingredients and savoury porridges (e.g. tomato-broccoli porridge) were excluded. Furthermore, breakfast cereals consisting mainly of nuts and seeds rather than cereals and declared as “protein”, “power”, or “sport” were eliminated, as a significantly different nutritional composition was expected. All included products were grouped into one of three categories (see Table 1 ) based on the product name and/or ingredient list. Section title: Inclusion criteria and definition of food categories Educational score: 1.7339259386062622 Domain: other Document type: Other Language: en Table 1 Description of breakfast cereal categories Category Definition Examples Muesli and porridge Contain quick cooking or rolled cereal grains, pseudo cereals, or soya flakes as main ingredients. May contain dried fruits, nuts, chocolate, or other ingredients. Crunchy nut and fruit muesli; apple cinnamon porridge Flakes Made from corn, oats, rice, or other cereal grains. Not containing any fruits, nuts or chocolate, except for in the coating. Cornflakes; frosted flakes Other cereal products Made from toasted, puffed or popped cereal grains shaped into biscuits, as well as flakes with additional ingredients such as fruits, nuts or chocolate. May contain chocolate or cocoa. Nougat bits; cinnamon chips; choco balls Section title: Inclusion criteria and definition of food categories Educational score: 1.7927058935165405 Domain: other Document type: Other Language: en Given the importance of breakfast cereals in children’s diets, a distinction was made between children’s breakfast cereals and those not targeted at them, in order to look separately at the nutrient composition and product reformulation. Within the three categories, the differentiation was based on the design of the packaging or the product itself. Table 2 describes the four criteria, of which a given product must fulfil at least one in order to be classified as children’s breakfast cereal. Section title: Inclusion criteria and definition of food categories Educational score: 1.281786561012268 Domain: other Document type: Other Language: en Table 2 Criteria for products classified as “children’s” Criteria Examples The product name includes “child(ren)” or “kids” etc. or appeals directly to children “Chocolate Bears” The packaging is attractively designed for children Displaying smiling animals or cartoon characters The food product itself or its components is/are designed for children Cereals in the shape of bears or letters The packaging includes information aimed at parents or children “for your little ones”, notes on games for children, learning effects, or information about free toys or collectible picture cards inside Section title: Product pairing Educational score: 3.284764051437378 Domain: biomedical Document type: Study Language: en In order to assess whether the nutrient composition of a given breakfast cereal changed over time, a subsample of breakfast cereals present on the market in both survey years was analysed. Such pairing of products over time is not performed in the German monitoring of packaged food but was carried out as part of the EU Joint Action Best-ReMaP, in which Germany took part . Pairing was done manually as follows: (1) GTIN for each product of 2022 was used to identify a matching GTIN in the 2019 data. (2) if there was no result of matching GTINs or no GTIN available, product information, including brand name, product name, legal name, flavour, and net weight were used to search for a match. The ingredient list and the nutritional values could be different, but product or legal name had to be the same or close. If two products of 2022 could be assigned to one product of 2019, the product nearest in net weight was counted as a match. Breakfast cereals that had changed the design over the years, resulting in a different classification of a product as child-targeting or not in 2019 and 2022, were classified according to the 2022 design for the subsample of paired products. Section title: Calculation of the Nutri-Score Educational score: 4.07613468170166 Domain: biomedical Document type: Study Language: en The 2023 algorithm of the Nutri-Score for general foods was used to compute the overall nutritional value of breakfast cereals . In brief, ‘unfavourable’ points were allocated for energy (0 to 10 points), saturated fats (0 to 10 points), sugar (0 to 15 points), and salt (0 to 20 points), whereas ‘favourable’ points were allocated to proteins (0 to 7 points) as proxy for iron and calcium, fibres (0 to 5 points), and the percentage of fruits, vegetables, and legumes (henceforth described as F&V component) (0 to 5 points). The ‘unfavourable’ (0 to 55 points in total) and ‘favourable’ points (0 to 17 points in total) are balanced out in the computation of the final nutritional score (FNS). However, ‘favourable’ points for protein are disregarded, if a product scores more than 11 ‘unfavourable’ points. In consideration of this rule, the FNS may range from − 17 (best rating) to 45 points (worst rating). The FNS is attributed a Nutri-Score ranging from A to E (Table 3 ). In our study, products classified as C, D, or E (FNS ≥ 3) are defined as ‘less favourable’. The computation of the Nutri-Score is based on the declared nutritional values per 100 g for energy, saturated fats, sugar, protein, salt, and fibre. Based on the ingredient list, the percentage of F&V was estimated by an experienced nutritionist using the Nutri-Score FAQ document . If the ingredient list was not available on the manufacturer’s website or fibre content was not declared, the product was excluded. Section title: Calculation of the Nutri-Score Educational score: 2.0728628635406494 Domain: biomedical Document type: Other Language: en Table 3 Final nutritional score (FNS) ranges with their corresponding Nutri-Score classification and colour code Section title: Statistical analysis Educational score: 4.003314971923828 Domain: biomedical Document type: Study Language: en All statistical analyses were performed using the software R, version 4.3.2. The normality of the data distribution was tested using the Shapiro-Wilk test and rejected. Consequently, the non-parametric Mann-Whitney test for two independent samples was used to compare energy and nutrient contents as well as the FNS of children’s breakfast cereals respectively non-children’s breakfast cereals between survey years 2019 and 2022. For all results, p-values < 0.05 were considered as statistically significant. Furthermore, shares of pairs that showed a change in FNS were calculated. This subsample is further analysed as the individual scores of the FNS in 2019 and 2022 were plotted against each other and the effects on the Nutri-Score classification described. Section title: Changes in the nutritional composition of the breakfast cereal offer on the German market Educational score: 1.481540560722351 Domain: other Document type: Study Language: en A total of 888 and 1473 breakfast cereals were included in the sample of 2019 and 2022, respectively. Overall, 14% of the breakfast cereals in 2019 ( n = 127) and 15% of the breakfast cereals in 2022 ( n = 225) were classified as children’s breakfast cereals. Figure 1 shows that in particular more mueslis and porridges (hereafter referred to as muesli) have been recorded over the years. However, only for children’s breakfast cereals, the distribution across the categories shifted clearly to muesli. The category of flakes constituted the smallest group in each year. Section title: Changes in the nutritional composition of the breakfast cereal offer on the German market Educational score: 1.5238182544708252 Domain: other Document type: Study Language: en Fig. 1 Distribution across the subcategories differentiated into non-children’s and children’s breakfast cereals for data collection years 2019 and 2022 Section title: Changes in the nutritional composition of the breakfast cereal offer on the German market Educational score: 4.110000133514404 Domain: biomedical Document type: Study Language: en The comparison between the subsamples revealed significantly lower sugar and significantly higher fat contents in 2022 than in 2019. Children’s breakfast cereals demonstrated a greater percentage change, with a 25.5% reduction in sugar ( p < 0.001) and a 32.0% increase in fat ( p < 0.001) than non-children’s breakfast cereals with − 8.7% ( p < 0.001) and + 7.0% ( p = 0.036), respectively. For energy, no significant change was found ( p = 0.913; p = 0.844). Overall, mean FNS was only significantly reduced in children’s breakfast cereals, by 3 points ( p < 0.001) (see Table 4 ). The share of products with a ‘green’ (A or B) classification as well as products classified as Nutri-Score C was higher in 2022 than in 2019. At the same time, the share of Nutri-score D products was lower. The share of breakfast cereals classified as less favourable (Nutri-Score C-E) was higher for children’s breakfast cereals . At category level of children’s breakfast cereals, a reduction of sugar content was found in other cereal products (12.4%, p = 0.003) and an increase of fat content in muesli (39.0%, p = 0.016) and flakes (149.3%, p = 0.016). For non-children’s breakfast cereals, a reduction of sugar content was shown in muesli (9.2%, p < 0.001) and flakes (25.9%, p = 0.032). The latter also showed an increase in fat content (99.5%, p = 0.031). For details on changes at category level, see Supplement File 1. Section title: Changes in the nutritional composition of the breakfast cereal offer on the German market Educational score: 4.080698013305664 Domain: biomedical Document type: Study Language: en Table 4 Mean energy and nutrient contents of breakfast cereals surveyed in Germany in 2019 ( n = 888) and corresponding changes in 2022 Breakfast cereals Non-children‘s Children‘s mean in 2019 ( n = 761) mean change in 2022 mean in 2019 ( n = 127) mean change in 2022 ( n = 225) abs. rel. (%) sig. 1 abs. rel. (%) sig. 1 Energy [kcal/100 g] 400.7 + 0.8 + 0.2 390.3 + 1.0 + 0.3 Fat [g/100 g] 10.7 + 0.7 + 7.0 * 5.5 + 1.8 +32.0 *** Saturated Fat [g/100 g] 3.1 0.0 + 0.5 1.6 + 0.5 +33.7 ** Carbohydrate [g/100 g] 60.9 -2.5 -4.1 *** 73.9 -5.6 -7.6 *** Sugar [g/100 g] 15.7 -1.4 -8.7 *** 22.9 -5.8 -25.5 *** Protein [g/100 g] 10.9 + 1.0 + 8.9 *** 8.4 + 1.2 +14.0 *** Salt [g/100 g] 0.25 -0.03 14.1 ** 0.43 -0.20 -45.5 *** Fibre [g/100 g] 2 8.8 + 0.4 + 4.1 * 6.1 + 1.4 +22.8 *** Final nutritional score 3 (FNS) 4.7 -0.3 -5.5 8.7 -3.1 -35.9 *** 1 Statistically significant change in mean content in 2022 compared to 2019 (* p < 0.05; ** p < 0.01; *** p < 0.001) 2 Sample size differs, as the fibre content was not available for 123 resp. 154 non-children’s breakfast cereals and 29 resp. 22 children’s breakfast cereals 3 Sample size differs, as the fibre content and/or ingredient list was not available to calculate the FNS for 123 resp. 156 non-children’s breakfast cereals and 29 resp. 22 children’s breakfast cereals Section title: Changes in the nutritional composition of the breakfast cereal offer on the German market Educational score: 1.688778042793274 Domain: biomedical Document type: Study Language: en Fig. 2 Shares of Nutri-Score classification differentiated into non-children’s and children’s breakfast cereals in Germany for data collection years 2019 ( n = 736) and 2022 Section title: Reformulation of breakfast cereals Educational score: 2.03568696975708 Domain: biomedical Document type: Study Language: en In order to examine whether a given product underwent reformulation, the FNS and Nutri-Score of a subsample of paired breakfast cereals ( n = 424), i.e. marketed under the same name in 2019 and 2022, was further analysed. Section title: Reformulation of breakfast cereals Educational score: 2.290595054626465 Domain: biomedical Document type: Study Language: en A change in the FNS occurred in 34.6% of all pairs (Table 5 ). The share of pairs showing an improved nutritional value was higher than the share of pairs which deteriorated. For children’s breakfast cereals the share of pairs that deteriorated was higher compared to non-children’s breakfast cereals (20.0% vs. 9.9%). Section title: Reformulation of breakfast cereals Educational score: 2.5535480976104736 Domain: biomedical Document type: Study Language: en Table 5 Changes of the final nutritional score (FNS) and Nutri-Score for all pairs of breakfast cereals on the German market in 2019 and 2022 stratified into non-children’s and children’s products Pairs n (%) FNS change n (%) More favourable Less favourable Nutri-Score change n (%) More favourable Less favourable All pairs 424 (100%) 147 (34.6%) 99 (23.3%) 48 (11.3%) 60 (14.2%) 44 (10.4%) 16 (3.8%) Non-children’s 364 (100%) 119 (32.7%) 83 (22.8%) 36 (9.9%) 46 (12.6%) 36 (9.9%) 10 (2.7%) Children’s 60 (100%) 28 (46.7%) 16 (26.7%) 12 (20.0%) 14 (23.3%) 8 (13.3%) 6 (10.0%) Section title: Effects of reformulated breakfast cereals on the Nutri-Score classification Educational score: 2.7854583263397217 Domain: biomedical Document type: Study Language: en Only for a small number of pairs (60 out of 424 products) did reformulation have an effect on the Nutri-Score classification (Table 5 ). Of these, most products (45%; n = 27) shifted from Nutri-Score D to Nutri-Score C . In the case of children’s breakfast cereals, these were exclusively products belonging to the category of other cereal products, while for non-children’s breakfast cereals these were mainly flakes and muesli products. Only few products (13%; n = 8) improved their rating to ‘green’ (Nutri-Score A or B) (especially mueslis) . In 27% of products ( n = 16), the Nutri-Score classification deteriorated, with shifts mostly from class A to B or C (only non-children’s products) or from C to D (only children’s products) . Section title: Effects of reformulated breakfast cereals on the Nutri-Score classification Educational score: 2.024162530899048 Domain: biomedical Document type: Study Language: en Fig. 3 Shifts in Nutri-Score classification of pairs showing a change ( n = 60); arrows indicate an improvement Section title: Effects of reformulated breakfast cereals on the Nutri-Score classification Educational score: 4.053516864776611 Domain: biomedical Document type: Study Language: en Changes in the FNS that led to a change in the Nutri-Score classification were in the range of -11 to -1 (improvement) and in the range of + 1 to + 9 (deterioration) points, respectively. The most often observed changes in FNS were in the range of -1 to -4 points. Most products achieved an improvement in FNS by reducing sugar (-0.1 to -10 g/100 g), often accompanied by FNS-affecting changes in fibre (+ 0.1 to + 3.0 g/100 g), protein (+ 0.1 to + 3.6 g/100 g), saturated fat (-0.1 to -5.2 g/100 g), or salt (-0.09 to -1.3 g/100 g), or a combination of these. The largest FNS improvements were primarily driven by reducing saturated fat, often by substituting palm oil with sunflower oil, or by reducing salt content. For most products with a reduction of at least 5 FNS points, the number of ‘unfavourable‘ points was below the limit of 11 points, resulting in considering the ‘favourable‘ points for protein content in the product of 2022, which were not counted in the product of 2019. Section title: Effects of reformulated breakfast cereals on the Nutri-Score classification Educational score: 2.585921049118042 Domain: biomedical Document type: Study Language: en For products with a large increase in FNS (deterioration), increases in sugar content (+ 4 to + 6.6 g/100 g) and saturated fat content (+ 0.1 to + 4.4 g/100 g) in particular forced the disregard of the positive points for the protein content. Section title: Effects of reformulated breakfast cereals on the Nutri-Score classification Educational score: 3.208552837371826 Domain: biomedical Document type: Study Language: en As Fig. 4 illustrates, not only the magnitude of the change in the FNS score was decisive for a change in Nutri-Score classification, but also the initial position of a product (whether it was close to a class threshold or not) as well as the initial class itself. Since many of the products in category D were close to the threshold to category C, relatively small changes of 1–2 FNS points were already sufficient to change the Nutri-Score classification to C. Section title: Effects of reformulated breakfast cereals on the Nutri-Score classification Educational score: 2.173093557357788 Domain: biomedical Document type: Study Language: en Fig. 4 Final nutritional score (FNS) and corresponding Nutri-Scores for ( a ) all non-children’s and ( b ) all children’s pairs of breakfast cereal products with a change in FNS ( n = 147). Sorted in ascending order by FNS of 2019 (complete FNS scale ranges from − 17 to 45 points) Section title: Discussion Educational score: 2.2718141078948975 Domain: biomedical Document type: Study Language: en This study investigated the nutrient composition of children’s and non-children’s breakfast cereals on the German market in 2019 and 2022, particularly with regard to reformulation and the potential of the Nutri-Score to visualise such. Section title: Developments on the breakfast cereal market in Germany Educational score: 4.058358192443848 Domain: biomedical Document type: Study Language: en Our sample indicated that, between 2019 and 2022, the number of breakfast cereal products on the German market grew substantially, with concomitant changes in the average nutrient content. Whereas the average sugar content decreased significantly, especially in the group of children’s breakfast cereals, the average fat content increased significantly. Our results are in line with findings from other countries that showed declining sugar contents in this product group in the period between 2004 and 2020 [ 27 – 29 ]. A concomitant significant increase in average fat content as in our study was only found for the Australian market in different subcategories (granola and clusters, hot flavoured and plain cereal (porridge), and muesli) as well as overall in a sample of paired products . Our results on subcategory level showed these concomitant changes only for flakes. This diverse subcategory ranges from plain, unsweetened flakes to sugar- or chocolate-coated flakes high in sugar and fat, respectively. Whether this interaction (fat up, sugar down) shown for the overall non-paired sample is also evident on the single product level was outside the scope of this study but changes in Nutri-Score could help answer this question. A first look at the subsample of paired breakfast cereals with changes in Nutri-Score due to a decrease of negative points by a sugar reduction (e.g. -8.0 g/100 g) indicates that this interaction exists, although the increase in fat was rather small (e.g. 0.5 g/100 g). Section title: Developments on the breakfast cereal market in Germany Educational score: 3.010948419570923 Domain: biomedical Document type: Study Language: en Pairing the products from different time points revealed a fast-changing breakfast cereal market in Germany: out of 888 products in 2019, only 424 (47.7%) were still on the market in 2022. The product monitoring in France specifically analyses market changes and also shows that the overall market is mainly influenced by the introduction of new products and products that are withdrawn from the market. Between 2011 and 2018, 63% of the sample were newly introduced products . Therefore, the observed changes in average nutrient content are explainable by shifts in the portfolio of products over the years rather than the reformulation of existing products. Compared to 2019, the 2022 subsample contained substantially more mueslis (especially porridges), and these typically have lower sugar content but higher fat content than other categories . On the one hand, these market developments do appear beneficial for public health in terms of improving the food offer, as many mueslis in particular had come onto the market, many of which have a nutritionally favourable composition. Increasing demand for alternatives to classic cereals such as froot loops, honey pops or chocolate crispies (other cereals category), as well as more opportunities for product variation in the muesli category, could be the reason for the growing muesli market, which has had a major impact among children’s cereals in particular. This is supported by a pronounced shift in consumption shares away from other cereals towards muesli, but also rolled oats and cornflakes, in 2021 . Section title: Visualisation of food reformulation Educational score: 2.3310153484344482 Domain: biomedical Document type: Study Language: en FoPL is discussed as a lever for reformulation and thus for improving the product composition . However, our findings for paired products and based on the Nutri-Score algorithm as updated in 2023 demonstrate that little progress has been made regarding the improvement of the overall nutritional composition on the single product level. Only for a few products did reformulation have an impact on the Nutri-Score classification; this observation may have been different with the more lenient 2015 algorithm in force in the survey years. Particularly in the case of children’s products, a comparatively large share has actually deteriorated in the Nutri-Score. While this may be an artefact from using the stricter 2023 algorithm, it emphasises the gap between actual product formulation and what would be desirable in terms of public health. Section title: Visualisation of food reformulation Educational score: 4.095797061920166 Domain: biomedical Document type: Study Language: en Moreover, if a product had been reformulated, the product often fell just below the threshold of the next better Nutri-Score class, which is particularly noticeable in children’s products. One might argue that this again could be an artefact from using the stricter 2023 algorithm of the Nutri-Score, when the 2015 algorithm was the point of reference in 2019 and 2022. However, our observation is paralleled by recent results for the French market and based on the 2015 algorithm: even though the introduction of the Nutri-Score brought breakfast cereals with a more favourable nutritional composition to the market, an accumulation of products just below the threshold of the next better Nutri-Score class was recognised . Hence, products are not necessarily reformulated in a way that would make their being nutritionally favourable visible through a better Nutri-Score. Where improvements in Nutri-Score emerged, this was explained largely by decreases in sugar and salt content and increases in fibre and protein content . Our study extends these findings by exploring how changes in the individual nutrients interrelate to improve the Nutri-Score classification in the end. We found that the improvement was primarily due to a combination of sugar reduction and increase in protein or fibre content. Our analyses also revealed that the number of negative points can be considerably reduced, particularly by lowering saturated fat, as the range between two thresholds (1 g/100 g per level) are comparatively lower than for sugar (4.5 g/100 g per level). In this regard, substituting palm oil through e.g. sunflower oil, seems to be an easy leverage point for mueslis in particular to improve their overall rating, as we could determine for some crunchy mueslis. Further modelling studies at the individual food level are necessary to provide information as to what compositional changes would be the most feasible in order to achieve a better overall Nutri-Score. Section title: Visualisation of food reformulation Educational score: 3.192885637283325 Domain: biomedical Document type: Study Language: en Although we were able to demonstrate that reformulation within breakfast cereals could be visualised via the Nutri-Score and thus can be an opportunity for food producers to positively highlight their own products in comparison to competing products receiving a less favourable rating, the visibility for consumers appears to remain limited so far. As per current EU regulation , usage of the Nutri-Score is and can only be voluntary. A first survey, conducted just after the implementation of the Nutri-Score in Germany, showed a labelling of less than 10% of the included cereals . A snapshot taken about a year later noted a coverage of 28% . Despite this increase, the data suggest that the vast majority of breakfast cereals on the German market do not carry a Nutri-Score. Unless the Nutri-Score is applied on all products, the empowerment of consumers to quickly identify nutritionally favourable products within a given food category remains impaired. Food manufacturers could send a strong signal of consumer support by adopting the Nutri-Score as the government-endorsed FoPL in Germany on all their products. This is supported by comprehensive evidence reviews concluding that summary indicators with or without colour-coding help consumers best in identifying and choosing the nutritionally favourable option . It must be borne in mind though that the Nutri-Score as a FoPL should not be used as an overall guidance on ‘healthy’ products. Section title: Reformulation policies Educational score: 3.8416378498077393 Domain: biomedical Document type: Study Language: en Food reformulation is often viewed as one of several means to reduce the prevalence of overweight and obesity. However, as observed in our data and already discussed, measures targeting only individual nutrients (e.g. sugar), can lead in some product groups to an increase in another nutrient with the same or even higher energy content , and may thus not achieve the overall goal of reducing energy density. Gressier et al. showed that reformulation strategies generally resulted in products with the same energy content and are hence unlikely to contribute to a lower energy intake in the population . Since simultaneous reduction in energy content is not always easy to achieve due to technological challenges, it is even more important that reformulation strategies focus on nutritionally favourable products overall, e.g. by increasing the proportion of wholegrain in breakfast cereals. Section title: Reformulation policies Educational score: 2.805671453475952 Domain: biomedical Document type: Other Language: en The food market is characterised by a rapidly changing variety of products with a high proportion of new products. Thus, not only reformulation of existing products but also the launch of new products with a more favourable nutrient composition can help shift the food offer towards healthier choices. However, from a public health perspective this is only effective, if consumers switch to these products. Especially if the improvement of the overall nutritional composition of existing products (e.g. best-selling products) remains at a level as low as shown for this sample, there will be little public health benefit. Connecting product monitoring data with specific sales and consumption data on the single product level would be necessary in order to draw concrete conclusions about the public health impact of specific product reformulation or of general changes within the market portfolio. Monitoring of paired products over time is essential for assessing the contribution of reformulation to an overall improved nutritional composition of the food supply. Section title: Strengths and limitations Educational score: 3.3895251750946045 Domain: biomedical Document type: Study Language: en Our study sheds light on several nutrition policy issues. We were able not only to show differences between single nutrients over time in breakfast cereals on the German market but also to compare the Nutri-Score classification, including possible shifts therein due to product reformulation. A strength of the study is the combined investigation of the overall market and the subgroup of paired products. This allows for concrete insights regarding nutritional changes on the overall market as well as for reformulated products . However, no clear conclusion could be drawn regarding the public health impact of the observed change in nutritional composition of the breakfast cereal supply, as the data could not be linked to sales and/or consumption data. Also, thorough as our approach for pairing products from different survey years was, we may have missed products where a change in name or presentation obscured the link. Section title: Strengths and limitations Educational score: 1.2122758626937866 Domain: other Document type: Other Language: en Although the survey method was the same in both survey years, the large differences in the sample size and composition could, beside a changed market portfolio, also be due to the fact that products became more easily accessible via the internet as a result of the Covid-19 pandemic, during which online retail got more important . Section title: Strengths and limitations Educational score: 1.8824689388275146 Domain: biomedical Document type: Study Language: en The current investigation is based on the mandatory declared nutritional information. While we were able to draw on a large sample of breakfast cereals available on the German market, our analysis relies on the precision of the data presented on these labels or on the manufacturer’s website. It is presumed that manufacturers have adhered to EU regulations by supplying precise and up-to-date information. We carried out extensive plausibility checks to minimise erroneous data. Section title: Strengths and limitations Educational score: 2.168123483657837 Domain: biomedical Document type: Study Language: en For products lacking specific details on the fruit content, estimations were necessary for computing the FNS. These estimations were made by a trained nutritionist on the basis of comparable products for which sufficient information was available. Therefore, it is assumed that any bias resulting from the estimation was kept to a minimum. Section title: Conclusion Educational score: 4.052974700927734 Domain: biomedical Document type: Study Language: en This study showed a reduction in sugar of over 25% in children’s breakfast cereals on the German market between 2019 and 2022, along with a significant improvement of the FNS. Since there were hardly any specific product reformulations, particularly ones that led to an improved Nutri-Score, it can be inferred that changes in the overall sample were mostly driven by shifts in the product portfolio. Unless improvements in nutrient content and FNS are reflected in widely purchased and consumed breakfast cereals, benefit for consumer health will remain limited. Reformulation guided by holistic product monitoring approaches and FoPLs like the Nutri-Score can be seen as synergistic public health tools to improve the nutritional quality of the packaged food supply and enable consumers to make nutritionally more favourable food choices. Using these tools to their fullest potential appears a worthy ambition towards the goal of reducing the prevalence of overweight and obesity and with it the burden on health care systems and people’s health. Section title: Electronic Supplementary Material Educational score: 0.9769887328147888 Domain: other Document type: Other Language: en Below is the link to the electronic supplementary material. Section title: Electronic Supplementary Material Educational score: 1.2997462749481201 Domain: biomedical Document type: Other Language: en Supplementary Material 1 | Other | biomedical | en | 0.999995 |
PMC11699685 | Section title: Background Educational score: 4.224052429199219 Domain: biomedical Document type: Review Language: en Sarcoglycanopathies (SGPs) are autosomal recessive limb-girdle muscular dystrophies (LGMDs) caused by mutations in four genes: SGCA , SGCB , SGCG , and SGCD , which lead to α-SGP (LGMDR3), β-SGP (LGMDR4), γ-SGP (LGMDR5), and δ-SGP (LGMDR6), respectively . Since the protein products of SGCA , SGCB , SGCG , and SGCD form a sarcoglycan complex and work together, the symptoms among SGPs are almost similar. However, there are slight differences. For instance, LGMDR4 are more likely to be associated with cardiomyopathy, and LGMDR3 may have a milder symptom and slightly later onset and slower progression . Section title: Background Educational score: 3.988024950027466 Domain: biomedical Document type: Study Language: en Genetic analyses of patients with SGP from Algeria, China, and Europe have been reported [ 2 – 4 ], demonstrating that many share the same variants, and that alleles harboring variants are carried on specific haplotypes, indicating founder effects underlying SGPs in various countries [ 5 – 8 ]. In contrast, SGPs are relatively rare in Japan, although there have been a few reports of patients with LGMDR3, LGMDR4, or LGMDR5 [ 9 – 11 ]. Section title: Background Educational score: 4.120145320892334 Domain: biomedical Document type: Study Language: en Previous genetic research into SGPs has focused on variants within exons and their flanking intronic regions; however, technological advances in analysis techniques, including multiplex ligation-dependent probe amplification (MLPA), RNA-seq, and long-read sequencing, have enabled identification of genetic variations in intronic regions, as well as structural genomic changes . Further, the development of artificial intelligence-based prediction tools has contributed to more accurate and cost-effective identification of splice changes . The aims of this study were to profile the genetic variation in Japanese patients with SGP and identify variant founder effects. Section title: Patients Educational score: 4.0551042556762695 Domain: biomedical Document type: Study Language: en This retrospective cohort study was performed using samples and datasets sent to the National Center of Neurology and Psychiatry between January 1979 and June 2023. Cases with absent or reduced expression of sarcoglycan, detected by immunohistochemistry, and biallelic variants in SGCA , SGCB , SGCG , or SGCD , were selected for inclusion in these analyses. This study cohort included 39 patients with negative Western blotting result for SGCA . Section title: Muscle biopsy Educational score: 3.959643602371216 Domain: biomedical Document type: Study Language: en Muscle samples for histological examination were frozen in isopentane cooled with liquid nitrogen. A series of histochemical and immunohistochemical analyses, including assessment of sarcoglycan, were performed for diagnosis. Routine histochemical and immunohistochemical stainings were conducted using standard procedures . Section title: Genetic analysis Educational score: 4.128267288208008 Domain: biomedical Document type: Study Language: en Genomic DNA was extracted from peripheral blood or muscle samples from patients, using standard procedures . All exons and flanking intron regions of SGCA , SGCB , SGCG , and SGCD were amplified; some cases were also analyzed by whole genome sequencing. All variants were confirmed by Sanger sequencing. MLPA was conducted to analyze suspected large deletions, as previously described , using SALSA MLPA Probemix P116 SGC (MRC Holland, Amsterdam, The Netherlands), according to the manufacturer’s instructions. Section title: RNA-seq Educational score: 4.086603164672852 Domain: biomedical Document type: Study Language: en We applied RNA-seq for 2 patients. As previously reported , total RNA was extracted from frozen muscle using the RNAeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. Libraries were prepared from total RNA using Ribo-Zero Gold (Illumina), according to the manufacturer’s instructions, and 100 bp paired-end sequencing was performed on the NovaSeq 6000 platform (Macrogen, Seoul, Korea). RNA-seq reads were mapped using Spliced Transcripts Alignments to Reference software ( https://github.com/alexdobin/STAR ). Section title: RT-PCR Educational score: 4.085414886474609 Domain: biomedical Document type: Study Language: en We confirmed the splicing abnormality by RT-PCR for 5 patients. Total RNA was extracted from frozen muscle samples using an RNAeasy mini kit. Complementary DNA was generated by reverse transcription of RNA using SuperScript IV VILO Master Mix (ThermoFisher Scientific, Waltham, MA, USA). Primers are listed in Table S1 (see Additional file). The sequences of the products were confirmed by Sanger sequencing. Section title: In silico prediction Educational score: 4.352808952331543 Domain: biomedical Document type: Study Language: en The pathogenicity of missense variant is predicted to be pathogenic by more than one in silico tool (PolyPhen-2 ( http://genetics.bwh.harvard.edu/pph2/ ), MutationTaster ( http://www.mutationtaster.org/ ), or CADD ( http://cadd.gs.washington.edu/ )). The criteria for pathogenicity by prediction tool include ‘Probably Damaging’ for PolyPhen-2, ‘Disease-causing’ for MutationTaster, and a CADD score of ≥ 20. SpliceAI and MaxEntScan were used to predict the effects of identified single nucleotide variants (SNVs) on aberrant splicing. SpliceAI calculates the changes in activities of splicing sites (AG, gain of acceptor site; AL, loss of acceptor site; DG, gain of donor site; DL, loss of donor site) as delta scores, where higher scores indicate that SNVs are more likely to alter splicing. MaxEntScan assigns a log odds ratio (MaxEnt score) to the sequences of 5' and 3' splice sites, where higher scores indicate higher probabilities that splice sites are used. PDIVAS was used to predict pathogenicity of deep-intronic variants . To verify the protein 3D structure, we applied the software AlphaFold ( https://alphafold.ebi.ac.uk/ ). We also applied the prediction software iPSORT ( https://ipsort.hgc.jp/ ) and WoLF PSORT ( https://wolfpsort.hgc.jp/ ) to predict the subcellular localization of protein based on amino acid sequencing. Section title: Nanopore CRISPR/Cas9-targeted long-read resequencing Educational score: 4.167252540588379 Domain: biomedical Document type: Study Language: en Long-read sequencing was performed using genomic DNA extracted from patient blood. Library preparation was performed using a Ligation Sequencing Kit (SQK-LSK109; Oxford Nanopore Technologies Inc., Oxford, UK) with CRISPR-Cas9. Guide RNA (crRNA) sequences are listed in Table S2 (see Additional file). Prepared libraries were applied to MinION flow cells and sequenced on an MK1C instrument (Oxford Nanopore Technologies Inc., Oxford, UK). Real-time base calling was performed using MinKnow integrated in Guppy. Sequences were mapped to the human genome reference sequence (GRCh38) using Minimap2 ( https://github.com/lh3/minimap2 ). Section title: Haplotype analyses Educational score: 4.080956935882568 Domain: biomedical Document type: Study Language: en Single nucleotide polymorphisms (SNPs) around the variants, c.229C > T in SGCA , c.325C > T in SGCB , and exon 6 deletion in SGCG , were selected as neutral markers for haplotype reconstruction, based on their genomic position and allele frequency (< 0.2). Primers are shown in Table S3 (see Additional file). Section title: Results Educational score: 4.242819309234619 Domain: biomedical Document type: Study Language: en We identified 53 Japanese families and 55 patients who harbored biallelic variants in any of the SGC genes analyzed . Variants in SGCA , SGCB , SGCG , and SGCD were identified in 30 (56%), 9 (17%), 11 (21%), and 3 (6%) families, respectively . Among 32 identified variants, 14 had previously been reported and 18 were novel. In previous reports, limited types of variants have been identified in the different SGC genes, regardless of the causative gene involved and in the current study, we identified 18, 6, 4, and 4 types of variants in SGCA , SGCB , SGCG and SGCD , respectively. Some recurrent variants were identified in multiple patients . Notably, allele frequencies of the most common variants in SGCA , SGCB , and SGCG in our study population reached 32%–64%. Fig. 1 STRAD flow diagram showing the genetic analysis carried out. We started with sequencing analyses, such as Sanger sequencing, panel analysis, whole-exome sequencing, or whole-genome sequencing. Aberrant splicing was detected using RNA-seq or RT-PCR in patients with exon–intron boundary or intronic variants. MLPA was applied to patients who were not diagnosed by sequencing analysis, and the breakpoints of a large SGCG deletion were identified using long-read sequencing Table 1 Variants found in Japanese patients with sarcoglycanopathy Family Age at onset Age at biopsy Sex Serum CK (UI/L) Symptoms Causative gene Variants F1 2y 4y F 5393 High CK SGCA c.37 + 6 T > C c.725 T > C, p.V242A F2 5 y 5 y M 4538 High CK SGCA c.38-2A > C c.320C > T, p.A107V F3 4 y 37 y F 206 Arm and leg weakness, respiratory failure SGCA c.158-2_167del c.626dupG, p.C209Wfs*10 F4 58 y 59 y M 14620 Muscle pain SGCA c.190G > A, p.A64T (homozygous) F5 50 y 72 y F 1141 Arm and leg weakness, calf hypertrophy SGCA c.190G > A, p.A64T (homozygous) F6 3 y 56 y M No data Leg weakness, respiratory failure, heart failure SGCA c.190G > A, p.A64T (homozygous) F7 9 y 42 y M 2630 Arm and leg weakness SGCA c.190G > A, p.A64T c.626dupG, p.C209Wfs*10 F8 6 y 18 y F 706 Leg weakness, calf hypertrophy SGCA c.220C > T, p.R74W (homozygous) F9 3 y 12 y M 7244 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F10 3 y 7 y M 13299 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F11 6 y 15 y M 5159 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F12 10 m 11 y F 7581 Leg weakness, muscle pain, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F13 6 y 8 y M 9000 Leg weakness, calf hypertrophy SGCA c.229C > T, p.R77C (homozygous) F14 6 y 17 y F 3746 Leg weakness SGCA c.229C > T, p.R77C (homozygous) F15 1 y 9 y M 8415 Arm and leg weakness SGCA c.229C > T, p.R77C (homozygous) F16 2 y 6 y F 2599 Muscle pain SGCA c.229C > T, p.R77C c.320C > T, p.A107V F17 5 m 13 y M 1775 Arm and leg weakness muscle pain, calf hypertrophy SGCA c.229C > T, p.R77C c.320C > T, p.A107V F18 28 y 35 y M 1357 Muscle pain SGCA c.229C > T, p.R77C c.320C > T, p.A107V F19 2 y 2 y F 6280 Equinus foot SGCA c.229C > T, p.R77C c.409_410delinsCCTGGTGCGCAGCCAGG, p.E137delinsPGAQPG F20 2 y 2 y F 13766 Equinus foot SGCA c.229C > T, p.R77C c.626dupG, p.C209Wfs*10 F21-1* 3 y 9 y M 14418 Leg weakness, muscle pain, calf hypertrophy, equinus foot SGCA c.266 T > C, p.L89P c.749 T > G, p.V250G F21-2* 2 y 7 y M 7455 Leg weakness, muscle pain F22 1 y 6 y M 516 Muscle pain, equinus foot SGCA c.271G > A, p.G91S c.320C > T, p.A107V F23 6 y 48 y M 1328 Muscle pain, trunk and leg weakness SGCA c.320C > T, p.A107V c.409_410delinsCCTGGTGCGCAGCCAGG, p.E137delinsPGAQPG F24 62 y 70 y F 1115 Leg weakness SGCA c.320C > T, p.A107V c.662G > C, p.R221P F25 No data 71 y F No data No data SGCA c.584 + 1G > A c.662G > A, p.R221H F26 6 y 54 y M 887 Arm and leg weakness, calf hypertrophy SGCA c.584 + 5G > T (homozygous) F27 3 y 29 y M 465 Arm and leg weakness, respiratory failure, heart failure SGCA c.626dupG, p.C209Wfs*10 (homozygous) F28 6 y 80 y F 342 Arm and leg weakness SGCA c.634A > G, p.M212V (homozygous) F29 5 m 3 y M 555 Calf hypertrophy SGCA c.662G > A, p.R221H (homozygous) F30 59 y 67 y M 333 Arm and leg weakness SGCA c.662G > A, p.R221H (homozygous) F31 1 y 2 y M 42630 Leg weakness, calf hypertrophy SGCB c.214_221del, p.L72Pfs*24 (homozygous) F32 4 y 11 y M 8022 Leg weakness, calf hypertrophy, equinus foot SGCB c.214_221del, p.L72Pfs*24 c.325C > T, p.R109* F33-1* 3 y 3 y M 3994 Leg weakness, equinus foot, intellectual disability SGCB c.214_221del, p.L72Pfs*24 c.325C > T, p.R109* F33-2* 4 m 4 m M 2420 High CK F34 1 y 7 y F 9920 Arm and leg weakness, intellectual disability, respiratory failure SGCB c.325C > T, p.R109* (homozygous) F35 9 m 10 m F 32400 Muscle weakness, calf hypertrophy, equinus foot SGCB c.325C > T, p.R109* (homozygous) F36 8 y 9 y F 21944 Leg weakness, calf hypertrophy, equinus foot SGCB c.325C > T, p.R109* c.607G > C, p.A203P F37 4 y 6 y M 24880 Leg weakness, calf hypertrophy, equinus foot SGCB c.325C > T, p.R109* c.753 + 5G > A F38 3 y 4 y M 3254 Calf hypertrophy, equinus foot SGCB c.390_429dup, p.I144Afs*3 (homozygous) F39 3 y 7 y M 3370 Leg weakness, intellectual disability SGCB c.499G > A, p.G167S c.753 + 5G > A F40 1 y 8 y F 15772 Leg weakness, calf hypertrophy SGCG c.320C > T, p.S107L Exon 1–6 deletion F41 0 m 2 y F 17770 Leg weakness, calf hypertrophy, equinus foot SGCG c.320C > T, p.S107L (homozygous) F42 1 y 3 y M 8557 Leg weakness, calf hypertrophy SGCG c.320C > T, p.S107L Exon 6 deletion F43 2 y 6 y M 28300 Leg weakness, calf hypertrophy, equinus foot SGCG Exon 6 deletion (homozygous) F44 4 y 4 y M 10176 Leg weakness, calf hypertrophy SGCG Exon 6 deletion (homozygous) F45 2 y 5 y M 5524 Leg weakness, calf hypertrophy, equinus foot SGCG Exon 6 deletion (homozygous) F46 6 y 34 y F 1240 Leg weakness, calf hypertrophy, equinus foot SGCG Exon 6 deletion (homozygous) F47 7 y 8 y M 15336 Leg weakness SGCG Exon 6 deletion (homozygous) F48 6 y 25 y M 515 Leg weakness, calf hypertrophy, respiratory failure SGCG Exon 6 deletion Whole gene deletion F49 1 y 3 y M 9760 Leg weakness, calf hypertrophy SGCG Exon 6 deletion Whole gene deletion F50 1 y 1 y F 18630 Calf hypertrophy SGCG Exon 6 deletion Whole gene deletion F51 2 y 2 y F 17391 Calf hypertrophy, respiratory failure SGCD c.94A > G, p.K32E c.246_247del, p.S83* F52 4 y 7 y F 3290 Leg weakness, calf hypertrophy, equinus foot SGCD c.102delC, p.L35Cfs*9 c.246_247del, p.S83* F53 6 y 44 y F 1235 Leg weakness, intellectual disability SGCD c.502 + 24695G > T (homozygous) Bold characters indicate novel variants. y, years; m, months; CK, creatinine kinase *F21-1 and F21-2, and F33-1 and F33-2 are two pairs of siblings Fig. 2 Locations of amino-acid changes caused by identified genetic variants in predicted protein structures. Predicted three-dimensional structures of α-sarcoglycan (Q16586 (SGCA_HUMAN)), β-sarcoglycan (Q16585 (SGCB_HUMAN)), γ-sarcoglycan (Q13326 (SGCG_HUMAN)), and δ-sarcoglycan (Q92629 (SGCD_HUMAN)), obtained from Alphafold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ). Red circles indicate the positions of missense variants. Red squares indicate in-frame deletions or insertions. Gray circles denote the positions of truncation of normal sequences in truncation variants. Yellow and white arrowheads show the 4 amino-acid deletion (13–16) caused by c.38-2A > C in SGCA and p.K32E encoded by c.94A > G in SGCD , respectively Fig. 3 Frequencies of variants and variant types in patients with sarcoglycanopathies. The frequency of ( a ) causative genes in Japanese patients with sarcoglycanopathies, and ( b )–( e ) variants in each causative gene: b SGCA , c SGCB , d SGCG , and e SGCD . Gray sections in pie charts indicate variants detected one family. f Effects of gene variants on protein products Section title: Results Educational score: 4.2253947257995605 Domain: biomedical Document type: Study Language: en Two patients with a homozygous c.220C > T variant, the most common variant in the SGCA , tended to manifest symptoms in childhood and lost ambulation. However, 5 patients carrying a c.220C > T variant in combination with another variant exhibited milder symptoms, such as myalgia or elevated CK levels, compared to the homozygous patients. Patients with the c.190G > A, c. 320C > T and c.662G > A variants in the SGCA exhibited relatively mild symptoms like only high CK levels, maintaining ambulation into advanced age. All patients with the variants in the SGCB and SGCG showed childhood-onset. Adult patients with whole deletion of SGCG gene had lost ambulation and required respiratory support. A patient carrying the c.246_247del variant in the SGCD required respiratory support from an early age, while a patient with c.502 + 24695G > T variants preserved ambulation but presented with comorbidities such as intellectual disabilities. Section title: Results Educational score: 4.334097862243652 Domain: biomedical Document type: Study Language: en We identified three kinds of large deletions of the SGCG gene in nine families by MLPA, including deletions of exon 6, exon 1 to 6 and the entire gene (Table 1 ). To more accurately delineate the deleted regions, we applied Nanopore CRISPR/Cas9-targeted long-read resequencing. Genomic DNA samples from individuals 42–46 (F42-46) had deletions of precisely the same chromosome 13 region , which included exon 6 of SGCG . The 5'-breakpoint was within a short interspersed nuclear element (SINE) and the 3' breakpoint was within a long terminal repeat (LTR). In F40, 1.4-Mb sequence from downstream of LINC00424 to intron 6 of SGCG was deleted. Both 5’- and 3’- breakpoints were within SINE . In F49, a 1.6 Mb sequence from downstream of LINC00621 to upstream of PPAR4 , including the whole SGCG gene, was deleted. Similar sequences (approximately 7.3 kb) containing repeat elements (SINE, LTR, and long interspersed nuclear elements) were present in sequences flanking both ends of the 1.6 Mb deletion; therefore, we could not determine the exact breakpoints . In other cases, who had entire SGCG deletion, long genomic DNAs, which were required for Nanopore CRISPR/Cas9-targeted long-read resequencing, were unavailable. Fig. 4 Genome structures in patients with deletion of SGCG exon 6, SGCG exon 1 to 6 and the whole SGCG gene. a SGCG exon 6 deletion. The breakpoints were the same in all patients. b SGCG exon 1 to 6 deletion. c Whole gene deletion of SGCG ; similar sequences (identity: 94%) were present in the 5' and 3' breakpoints. Repeat sequence schema were retrieved from Ensembl ( https://asia.ensembl.org/index.html ). SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; LTR, long terminal repeat Section title: Results Educational score: 4.4377288818359375 Domain: biomedical Document type: Study Language: en Intronic single nucleotide variants were identified in five, two, and one families with LGMDR3, LGMDR4, and LGMDR6, respectively (Table 1 ) and we analyzed skeletal muscle transcripts from these patients to search for splicing abnormalities. We found a 59 bp extension to the 3’ end of exon 1 by RNA-seq in F1, since we could not obtain abnormal product by RT-PCR . One additional band from SGCA transcript was detected with some additional non-specific bands denoted by * in each of F2 and F3 by RT-PCR with primer pairs targeting SGCA exons 1–2 and 1–5, respectively; both products were amplified at comparable levels to those of the normal transcript. In The abnormal PCR product detected in F2 was truncated by 12 bp at the 5' end of exon 2, while that detected in F2 skipped exon 3 . RT-PCR analysis of samples from F25 and F26 with primers targeting exon 5–7 of SGCA generated the same longer product, which contained an 85 bp extension to the 3' end of exon 5, with an additional non-specific band denoted by *, in addition to the normal amplicon . In F37, which carried an SGCB variant, a smaller PCR product lacking exon 5, amplified at a comparable level to that of the normal product in control samples, was obtained by amplification of exon 4–6 . A deep intronic variant in SGCD was identified in F53 and found to result in transcription of a pseudoexon containing 91 bp of intron 6 . Fig. 5 Splicing abnormalities and MaxEnt scores generated by analysis of intronic variants. a F1 SGCA intron 1: c.37 + 6 T > C. Sashimi plots of RNA-seq data using control and patient muscles are shown. b – e RT-PCR products obtained from control and patient muscle samples separated on agarose gels and schema showing aberrant splicing events with MaxEnt scores. White squares are normal-sized exons and gray squares are altered-sized exons. In the right schema, genetic variants are shown in red and new splicing sites are shown in light blue. * indicate the non-specific products which were not related to SGC genes. b F2 SGCA intron 1: c.38-2A > C. c F3 SGCA intron 2/exon 3: c.158-2_167del. d F25 SGCA intron 5: c.584 + 1G > A and F26 SGCA intron 5: c.584 + 5G > T. e F37 SGCB intron 5: c.753 + 5G > A. f F53 SGCD intron 6: c.502 + 24695G > T. RT-PCR and Sashimi plots of RNA-seq data are shown; sequences 5' (upstream) of the variants included a possible branch point site, a polypyrimidine tract, and a splice acceptor site. ctrl, control sample; Ref, reference sequence Section title: Results Educational score: 4.5528950691223145 Domain: biomedical Document type: Study Language: en To evaluate the pathogenicity of the identified intronic variants as determinants of aberrant splicing, we calculated the scores of the splicing acceptor and donor sites of affected exons using SpliceAI and MaxEntScan in silico . In F1, F2, F3, F25, F26, and F37, losses of splice sites near the genetic variants were predicted by SpliceAI. Similarly, the scores for those sites were greatly reduced in MaxEntScan analysis. Both software packages predicted the creation of alternative splice sites, including a splice acceptor site 12-bp downstream of the normal site in F2 and a splice donor sites, 59-bp downstream in F1, and 85-bp downstream in F25 and F26, with high delta scores for AG and DG, respectively. SpliceAI and MaxEntScan also successfully predicted the pseudoexon-creation observed in F53, with high scores for donor and acceptor site creation (DG = 1.00 and AG = 0.95) in SpliceAI, and a high score of 9.31 at the new donor site using MaxEntScan against 1.67 of the reference sequence. In addition, the pathogenicity of the pseudo-exon creation was also predicted by PDIVAS with a high score of 0.971 . Interestingly, this pseudoexon preserved the functional upstream sequence in the flanking intron; a branch-point-like structure at –29 nucleotides (nt), a polypyrimidine track at –22 to –5 nt, and a pseudoexon acceptor site. Together with the results of RT-PCR, these in silico predictions indicated that all variants identified in introns were pathogenic and the causes of aberrant splicing. Table 2 Summary of splicing abnormalities and SpliceAI predictions Family Gene Variant Effect on transcript Acceptor loss Donor loss Acceptor gain Donor gain ORF F1 SGCA Intron 1: c.37 + 6 T > C Exon 1 extension 0 0.51 0 0.25 Out of frame F2 SGCA Intron 1: c.38-2A > C Exon 2 shortening 0.96 0 0.68 0.09 In frame F3 SGCA Intron 2/exon 3: c.158-2_167del Exon 3 skipping 0.99 0 0.49 0.01 Out of frame F25 SGCA Intron 5: c.584 + 1G > A Exon 5 extension 0 0.99 0.01 0.54 Nonsense codon F26 SGCA Intron 5: c.584 + 5G > T Exon 5 extension 0 0.58 0.01 0.48 Nonsense codon F37 SGCB Intron 5: c.753 + 5G > A Exon 4 skipping 0 0.86 0.02 0 In frame F53 SGCD Intron 6: c.502 + 24695G > T Pseudoexon creation 0 0 0.95 1.00 Nonsense codon ORF, open reading frame Section title: Results Educational score: 4.205074787139893 Domain: biomedical Document type: Study Language: en Founder effects in patients with SGPs worldwide have been suggested in several previous reports [ 8 , 19 – 21 ]. Therefore, we analyzed the haplotypes containing the following variants: SGCA : c.229C > T, SGCB : c.325C > T, and SGCG : exon 6 deletion . We identified 5–10 SNPs in a homozygous state in individuals homozygous for the pathogenic variants, F11 and F12 with SGCA c.229C > T, F34 with SGCB c.325C > T, and F47 with SGCG exon 6 deletion. Among families with the SGCA c.229C > T variant, F11, F12, and F15 had the same haplotype, while F9, F13, and F14 had a distinct haplotype , indicating the presence of at least two haplotypes. Heterozygous individuals had compatible haplotypes, but were not informative, because they were hybrid for both genotypes. Families homozygous for SGCB c.325C > T (F34 and F35) shared the same haplotype, and those heterozygous for this variant (F32, F36, and F37) also possessed the same haplotype, indicating a single haplotype carrying SGCB c.325C > T. In families with SGCG exon 6 deletion, the homozygous individuals, F44, F45, and F47, and those hemizygous for the deletion, F48–F50, had the same haplotype; further, one heterozygous individual, F42, also showed compatible results, while the homozygotes, F43 and F46, had haplotypes with evidence of genetic recombination. Fig. 6 Haplotype analyses of alleles containing common single nucleotide polymorphism (SNP) variants. a Patients with SGCA c.229C > T. b Patients with SGCB c.325C > T. c Patients with SGCG exon 6 deletion. Bases in blue font indicate minor SNP alleles and bases in red font indicate variants. Orange shaded cells indicate homozygosity for minor SNP alleles. Yellow shaded cells indicate heterozygosity for minor SNP alleles. Blue shaded cells indicate homozygosity for major SNP alleles Section title: Results Educational score: 4.120628833770752 Domain: biomedical Document type: Study Language: en Notably, the frequencies of missense and truncation variants differed among causative genes of SGPs, as reported previously [ 2 – 4 ]. Missense variants in SGCA were frequent at 78.3%, while they were relatively rare in SGCB , SGCG , and SGCD , at 11.1%, 18.2%, and 16.7% respectively. By contrast, truncation variants were common in SGCB , SGCG , and SGCD. Section title: Discussion Educational score: 4.134275436401367 Domain: biomedical Document type: Study Language: en In this study, we conducted genetic profiling of 53 Japanese families with SGP. In Japan, SGCA was the most common causative gene, followed by SGCG , SGCB , and SGCD . The proportion of causative genes varies by region, with SGCA being the most common in Europe, the USA and Brazil , SGCB in Iran and SGCG in Algeria and India . No patients with LGMDR6 have previously been reported in Japan; here, we report the first identification of Japanese patients with LGMDR6, with biallelic variants in SGCD . Although the reason for the low prevalence of LGMDR6 is unclear, it has been suggested that SGCD is expressed in the heart, and that individuals with variants in SGCD may die in utero ; however, no cardiac disease was observed in our patients with LGMDR6, although genetically undiagnosed siblings of F53 are recorded to have died of cardiac disease. Section title: Discussion Educational score: 4.214931488037109 Domain: biomedical Document type: Study Language: en A few types of variants accounted for more than half of alleles in all causative genes. In particular, c.229C > T (R77C) in SGCA , which is also reported to be common in other countries, and has been suggested as a founder variant [ 3 , 25 – 27 ]. Analysis of the genotypes of Japanese patients with c.229C > T indicated the presence of two distinct haplotypes, suggesting that SGCA : c.229C > T can be considered a recurrent variant. The alleles harboring c.325C > T in SGCB and exon 6 deletion in SGCG , shared common haplotypes and were likely each inherited from a single common ancestor. This concept is significant, as genomic screening that prioritizes founder and recurrent variants enhances diagnosis efficacy and provide a foundation for the development of gene therapies. Section title: Discussion Educational score: 4.2957963943481445 Domain: biomedical Document type: Study Language: en Interestingly, large deletions of SGCG are common in Japan, and SGCG contains numerous repetitive sequences in its introns, which may be prone to genetic recombination. The breakpoints of the Exon 6 deletion and Exon 1 to 6 deletion were within an LTR and SINE respectively, and the breakpoints flanking whole SGCG deletions contained similar repetitive elements, suggesting non-allelic homologous recombination as the likely mechanism underlying both types of deletion . Additionally, individuals with deletion of the entire SGCG gene also lacked five other genes, among which SACS and MIPEP are reported to cause spastic ataxia and combined oxidative phosphorylation deficiency 31, respectively . Thus far, no patient with SGP and homozygous deletion of the entire SGCG gene has been identified, as combined oxidative phosphorylation deficiency is a very severe condition. MLPA combined with long-read sequencing proved useful for the diagnosis of these variants. Section title: Discussion Educational score: 4.298035621643066 Domain: biomedical Document type: Study Language: en We also predicted the pathogenicity of detected intronic variants and altered transcript structures in silico, as well as successfully identifying their splicing abnormalities in all cases by RT-PCR and RNA-seq. The shifted PCR products detected in F2 and F37 had in-frame changes and were not subject to nonsense-mediated mRNA decay; therefore, they appeared to be expressed in comparable amounts to normal transcripts in control samples. In SGCD , creation of a pseudoexon deep in intron 6 is pathogenic. Exon-skipping therapy is an approved treatment for Duchenne muscular dystrophy and recently, we developed a therapeutic strategy using branchpoint-targeted antisense oligonucleotides for patients with Fukuyama congenital muscular dystrophy caused by an FKTN transcript involving a pseudoexon . Accurate diagnosis of SGP may also provide therapeutic opportunities. Section title: Discussion Educational score: 4.55246114730835 Domain: biomedical Document type: Study Language: en We found that many of the SGCA variants detected were predicted to be missense and to cause amino-acid substitutions in the protein, while most SGCB , SGCG , and SGCD variants were predicted to cause truncation of their protein products; this trend has been also reported in other cohort studies . Interestingly, the rates of missense/truncated variants in SGCA-SGCD may be related to the roles of each protein subunit in the formation and function of the sarcoglycan complex. Recent advances in the prediction of protein structures using Alphafold have allowed us to consider the pathogenicity of identified variants in the context of whole protein structures . α-Sarcoglycan has a globular domain in its central extracellular region and missense variants in SGCA are concentrated in this globular domain. Additionally, the central extracellular region of α-sarcoglycan has been reported to exhibit ecto-ATPase activity . By contrast, the extracellular regions of β-, γ-, and δ-sarcoglycans are predicted to have simple structures, without globular domains. The majority of variants in SGCB , SGCG , and SGCD cause protein truncation. Our previous model of sarcoglycan complex formation suggested that β-, γ-, and δ-sarcoglycans make up the core of the complex, while α-sarcoglycan binds later and is primarily on the outside of the complex . On the other hand, other reports demonstrated core structure of sarcoglycan complex consists of β- and δ-sarcoglycans and later incorporates α- and γ-sarcoglycan . Thus, amino-acid substitutions caused by missense variants in SGCA most probably affect the ecto-ATPase activity of α-sarcoglycan and also cause instability of the sarcoglycan complex, while truncations of β-, γ-, and δ-sarcoglycans might affect the de novo formation of the sarcoglycan complex. Section title: Discussion Educational score: 4.385463714599609 Domain: biomedical Document type: Study Language: en Additionally, we identified two novel variants which may prevent the resulting mutated proteins from undergoing normal intracellular processing. One of these novel variants is c.38-2A > C in SGCA , detected in F2, which causes aberrant splicing resulting in in-frame deletion of four hydrophobic amino acids in the signal peptide of α-sarcoglycan ; this deletion is predicted by iPSORT ( https://ipsort.hgc.jp/ ) to render the signal peptide non-functional . The other novel variant is c.94A > G in SGCD in F51, which is predicted to lead to a p.K32E substitution . This substitution decreases the positive charges on the cytosolic side of the transmembrane domain by replacing a basic amino acid, which was predicted to influence the membrane topology of δ-sarcoglycan using WoLF PSORT ( https://wolfpsort.hgc.jp/ ) . Further structural and functional studies on mutated proteins are required to clarify the exact roles and pathogenicity of the identified variants. Section title: Conclusions Educational score: 4.118749141693115 Domain: biomedical Document type: Study Language: en In conclusion, both muscle biopsy and genetic analysis are essential for accurate diagnosis of SGPs; LGMDR3 is the most common type in Japan, while LGMDR6 is very rare. Patients with SGPs share relatively few variants. Missense variants are the most common type of change in SGCA , while truncation mutations account for a major proportion of SGCB , SGCG , and SGCD variants. Tools for prediction of splicing changes are useful for the identification of pathogenic intronic variants. Section title: Supplementary Information Educational score: 2.727313756942749 Domain: biomedical Document type: Other Language: en Additional file 1.Table S1. Primers use in RT-PCR.Table S2.Guide RNA-targeted sequences for long-read sequencing. Table S3. Primers for haplotype analysis. | Review | biomedical | en | 0.999996 |
PMC11699709 | Section title: Introduction Educational score: 3.9647672176361084 Domain: biomedical Document type: Other Language: en A stroke is caused by an arterial blockage or hemorrhage in one part of the brain tissue, which leads to focal neurological damage in the adjacent area. Ischemic stroke is induced by ischemia and hypoxia in the brain, which leads to limited ischemic necrosis or softening of the brain tissue, and it will cause severe neurological deficits, which can result in cell death if reperfusion is not possible in a short period, this type of stroke accounts for about 70% of all strokes . The incidence and prevalence of stroke have been reported to increase year by year, with high rates of disability and mortality, and is a major cause of death and disability in the world’s population, seriously jeopardizing human life and health . Section title: Introduction Educational score: 3.968304395675659 Domain: biomedical Document type: Review Language: en Currently, thrombolysis applied to achieve early reperfusion is the most valid method of treating acute cerebral ischemia. Still, this therapy is limited by the time window of thrombolysis, and successful thrombolysis causes substantial damage to brain tissue with cerebral ischemia (ischemia/reperfusion injury (I/R)), which may be related to inflammatory responses, mitochondrial dysfunction, increased production of ROS, oxidative stress, and activation of cell death pathways [ 3 – 8 ]. For many years, despite many studies in this field, we did not significantly improve the prognosis and treatment of ischemic stroke patients. Therefore, exploring new targets and therapies to reduce re-injury in cerebral ischemia remains a pivotal challenge for treating cerebral ischemia. Section title: Introduction Educational score: 4.9611124992370605 Domain: biomedical Document type: Study Language: en Ferroptosis was a new mechanism of programmed cell death, a non-apoptotic programmed death pathway reliant on iron ions and ROS . Ferroptosis is characterized by a significant elevation of iron content and accumulation of lipid peroxides, and lead to cell death and neurological damage after stroke. During ischemia/reperfusion injury, the abnormal increase of cerebral microvascular endothelial cell permeability severely damages the blood–brain barrier (BBB), which allows a large amount of iron to enter the brain parenchyma and trigger iron overload. The increased iron content results in the disturbance of cerebral iron metabolism and eventually contributes to the occurrence of cellular ferroptosis . During cerebral ischemia, local cerebral tissue blood supply is insufficient, mitochondrial dysfunction occurs, and ATP can not be produced. After reperfusion of cerebral blood flow, oxidative stress increases mitochondrial damage further and generates excessive ROS . The ROS accumulation-mediated oxidative stress and lipid peroxidation are the key driving forces triggering cellular ferroptosis . In addition, overaccumulation accumulation of extracellular glutamate is also a major cause of neuronal cell death, which exerts oxidative toxicity by inhibiting cystine absorption through restraining the activity of System Xc-. As the light chain of the Xc system, SLC7A11 translocates a molecule of glutamate out of the cell and exchanges a molecule of cystine into the cell in a 1:1 ratio. Cystine is necessary for the synthesis of glutathione (GSH) . Intracellular GSH depletion reduces the activity of GPX4 and further leads to the dysfunction of lipid peroxides metabolism . Excess Fe 2+ oxidizes lipids employing the Fenton reaction, generating massive amounts of ROS and eventually leading to ferroptosis [ 19 – 21 ]. As shown in many studies, inhibition of ferroptosis is pivotal in rescuing neuronal damage after brain I/R injury. Section title: Introduction Educational score: 4.19851541519165 Domain: biomedical Document type: Review Language: en Electroacupuncture (EA), integrates traditional acupuncture with modern electric stimulation and has been widely used in treating stroke and poststroke with significant clinical effects, and less undesirable effects. EA therapy has been demonstrated to be neuroprotective against ischemic stroke by effectively attenuating a wide range of pathologic processes. In addition, the co-application of EA at the “Baihui” point and the “Zusanli” point had a synergistic protective effect on mitigating neuronal damage in MCAO rats by curbing endoplasmic reticulum stress and ameliorating the defective mitochondrial function . Therefore, EA can effectively restrain the ischemic-hypoxic cascade response and neuronal injury . Recent studies have found that EA intervention in MCAO rats can also repress the formation of ferroptosis, which protects damaged neuronal cells in MCAO rats . However, the mechanism of the way by which EA inhibits ferroptosis is still obscure. Investigating the specific mechanism of EA for ischemic stroke is anticipated to yield a new scientific basis for targeted therapy of relevant neurological diseases in the future. Section title: Introduction Educational score: 4.7121686935424805 Domain: biomedical Document type: Study Language: en The etiopathogenesis of ferroptosis is sophisticated and involves multiple transcription factors including p53, NFE2L2, and Nrf2 . Among them, Nrf2, a crucial ferroptosis regulator, is known to transcribe several antioxidant response element (ARE)-containing genes to maintain redox homeostasis . Therefore, deficiency of Nrf2 causes increased susceptibility to brain injury [ 29 – 31 ]. Under normal conditions, Nrf2 binds to Kelch-like ECH-associated protein1 (Keap1), an interface protein for Cul3 E3 ubiquitin ligases, which is accountable for the ubiquitination and degradation of Nrf2 ; under oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus and activates the transcription of the ARE. The ARE transcribes and drives a variety of antioxidant genes, including heme oxygenase-1 (HO-1), to exert antioxidant effects [ 34 – 38 ]. As a transcription factor, Nrf2 can directly regulate the expression of several important genes in the process of ferroptosis in the nucleus, including SLC7A11, GPX4, FTH1, etc. , which in turn regulates intracellular iron metabolism, GSH levels, GPX4 synthesis, lipid oxidation, etc. . Therefore, activation of Nrf2 is a valuable target for treating cerebral ischemia . Section title: Introduction Educational score: 4.120477199554443 Domain: biomedical Document type: Study Language: en The purpose of this research was to explore the molecular mechanisms regarding the beneficial efficacy of EA in the rat model of MCAO/R-induced brain injury, and it was found that EA could prevent neuronal ferroptosis after ischemic stroke by facilitating the Nrf2 nuclear translocation and activating the Nrf2/SLC7A11/GPX4 pathway, which in turn achieves a neuroprotective effect on the nervous system. Section title: Preparation of MCAO/R rat models and group Educational score: 4.13295841217041 Domain: biomedical Document type: Study Language: en Male SPF Sprague–Dawley (SD) rats (200 ± 20 g) were obtained from Hunan Slack (Certificate No.: SCXK (Xiang) 2019–0004) and reared in the Laboratory Animal Center under a 12 h light/dark cycle at 21 ± 2 °C and 60–70% humidity. These rats had unlimited access to food and water. All animal experiments were granted by the Animal Ethics Committee of Yunnan University of Traditional Chinese Medicine . The middle cerebral artery occlusion/reperfusion (MCAO/R) model was constructed with the Longa method . In brief, the rats were sterilized by intraperitoneal injection of 3% sodium pentobarbital (40 mg/kg) anesthesia and fastened to an operating platform, and the neck hair was removed, and the muscles and connective tissues were separated. Then the left common carotid artery, external carotid artery, and internal carotid artery. The external carotid artery was tied with a thin line and a minor incision was cut in the external carotid artery. A nylon monofilament (Beijing Sinon Technology Co., Ltd., Beijing, China) with a silicone coating at the tip was then plunged into the internal carotid artery through a tiny incision to a depth of approximately 18–20 mm at the bifurcation. During ischemia, these rats were placed on an insulating pad at 37 °C for 60 min. 60 min later, the nylon monofilament was withdrawn to establish reperfusion and the wound was sutured. Sham rats underwent all steps apart from insertion and extraction of the nylon monofilament. Section title: Preparation of MCAO/R rat models and group Educational score: 3.979736089706421 Domain: biomedical Document type: Study Language: en After 24 h of surgery, the success of MCAO/R was appraised with a neurobehavioral score. 72 rats with successful MCAO/R were randomly numbered and categorized into four groups ( n = 18): the MCAO/R group, the EA + MCAO/R group, the EA + MCAO/R + DMSO group, and the EA + MCAO/R + Brusatol group, the Sham group was used for control ( n = 18). Section title: Electroacupuncture intervention Educational score: 4.136346340179443 Domain: biomedical Document type: Study Language: en EA intervention on Quchi (LI11), Baihui (GV20), Dazhui (GV14), and Neiguan (P6) was performed in the MCAO/R + EA group, the EA + MCAO/R + DMSO group, and the EA + MCAO/R + Brusatol group at 24 h after modeling . The Baihui point is located at the top of the head, where the Mai qi converges. Stimulating the Baihui point can directly or indirectly connect with the meridian system of the whole body, thus achieving the effect of nourishing the blood. The Dazhui point belongs to the Governor's Chakra, which has the effect of relieving spasms, relaxing contracture, calming the mind, and strengthening the body; Quchi point applies to all clinical disciplines and is particularly good in Chinese medicine internal diseases, among which paralysis in limb meridian diseases is the most specialized; Neiguan point has the effect of enlightening the mind and tranquilizing the mood, so Neiguan point can treat the diseases of the mind. Therefore, these four acupoints we have chosen are indicated for ischemic stroke treatment. The specific parameters of acupoint were as follows: Baihui point: located in the middle of the parietal bone, and obliquely stabbed forward for 2 mm; Dazhui point: located in the posterior midline and depression below the spinous process of the seventh cervical vertebrae, and stabbed straightly for 5 mm; the right Quchi point: located in the depression in the proximal end of the radius just anterior to the lateral side of the elbow joint, and stabbed straightly for 4 mm; and the right Neiguan point: the medial side of the forelimb, the About 3 mm from the wrist joint, between the ulnar-radial suture, straight stabbing 1 mm. The disposable acupuncture needle (Beijing Zhongyan Taihe Medical Instrument Co., Ltd., Beijing, China) was used for stabbing and then connected to the EA instrument (Changzhou Indy Electronic Medical Instrument Co., Ltd., Changzhou, China), with a current of 2 mA, the frequency of 2 Hz, sparse and dense waves, 30 min/time, 1 time/day for consecutive 7 days . Section title: Electroacupuncture intervention Educational score: 3.873349905014038 Domain: biomedical Document type: Study Language: en Rats in the EA + MCAO/R + Brusatol group were injected intraperitoneally with the Nrf2 inhibitor Brusatol ((2 mg/kg; Shanghai Yuanye Bio-Technology, CAS number: 14907–98-3, (Shanghai, China)) at 30 min before EA treatment on the first day after modeling, and every other day until the seventh day. Section title: Neurological severity scores Educational score: 3.968384027481079 Domain: biomedical Document type: Study Language: en On the seventh day after MCAO/R, all rats were scored for neurologic severity. Neurological scoring was performed by the same investigator in a blinded manner, and the rats were rated for neurological deficits using a 7-point NSS scale. Higher NSS indicated poorer neurological function . Section title: Garcia score Educational score: 3.9225215911865234 Domain: biomedical Document type: Study Language: en All rats were blinded by the same researcher, and the Garcia score was based on six main characteristics: voluntary locomotion, body symmetry, forelimb extension, grasp, and climb ability, tactile reflexes on both sides of the body, and whisker touch response on both sides of the body. The total score is 18, with higher scores meaning less nerve impairment . Section title: Foot-fault test Educational score: 3.7854225635528564 Domain: biomedical Document type: Study Language: en Motor coordination in rats was evaluated by the foot fault test. The test was performed three times consecutively at 5-min intervals. The experiment was scored by the state of the rat's right forefoot grasping the horizontal ladder each time , and higher scores represented the better walking ability of the rats. Section title: Rotarod test Educational score: 3.858421564102173 Domain: biomedical Document type: Study Language: en The Rotarod test can assess the recovery of motor function and tolerance level of rats, the operation is as follows: the rats were placed on the rotating rod, the velocity of which was raised from 0 to 10 rpm in 10 s for 5 min. The test was performed three times, recording the time of the rats running on the rotating rod, and average values were taken for statistical analysis . Section title: TTC staining Educational score: 4.144153594970703 Domain: biomedical Document type: Study Language: en On postoperative day 7, rats were dosed intraperitoneally with 3% sodium pentobarbital, and the brains were immediately removed and refrigerated for 5 min at −20 °C. The brains were then sliced into 2 mm thick sections. There were 6 slices in total, which were put into a Petri dish containing 2% TTC staining solution (Solarbio Company, Beijing, China), and then covered with tin foil for light protection, and then put into a 37 ℃ incubator to incubate for 15 min, and then brain slices were taken out and arranged in an anterior–posterior order, photographs were taken of these sections. The pale areas were characterized as infarcted. Infarct volume was counted using Image J software (Media Cybernetics, Rockville, MD, USA). Percentage of infarct volume (%) = (infarct volume/total volume) × 100%. Section title: Nissl staining Educational score: 4.193747520446777 Domain: biomedical Document type: Study Language: en Rats were perfused transcardially with 0.9% NaCl and 4% paraformaldehyde solution. The brains were taken out and fixated with paraformaldehyde solution for 24 h before being immersed in 30% sucrose solution at 4 °C for 24 h. After removal, the brains were embedded in paraffin and cut into 4 μm thick slices on a paraffin slicer in preparation for Nissl staining. Briefly, slices of each group were degreased in graded alcohol (70%, 95%, and 100% alcohol) for 3 min, followed by hydration in graded alcohol (95%, 70%, and 50% alcohol) for 3 min. Next, the slices were immersed in a toluidine blue solution at 50–60 °C for 40 min. Following washing with distilled water, the slices were dehydrated once in 70%, 80%, and 95% ethanol for a total of 3 min each, and then twice in 100% ethanol solution. Finally, the slices were immersed in 100% dimethylbenzene solution for 5 min and then sealed with a drop of neutral resin. Section title: Iron content Educational score: 3.8204479217529297 Domain: biomedical Document type: Study Language: en Fe 2+ levels in brain tissue were measured by use of an iron content assay kit . Measure the absorbance at a wavelength of 593 nm with a spectrophotometer based on the manufacturer's instructions. Section title: ROS, SOD, and MDA assays Educational score: 3.9863150119781494 Domain: biomedical Document type: Study Language: en ROS levels were assayed by a ROS kit (E004-1-1, Nanjing Jianjian Biotechnology Co., Ltd.) according to the manufacturer's instructions. Fluorescence intensity was measured with a luciferase marker (Spectra Max Gemini EM, Molecular Devices, USA) with 485 nm excitation wavelength and 530 nm emission wavelength. The MDA assay kit and SOD assay kit were performed to detect MDA and SOD levels. Section title: Extraction of nuclear and cytoplasmic proteins Educational score: 3.946589469909668 Domain: biomedical Document type: Study Language: en Nucleoprotein and cytoplasmic proteins were isolated using the Nucleoprotein Extraction Kit following the manufacturer's instructions. After obtaining nuclear and cytoplasmic proteins, Nrf2 was quantified in the nucleus and cytoplasm by Western blotting and Lamin-B1 was applied as an up-loading control for nuclear proteins. Data were resolved by Image J. Section title: Western blot Educational score: 4.108119964599609 Domain: biomedical Document type: Study Language: en Rat brain samples were cleaved with RIPA lysis solution (Solarbio, Beijing, China) containing PMSF and phosphatase inhibitors. Total proteins were collected and quantified by BCA protein assay kit (Biosharp, Beijing, China). Total proteins were detached in SDS-PAGE gels and shifted to PVDF (Merck, USA) membranes. The membranes were occluded with 5% skimmed milk (Biofroxx) for 2 h at room temperature and then incubated with primary antibodies at 4 °C overnight. Primary antibodies included Anti-Nrf2 , Anti-pNrf2 , Anti-GPX4 , Anti-HO-1 , Anti-FTH1 , Anti-SLC7A11 , Anti-β-actin , and Anti-Lamin-B1 . The next day, the membranes were cleaned with TBST three times, and then incubated with HRP-coupled goat anti-rabbit IgG secondary antibody at room temperature for 1 h. After three washings, the membranes were visualized on a chemiluminescence detection system (Clink Science Instruments Co., Ltd., Shanghai, China) with ECL chemiluminescence reagent (Biosharp Life Science Co., Ltd.) and analyzed with Image J software. Section title: Immunofluorescence Educational score: 4.155169486999512 Domain: biomedical Document type: Study Language: en Rats were anesthetized intensively and perfused transcardially with 0.9% NaCl and 4% paraformaldehyde. Whole brains were removed and immobilized in 4% paraformaldehyde through the night, and then dehydrated with a sucrose gradient at room temperature. The brains were encapsulated in OCT and sectioned into 10 μm thick slices using a cryosectioner. Sections were incubated with 5% bovine serum albumin (BSA) blocked at room temperature for 1 h and then hatched overnight at 4 °C with Anti-GPX4 , Anti-Nrf2 , and Anti-NeuN . Subsequently, sections were rinsed three times with PBS and incubated with a fluorescent secondary antibody (Alexa Fluor 488/594 AffiniPure Goat Anti-Rabbit IgG (H + L); Jackson ImmunoResearch) and then in a 37 °C incubator. The sections were then incubated with DAPI for 5 min at room temperature. Positive signals in the ischemic penumbra were visualized and photographed using a Zeiss LSM-710 confocal microscope. Fluorescence intensity was analyzed using ImageJ software. Section title: Transmission electron microscopy Educational score: 4.189305305480957 Domain: biomedical Document type: Study Language: en Brain tissues of 1 × 1 × 2 mm size from the ischemic penumbra were stabilized in 3% glutaraldehyde solution for 1 h and then in 1% osmium tetroxide solution for 2 h. The sections were then dehydrated in acetone and finally imbedded in Epon812. Ultrathin sections of 60–90 nm were fabricated using an ultrathin sectioning machine. The sections were then colored with uranyl acetate for 10–15 min at room temperature, then with lead citrate for 1–2 min, and lastly, the morphology of mitochondria was investigated by transmission electron microscopy . Section title: Statistical analysis Educational score: 3.3533473014831543 Domain: biomedical Document type: Study Language: en Data were statistically analyzed using SPSS 26.0 software. All experimental data were reported as (mean ± SD). One-way ANOVA with a 95% confidence interval was used to assess the differences between three or more experimental groups. Tukey’s multiple comparison post-hoc test was then performed, p < 0.05 denoted a statistically significant difference. Graphs were plotted using GraphPad Prism 9.5 software. Section title: Neuroprotective effect of EA against cerebral MCAO/R induced injuries Educational score: 4.138856410980225 Domain: biomedical Document type: Study Language: en The neurofunctional recovery was measured by NSS and Garcia score on days 7 after MCAO/R. The results indicate that the neurological function of rats in the MCAO/R group was markedly corrupted. EA markedly improved the neurological deficits. The NSS in the MCAO/R group was significantly higher than that in the Sham group, EA treatment significantly improved the neurological deficits compared with the MCAO/R groups . Similarly, the Garcia score was significantly lower in the MCAO/R group than in the Sham group, and the EA treatment improved the Garcia score . The results of the Foot-fault Test and the Rotarod Test revealed that exercise capacity was enhanced in the MCAO/R + EA group as compared to the MCAO/R group. Major cerebral infarcts were noticed in MCAO/R operated rats by TTC staining compared to the Sham group. Yet, the volume of cerebral infarction was diminished in the EA + MCAO/R group compared with the MCAO/R group. . Meanwhile, Nissl staining indicated that compared with the Sham group, the neuronal cells in the MCAO/R group were disorganized or missing, and the Nissl body staining was incomplete or even dissolved, suggesting that neuronal damage was induced by MCAO/R modeling. The infarct volume was decreased and the neuronal cell recovery in the ischemic rats with EA treatment compared with that of the ischemic rats without treatment . Fig. 1 Neuroprotective effect of EA against cerebral MCAO/R induced injuries. A – D Neurological deficits of each group were scored with the NSS, Garcia score, Foot-fault test, and Rotarod test at 7 days after surgery ( n = 12). E , F Representative images of TTC stained section and quantitation ( n = 6). G Representative photomicrographs of Nissl stained sections (Scale bar: 1 mM, 200 μM, and 50 μM, n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 Section title: EA attenuates MCAO/R-induced ferroptosis Educational score: 4.104422569274902 Domain: biomedical Document type: Study Language: en MDA and Fe 2+ levels were elevated distinctly in the MCAO/R and EA + MCAO/R groups relative to the Sham group. Yet, the levels of MDA and Fe 2+ in the EA + MCAO/R group were decreased compared with those in the MCAO/R group. Similarly, the expression of ferroptosis-related proteins, GPX4 , SLC7A11 , and FTH1 was reduced substantially in the MCAO/R group despite the Sham group; nonetheless, the EA intervention counteracted this trend in the EA + MCAO/R group. Fig. 2 EA inhibits ferroptosis in rats following MCAO/R. A , B The concentration of Fe 2+ , and MDA in brain tissue ( n = 6). C The representative Western blotting results of GPX4, SLC7A11, and FTH1 expression. D – F Quantification of GPX4, SLC7A11, and FTH1 expressions ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 Section title: EA attenuates MCAO/R-induced ferroptosis Educational score: 4.085076332092285 Domain: biomedical Document type: Study Language: en Ferroptosis damages the structure and functional impairment in mitochondria. We observed the mitochondrial ultrastructure by TEM. The samples exhibited irregular mitochondrial morphology, proliferation of vacuoles, and invagination of the mitochondrial membrane after MCAO/R, whereas the mitochondrial morphology in the MCAO/R + EA group was standardized with fewer vacuoles. . Fig. 3 EA inhibits ferroptosis in rats following MCAO/R. The bottom panels display the magnified images of regions indicated by yellow rectangles in the top panels. Arrows labeled: representative images of mitochondria. (Scale bar: 5 μM, 2 μM, and 1 μM, n = 3) Section title: EA increases the expression of Nrf2, and promotes Nrf2 phosphorylation and nuclear translocation Educational score: 4.216953277587891 Domain: biomedical Document type: Study Language: en To probe the molecular mechanism by which EA anti-ferroptosis potential, a crucial factor of the Nrf2 pathway was determined by the researchers. The outcomes revealed that the expression and phosphorylation values of Nrf2 and HO-1 were pronouncedly under-regulated in the MCAO/R group. However, the decrease of total Nrf2 , p-Nrf2 and HO-1 was rescued by EA treatment. Intriguingly, nuclear Nrf2 was slightly elevated in the MCAO/R group and markedly elevated in the EA + MCAO/R group, whereas cytoplasmic Nrf2 was strikingly diminished in both the MCAO/R group and the EA + MCAO/R group. Compared to the MCAO/R group, the cytoplasmic Nrf2 was less while the nuclear Nrf2 was more. We further observed the localization of Nrf2 by confocal. Our findings confirmed that Nrf2 mainly accumulates in the cytoplasm in the MCAO/R group, and EA efficiently promotes the nuclear translocation of Nrf2 . These results implied that EA promoted the phosphorylation of Nrf2 and nuclear translocation. Fig. 4 EA increases the expression of Nrf2 and promotes Nrf2 phosphorylation and nuclear translocation. A The representative Western blotting bands of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2, and Cytoplasmic Nrf2 expression. B – F Quantification of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2 and Cytoplasmic Nrf2 expressions ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001. G The confocal images of Nrf2. Scale bar: 5 μm. Nrf2 and DAPI were excited at 488 and 405 nm, respectively. Exemplary XY plane of confocal z-stack acquisition of Nrf2 (green signal) and treated with DAPI for nuclear staining (blue signal). Orthogonal views (XZ and YZ planes) extracted from z-stack are reported Section title: Brusatol reduces the content of Nrf2, inhibits Nrf2 phosphorylation and nuclear translocation Educational score: 4.165668487548828 Domain: biomedical Document type: Study Language: en To further verify the effects of Nrf2 in MCAO/R-induced ferroptosis, we inhibited the Nrf2 by brusatol. The expression of Total Nrf2 , p-Nrf2 , HO-1 , Nuclear Nrf2 , and Cytoplasmic Nrf2 were examined by Western blotting, and the results indicated that brusatol reversed the effects of increased expression and promoted nuclear translocation induced by EA treatment. We further observed the location of Nrf2 by immunofluorescence . The results also revealed that EA treatment enhanced the content of Nrf2 and attracted the nuclear translocation of Nrf2 in the MCAO/R + EA group, while Brusatol suppressed the nuclear translocation of Nrf2 prominently. These observations confirm that Brusatol prominently suppressed the total content of Nrf2 and HO-1, and suppressed the phosphorylation and nuclear translocation of Nrf2 upon EA treatment. Fig. 5 Brusatol reduces the content of Nrf2 and inhibits Nrf2 phosphorylation and nuclear translocation. A The representative Western blotting results of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2, and Cytoplasmic Nrf2 expression. B – F Quantification of Total Nrf2, p-Nrf2, HO-1, Nuclear Nrf2 and Cytoplasmic Nrf2 expressions ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001. G The confocal images were performed to determine the protein expressions of Nrf2. Scale bar: 5 μm. Nrf2 and DAPI were excited at 488 and 405 nm, respectively. Exemplary XY plane of confocal z-stack acquisition of Nrf2 (green signal) and treated with DAPI for nuclear staining (blue signal). Orthogonal views (XZ and YZ planes) extracted from z-stack are reported Section title: Brusatol diminishes the attenuated ferroptosis induced by EA after MCAO/R Educational score: 4.1133880615234375 Domain: biomedical Document type: Study Language: en Compared with the MCAO/R + EA + DMSO group, the Fe 2+ content, ROS and MDA levels in MCAO/R + EA + Brusatol group were significantly higher, while, the SOD level in was significantly lower. Similarly, the results by Western blotting showed that Brusatol significantly down-regulated the expression of GPX4 , SLC7A11 and FTH1 proteins . The GPX4 expression level was further assessed by Double immunofluorescence staining. The findings displayed that GPX4 was predominantly co-localized with neurons (F i g. 6 I). Consistent with GPX4 protein levels, MCAO/R prominently degraded GPX4 expression. EA augmented GPX4 expression, while the Brusatol reversed the up-regulation of GPX4 induced by EA . These findings demonstrate that brusatol counteracts the protectiveness of EA through the Nrf2 pathway. Fig. 6 Brusatol diminishes the protective effect of EA that attenuates ferroptosis after MCAO/R. A – D The concentration of Fe 2+ , ROS, MDA, and SOD in each group's brain tissue ( n = 6). E The representative Western blotting results of GPX4, SLC7A11, and FTH1 expression. (F–H) Quantification of GPX4, SLC7A11, and FTH1 expressions ( n = 6). I , J Typical double immunofluorescence images (GPX4 (green), NeuN (red), DAPI (blue). Scale bar: 25 µm. n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 Section title: Brusatol diminishes the attenuated ferroptosis induced by EA after MCAO/R Educational score: 4.109766006469727 Domain: biomedical Document type: Study Language: en Next, TEM was used to observe the morphology of mitochondria. As shown in the images, mitochondria in the Sham group had an intact outer membrane, abundant cristae, and normal morphology, MCAO/R led to the production of fragmented mitochondria with decreased cristae, even appeared to be vacuolated, which is a characteristic of ferroptosis. However, EA dampened MCAO/R-induced morphological changes in mitochondria . In contrast, Brusatol inverted the advantageous effects of EA on mitochondria. Fig. 7 Brusatol diminishes the protective effect of EA that attenuates ferroptosis after MCAO/R. Mitochondrial morphology associated with ferroptosis was determined by TEM. The bottom panels display the magnified images of regions indicated by yellow rectangles in the top panels. Arrows labeled: representative images of mitochondria. (Scale bar: 5 μM, 2 μM, and 1 μM, n = 3) Section title: Brusatol reverses the neuroprotective effect of EA Educational score: 4.124593734741211 Domain: biomedical Document type: Study Language: en Behavioral tests and TTC staining were utilized to appraise the effects of brusatlo on the neuroprotection provided by EA. The results presented that the score of NSS was significantly greater and the Garcia score was below in the MCAO/R + EA + Brusatol group than in MCAO/R + EA + DMSO group. Similarly, The results of the Foot-fault Test and the Rotarod Test suggested that functional motor restoration was inferior in the MCAO/R + EA + Brusatol group compared to the MCAO/R + EA + DMSO group. TTC staining revealed that the volume of cerebral infarction in rats in the MCAO/R + EA + Brusatol group was noticeably larger than that in rats in the MCAO/R + EA + DMSO group. . Meanwhile, Nissl staining results showed that compared with the MCAO/R + EA + DMSO group, the MCAO/R + EA + Brusatol group had an increased volume of cerebral infarction, disorganized neuronal cell arrangement, and more severe brain damage . These results suggested that EA provided a neuroprotection against stroke, while the neuroprotective effect was diminished by the inhibition of Nrf2. Fig. 8 Brusatol reverses the neuroprotective effect of EA. A – D Neurological deficits of each group were scored with the NSS, Garcia score, Rotarod Test, and Foot-fault Test at 7 days after surgery ( n = 12). E , F Evaluation of cerebral infarction volume of rats by TTC staining and analysis ( n = 6). G Representative photomicrographs of Nissl stained sections (Scale bar: 1 mM, 200 μM, and 50 μM, n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 Section title: Discussion Educational score: 4.125479698181152 Domain: biomedical Document type: Study Language: en In this study, we established the MCAO/R model in rats and treated them with EA for 7 consecutive days starting from one day after modeling, which ultimately diminished infarct volume and improved neurological function and motor behavior scores. The possible neuroprotective mechanism was that EA alleviated MCAO/R-induced ferroptosis by promoting Nrf2 nuclear transposition and activating the Nrf2/SLC7A11/GPX4 pathway. Section title: Discussion Educational score: 4.584878444671631 Domain: biomedical Document type: Review Language: en Ischemic stroke is the prevalent type of stroke with high mortality and restricted functional recovery in survivors . Ischemic stroke occurs with ischemia and hypoxia, which drastically declines the provision of oxygen, glucose, and other nutrients, and blood supply to the brain, consequently leading to disruption of cellular energy metabolism and destruction of neuronal function . The region of infarction formed by a sharp diminution of blood flow to the brain in ischemic stroke is referred to as the ischemic core. Once the local blood flow supply is curtailed to below 20%, ischemia and hypoxia induce severe reactions such as ATP exhaustion, invalidation of Na + /K + pumps, proliferation of intracellular Ca 2+ , and discharge of neurotoxic substances in the ischemic core region within a short period, which results in speedy cell death. It is surrounded by the peri-infarct region or penumbra, which encloses brain tissue that is functionally damaged but potentially salvageable. Cells in the penumbra region are not electrically active but retain energy in the form of ATP, and thereby the cells are typically enabled to die in a modulated programmed manner, which prevents deleterious inflammation from occurring by rendering cellular contents from being released into the extracellular milieu . Hence, salvaging ischemic penumbral regions and facilitating cell survival are the primary goals of exploiting neuroprotective strategies to mitigate the severity of cerebral ischemic injury through immediate intervention . Section title: Discussion Educational score: 4.44188117980957 Domain: biomedical Document type: Study Language: en Recent research has revealed that ferroptosis is an influential factor in brain I/R injury, and its development is closely linked to various biological courses like iron, amino acid, polyunsaturated fatty acid metabolism, and GSH biosynthesis . Morphologically, ferroptosis occurs mainly within the cell, as evidenced by a reduction in mitochondrial volume, density of the bilayer membrane, and reduction or disappearance of the mitochondrial cristae, but with intact membranes and normal nuclear size . Biochemically, ferroptosis is manifested by intracellular GSH depletion, the reduction of GPX4 activity, and the inability of GPX4 to metabolize lipid peroxides to catalyze the reduction reaction, which generates a large amount of ROS and facilitates ferroptosis . Previous studies have indicated that high levels of GPX4 can safeguard neurons and mitochondria from oxidative damage . Meanwhile, reduced levels of GPX4 dramatically diminished the amount of NeuN-positive cells in the hippocampus and elicited neuronal ferroptosis , implying that GPX4 is a crucial factor in the modulation of neuronal ferroptosis. Similarly, diminished levels of SLC7A11 can generate a drop in intracellular cystine levels, leading to GSH depletion and inhibition of GPX4 activity, eventually activating ferroptosis [ 65 – 67 ]. Therefore, the expression level of SLC7A11 can directly mediate the susceptibility of cells to ferroptosis. In addition, FTH1 is known to carry out high levels of iron storage, thus ensuring normal biochemical reactions in vivo , and the genesis of ferroptosis is often accompanied by reduced levels of FTH1 . Our results showed that the levels of GPX4, SLC7A11, and FTH1 were decreased after MCAO/R, while EA treatment significantly augmented the expression of GPX4, SLC7A11, and FTH1. Section title: Discussion Educational score: 4.659289836883545 Domain: biomedical Document type: Study Language: en Iron is characterized by a diversity of metabolic roles and performs valuable physiological functions in vivo. Extracellular Fe 3+ combines with transferrin (TF) and is internalized to the nucleus through transferrin receptor 1 (TFR1) on the cell membrane, where Fe 3+ is reduced to Fe 2+ . Eventually, divalent metal transporter 1 (DMT1) discharges Fe 2+ from the nuclear endosome into the intracytoplasmic unstable iron pool in the cytoplasm. Furthermore, ferroportin 1 (FPN1) on the cell membrane frees surplus Fe 2+ to the exterior of the cell to ensure the maintenance of iron homeostasis . While the body's tissues or organs suffer damage, the cells are under stress and the intracellular redox balance is in jeopardy, with a resultant reduction of Fe 3+ to Fe 2+ . Overloaded Fe 2+ can catalyze the conversion of H 2 O 2 , a product of oxidative respiration in mitochondria, into hydroxyl radicals, which undergo nonenzymatic lipid peroxidation, eliciting membrane lipid peroxidation and mitochondrial damage, resulting in ferroptosis, which then exacerbates pathological damage . MDA as the terminal product of membrane lipid peroxidation reaction is a biomarker of lipid peroxidation and oxidized protein damage. SOD is among the prominent enzymes capable of scavenging ROS efficiently , and is liable for the conversion of superoxide anion, a generated ROS, into hydrogen peroxide, which is then converted to H 2 O by CAT (catalase), thus it is a crucial endogenous antioxidant factor for preserving cellular redox homeostasis [ 74 – 76 ]. Levels of MDA, SOD, and Fe 2+ have been recognized as signatures and pivotal indicators of ferroptosis; as yet, the definitive mechanisms governing ferroptosis downstream of lipid peroxidation remain elusive. Therefore, while the levels of MDA, SOD, and Fe 2+ cannot be regarded as the “gold standard” for ferroptosis, for the time being, these indicators can be one of the bases for determining if ferroptosis occurs in cells, and in the future, we will also further seek for more applicable indicators to refine the experiments. We showed that high accumulation of Fe 2+ in the brain of I/R-injured rats resulted in an imbalance of the antioxidant system, whereas EA treatment remarkably declined MDA levels, elevated SOD activity, and lessened ROS content, pointing to a striking amelioration of intracerebral lipid peroxidation and curtailing of ferroptosis, and that suppressing the origin and progression of cellular ferroptosis might be an equally valuable target for the exploration of novel therapeutic approaches for ischemic stroke in the future. Section title: Discussion Educational score: 4.364414215087891 Domain: biomedical Document type: Study Language: en The brain I/R injury belongs to the category of "stroke disease" in Chinese medicine, its cause is mainly due to the deficiency of healthy energy in the body, which leads to the inability to transport blood, so it stagnates in the body and produces stagnation. Although the vital signs have been stabilized during the recovery period, the blood stasis and phlegm turbidity have not been effectively eradicated , thus the treatment is centered on the principle of activating blood circulation, removing blood stasis, and regulating the meridians . Growing evidence indicates that EA is a potential strategy to promote neurological recovery in patients with ischemic stroke. As a safe and effective treatment, EA can be used to alleviate the symptoms of ischemic stroke and promote neurological recovery. Clinical trials and meta-analyses have revealed the effectiveness of EA in alleviating spasticity, reinforcing muscle strength, and achieving better overall health post-stroke, whilst improving patients' quality of life . It has been reported that EA not only protects neurovascular units by modulating cellular autophagy but also significantly reduces the level of oxidative stress and inhibits cellular ferroptosis . Compared to acupuncture alone, EA can produce a synergy effect through combining acupuncture and electrical stimulation . Four acupoints were chosen for this study: Baihui, Dazhui, Neiguan, and Quchi. Baihui and Dazhui have special therapeutic effects on brain disorders, and they are commonly used acupoints for emergencies as recorded in ancient Chinese medical literature, in the preventive period, acute period, relief period, and sequelae period of cerebral stroke, especially in the acute period of cerebral stroke in the state of coma is the preferred acupoints, which are worthy of in-depth study . Some research has found that acupuncture at Baihui and Dazhui points can reduce neuronal apoptosis in the ischemic area, inhibit oxidative stress, attenuate ischemic cerebral edema [ 83 – 85 ], and other mechanisms, that can promote the recovery of neurological function. In addition, acupuncture at the Quchi and Neiguan points has the property of replenishing vital energy and can cure hemiplegia, which are crucial points about health care. When we applied EA stimulation to these four acupoints, it significantly improved neurobehavioral scores and motor-behavioral outcomes after ischemic stroke and reduced cerebral infarct volume. We further confirmed that EA exerts this neuroprotective effect by inhibiting ferroptosis. Interestingly, we discovered that EA facilitated Nrf2 translocation to the nucleus, and thus we pursued further the correspondence between Nrf2 nuclear translocation and the preventive role of EA against stroke. Section title: Discussion Educational score: 5.063032627105713 Domain: biomedical Document type: Study Language: en Nrf2 is ubiquitously present in a broad range of cells and is redox-sensitive . At equilibrium, Nrf2 is bound to Keap1, continuously ubiquitinated by Cul3 E3 ubiquitin ligase, and subtracted by the proteasome, which maintains a high Nrf2 turnover rate. Under stress, Keap1 is oxidized and inactivated, resulting in the stability and translocation of Nrf2 to the nucleus, where it forms a heterodimer with the small Maf proteins, combines with the ARE, and triggers transcription of its target genes . Nrf2 regulates several downstream pathways, including apoptosis, inflammation, oxidative stress, calcium overload load, etc., assisting the body in upholding redox responses. Therefore, stabilizing Nrf2 activity is pivotal to maintaining redox balance and homeostasis in the brain. After a stroke, excess ROS activate Nrf2 , and incremental Nrf2 facilitates the expression of antioxidant genes and dampens ROS expression, which ultimately strengthens the mitochondrial antioxidant response and attenuates the destruction of the blood–brain barrier and neurological damage . HO-1, an ıncredibly essential downstream factor of Nrf2, which degrades heme to CO and biliverdin, products that are usually anti-inflammatory and antioxidant , and is a major mediator of the salutary effects of Nrf2 . By decomposing free radicals in the body into water and molecular oxygen, the Nrf2/HO-1 signaling pathway serves as a vital mechanism for the body's defense against oxidative stress, mitigating oxidative stress damage and curtailing the production of oxidative products, thereby rendering anti-inflammatory and antioxidant effects . As such, Nrf2 is located at the center of a sophisticated modulatory network that exerts protective effects against cerebral ischemia through multiple mechanisms . Section title: Discussion Educational score: 4.203816890716553 Domain: biomedical Document type: Study Language: en Our study found that EA promoted the translocation of Nrf2 from the cytoplasm to the nucleus and resulted in elevated HO-1 levels. To elucidate the protective mechanism of EA on MCAO/R, we applied Brusatol, an inhibitor of Nrf2, to block this pathway. The results showed that Brusatol decreased the content of Total-Nrf2, Nuclear-Nrf2, and HO-1 and decreased the locomotor ability, increased the infarct volume, increased the levels of lipid peroxides and Fe 2+ , inhibited the expression of GPX4, SLC7A11, and FTH1, and reversed the inhibitory effect of EA on ferroptosis. This finding suggests that EA therapy inhibited the occurrence of ferroptosis in cells by facilitating the nuclear translocation of Nrf2 and exerting its antioxidant effects. Section title: Discussion Educational score: 4.355591297149658 Domain: biomedical Document type: Study Language: en In the last decades, research on ischemic encephalopathy models using rodents has yielded very rewarding results, and many neuroprotective measures have been deemed as prospective therapies. Unfortunately, many drugs have mostly failed to show effectiveness in ischemic stroke patients in randomized clinical trials, however. Therefore, translating research findings into clinical practice is quite a challenging task, and we expect EA to become a more efficacious treatment modality in the clinical management of ischemic stroke. Additionally, modulation of iron metabolism and antioxidant pathways to inhibit ferroptosis is an encouraging goal for the future treatment of ischemic stroke. However, more studies are required to elucidate the functional alterations and molecular mechanisms of ferroptosis. In recent years, Nrf2 has become one of the hotspots in the research field due to its potent value in inhibitory inflammatory response and anti-oxidative stress, therefore, the detailed mechanism of EA on the Nrf2/SLC7A11/GPX4 axis needs to be further explored, such as how EA specifically regulates Nrf2 expression and entry into the nucleus to exert a neuroprotective effect. In addition, in vitro experiments are necessary to investigate the effect of activating Nrf2 in ferroptosis, which could help to further elucidate and verify this process. In this experiment, EA inhibited cellular ferroptosis by stimulating Nrf2 nuclear translocation, thus displaying a neuroprotective effect versus acute brain injury, which provides an emerging target for EA to remedy specific mechanisms of ischemic stroke. As our understanding of the mechanisms and pathology of stroke increases, neuroprotective strategies emerge as hopeful treatments. With multi-center, large-sample, well-quality laboratory, and clinical trials, further studies on the molecular mechanisms underlying the neuroprotective effects of electroacupuncture may provide a scientific basis for the selection of acupoints and therapeutic parameters for clinical treatment, as well as a more optimized strategy. Section title: Conclusion Educational score: 4.1288347244262695 Domain: biomedical Document type: Study Language: en The present study highlights the efficacy of EA at Baihui, Dazhui, Quchi, and Neiguan on MCAO/R rats, and simultaneously proposes a mechanism involving the promotion of Nrf2 nuclear translocation and activation of the Nrf2/SLC7A11/GPX4 pathway to suppress ferroptosis following ischemic stroke in rat, these suggesting that EA treatment could be applied as an effective therapeutic intervention to improve neurological function. These findings afford prospective therapeutic mechanisms for EA in the treatment of ischemic stroke at Baishui, Dashui, Quchi, and Neiguan, and render valuable doctrinal support for clinical application. | Other | biomedical | en | 0.999998 |
PMC11699711 | Section title: Introduction Educational score: 4.0656938552856445 Domain: biomedical Document type: Study Language: en In recent years, regenerative endodontic therapy (RET) has been suggested for the treatment of immature necrotic teeth . The primary aim of RET is apical healing; secondary aims include continued root development, demonstrated by both root elongation and an increase in the canal wall thickness, as well as a positive response to sensibility tests . In a follow-up of teeth one year after RET, apical healing and complete apical closure were observed in 100% and 55% of the teeth, respectively, which were accompanied by an increase in the canal’s width and lengthening of roots . Similar survival rates were reported in a recently published meta-analysis . Section title: Introduction Educational score: 4.120524883270264 Domain: biomedical Document type: Study Language: en Clinically, during the final steps of RET, a layer of bio-ceramic material barrier is applied on the scaffold within the root canal space . There is limited evidence as to the effect of the scaffold, either platelet-rich plasma (PRP), platelet-rich fibrin (PRF), platelet pellet, or blood clot, on the secondary outcomes, although all treatment modalities resulted in healing of the apical rarefaction . A retrospective study reported lower success and survival rates after RET in traumatized immature necrotic teeth. The type of traumatic injury had an effect on the treatment outcome, with teeth after avulsion having the lowest survival and success rates (66.7% and 33.3%, respectively) . The etiology of pulp necrosis, which led to treatment, either trauma, dens evaginatus, or caries, did not affect the success of the treatment, which ranged between 93.1% to 96% . Histological examinations of extracted human teeth that have undergone regenerative endodontic therapy (RET) have shown that the newly formed tissues primarily consist of fibrous connective tissue and cementum-like connective tissue . Section title: Introduction Educational score: 3.998387575149536 Domain: biomedical Document type: Study Language: en Failures after RET may be observed during follow-up examination. In a systematic review of failed RET cases, 39% of the 67 failures were detected more than two years after initiation of the RET . According to a study that analyzed 16 failed cases, 37.5% of the failures were identified within six months after the procedure. The main etiologic factors that can lead to failure are dental trauma, dens evaginatus, and dental caries . Interventions after failures include extraction, nonsurgical root canal treatment, repeated RET, and Ca(OH)₂ apexification , as well as a surgical approach . There is scarce clinical evidence regarding the outcome of treatment of failed RET cases . In a report of three failed root canal treatment (RET) cases, the teeth were retreated after repeated debridement of the root canals. All the cases were treated in a single visit using negative-pressure irrigation. Orthograde root canal treatment was performed in one case, and in a second case, orthograde retreatment was supplemented by an apical mineral trioxide aggregate (MTA) prior to obturation. In the third case, repeated RET was performed. In all three cases, resolution of the periapical lesion was observed . Successful repeated RET was observed in another case report describing the failure of a previously healed immature incisor tooth. The tooth was retreated in one visit and showed complete apical repair after two years . Section title: Introduction Educational score: 4.101737976074219 Domain: biomedical Document type: Study Language: en The main reason for failure is a persistent bacterial infection . A more significant decrease in primary microflora was found in successful RET cases than in failed cases treated with either Ca(OH)₂ or chlorhexidine. After dressing with Ca(OH)₂ or chlorhexidine, a greater reduction in microflora was observed in successful RET cases compared to failed treatments . It is assumed that lack of mechanical debridement during the regenerative procedure allows for relapse of the apical pathology after a temporary resolution of the signs and symptoms . This study aims to describe the outcome of retreatment in failed cases of regenerative treatment, describe the reasons for the failure, discuss the optional treatment options, and present a decision-making flowchart. Section title: Materials and methods Educational score: 4.02048397064209 Domain: biomedical Document type: Study Language: en The study was conducted in the Hebrew University Hadassah School of Dental Medicine in Jerusalem. Ethical approval was obtained from the Institutional Helsinki Ethics Committee . Data collection was based on the digital medical records of patients treated by endodontic post-grad students between 2015 and 2020. From patients aged six to 17, 414 endodontically treated immature teeth (Nolla stages 7-9) were screened to allocate patients with a failed RET. Data including patients' ages, sex, medical, and dental history were collected. Their dental records, specifically regarding the treated tooth, were compiled. Cases with incomplete records or insufficient follow-up were excluded. Around 48 teeth underwent RET and were followed for one year or more. Eight failed cases were observed among the teeth that underwent RET. Two teeth were referred for extraction due to a root fracture. Six patients (aged seven to 16 years) who were referred for the management of apical periodontitis that developed after a previous RET failed were retreated and followed for more than one year. The teeth included five maxillary incisor teeth and one maxillary second premolar (Table 1 ). Section title: Materials and methods Educational score: 2.3031163215637207 Domain: biomedical Document type: Study Language: en All patients underwent clinical and radiographic examinations. In cases where apical surgery was deemed necessary, CBCT scans were also conducted. After evaluating various treatment options, the teeth were successfully treated (Table 2 ). Section title: Results Educational score: 3.9390695095062256 Domain: biomedical Document type: Study Language: en Reviewing the medical records of the young patients treated in the endodontic post-grad clinic revealed 48 well-documented cases of RET, eight of which failed. Initially, 46 of the cases were successful, with one immediate failure recorded and one tooth extracted because of infection after one year. This resulted in an initial survival rate of 97.9% (47 out of 48 teeth) and an initial success rate of 95.8% (46 out of 48 teeth). Over time, one additional tooth was extracted due to root fracture, leading to a long-term survival rate of 95.8% (46 out of 48 teeth) and a long-term success rate of 83.3% (40 out of 48 teeth). Section title: Results Educational score: 1.1937052011489868 Domain: clinical Document type: Clinical case Language: pt Case 1 Section title: Results Educational score: 3.766143560409546 Domain: clinical Document type: Clinical case Language: en A seven-year-old female patient who was diagnosed with pulp necrosis and asymptomatic apical periodontitis of the left maxillary central incisor following a complicated crown fracture and traumatic extrusion was treated by RET according to the American Association of Endodontists (AAE) guidelines. The immature tooth was classified as Nolla stage 7, and the plug placed in the canal during the second visit was MTA ProRoot (Dentsply Sirona, Johnson City, TN). The girl was scheduled for follow-up appointments, and after 1.5 years, continued root development, and the periapical lesion has healed. Section title: Results Educational score: 3.78511381149292 Domain: clinical Document type: Clinical case Language: en Six years later, a periapical lesion reemerged, and her dentist referred her after unsuccessfully attempting to perform a root canal treatment. The root was fully developed and was now classified as Nolla 10, and pulp canal obliteration (PCO) was observed. During the orthograde retreatment, the root canal was negotiated using ReadySteel C+ files (Dentsply Maillefer, Switzerland) under the operative microscope. After ultrasonic irrigation, the canal was obturated with gutta-percha and AH Plus root canal sealer (Dentsply, DeTrey, Konstanz, Germany), and the access cavity was sealed with a composite restoration. After a one-year follow-up, the periapical lesion healed . Section title: Results Educational score: 1.0366506576538086 Domain: clinical Document type: Clinical case Language: pt Case 2 Section title: Results Educational score: 3.749444007873535 Domain: clinical Document type: Clinical case Language: en An eight-year-old male patient who was diagnosed with asymptomatic apical periodontitis for the right maxillary central incisor following lateral luxation was treated by RET according to the AAE guidelines. The immature tooth was classified as Nolla state 8, and the plug placed in the canal during the second visit was MTA ProRoot (Dentsply Sirona Johnson City, TN). The boy was scheduled for follow-up appointments, and after three years, the periapical lesion had healed, and a calcified barrier was observed apical to the MTA plug. The root canal remained without any additional change. Section title: Results Educational score: 3.4201509952545166 Domain: clinical Document type: Clinical case Language: en Seven years after the RET, he was referred for treatment by his orthodontist due to a reemerging periapical lesion. The MTA plug appeared to be disintegrated. After the composite restoration of the access cavity was removed, the remnants of the MTA plus were removed. Following chemomechanical preparation, and since the apical foramen diameter was very wide, an apical plug was placed using EndoSequence® RRM (Brasseler, USA). After a one-year follow-up, the periapical lesion healed . Section title: Results Educational score: 1.0525002479553223 Domain: clinical Document type: Clinical case Language: pt Case 3 Section title: Results Educational score: 3.224355459213257 Domain: clinical Document type: Clinical case Language: en A 16-year-old male patient was diagnosed with pulp necrosis and symptomatic apical periodontitis for the right maxillary central incisor following a traumatic dental injury that occurred two and a half years earlier. The exact nature of the dental injury was unknown. According to his dental records, the boy was treated by RET following the AAE guidelines. The immature tooth was classified as Nolla state 8, and the plug placed in the canal during the second visit was MTA ProRoot (Dentsply Sirona, Johnson City, TN). Section title: Results Educational score: 3.3837978839874268 Domain: clinical Document type: Clinical case Language: en The boy returned 2.5 years after completion of the RET due to the re-emergence of the periapical lesion. Considering the large MTA plug, the calcified barrier apical to the plug, and the thin dentinal walls, apical surgery was considered. The patient was scheduled for apical surgery. During the procedure, the canal was only minimally resected, and the canal walls were irrigated and scrubbed with a micro-brush with chlorhexidine 2%. The empty root canal space was filled with EndoSequence® RRM. At the two-year follow-up, incomplete healing was observed . Section title: Results Educational score: 1.041892170906067 Domain: clinical Document type: Clinical case Language: pt Case 4 Section title: Results Educational score: 3.090977430343628 Domain: clinical Document type: Clinical case Language: en A 15-year-old male patient who was diagnosed with asymptomatic apical periodontitis of the right maxillary second premolar was referred by his orthodontist before the beginning of an orthodontic treatment. RET was performed two years earlier, following the AAE guidelines using MTA, because of pulp necrosis that developed because of the irregular type II dens invaginatus anatomy. Section title: Results Educational score: 3.769101858139038 Domain: clinical Document type: Clinical case Language: en Upon arrival, it was evident that the root continued to develop, although a periapical lesion was now evident. A CBCT scan revealed that the dens had been only partially sealed, and the canal apical to the intra-coronal plug was obliterated. Following the replacement of the coronal seal, apical surgery was performed. The short root canal was only minimally resected. Debridement of the apical root canal was done using ultrasonic tips, and retrograde obturation was done using EndoSequence® RRM. After one year, complete healing of the apical radiolucency was observed, and orthodontic treatment was initiated. Three years after the completion of the RET, the periapical tissue remained normal . Section title: Results Educational score: 1.0837150812149048 Domain: clinical Document type: Clinical case Language: pt Case 5 Section title: Results Educational score: 3.0862271785736084 Domain: clinical Document type: Clinical case Language: en A 10-year-old male patient who suffered a crown fracture and pulp necrosis with asymptomatic apical periodontitis in the right maxillary central incisor was treated by RET using MTA following the AAE guidelines. Apical repair was observed during the follow-up examination, and the tooth remained asymptomatic. Section title: Results Educational score: 3.6026270389556885 Domain: clinical Document type: Clinical case Language: en Two years later, his orthodontist referred the patient due to an acutization of an apical radiolucency. A periapical radiograph and a CBCT scan demonstrated an increase in the thickness of the root canal walls, with the root end becoming blunt and almost closed. Repeated RET resulted in complete repair of the periapical lesion and a calcified apical tissue, observed two years later. After apical healing, the orthodontic treatment was continued . Section title: Results Educational score: 1.026190161705017 Domain: clinical Document type: Clinical case Language: pt Case 6 Section title: Results Educational score: 3.8346445560455322 Domain: clinical Document type: Clinical case Language: en A nine-year-old boy presented with a horizontal root fracture in the right central maxillary incisor following the tooth's extrusion. A periapical radiograph revealed a type II dens invaginatus. The tooth was diagnosed with pulp necrosis, and inflammatory root resorption was observed. RET was performed according to the AAE guidelines using calcium hydroxide dressing between visits. After bleeding was allowed into the canal space, a collagen plug was placed as a barrier covered by EndoSequence® RRM. Eight weeks after its initiation, treatment was completed, and healing of the radiolucent lesion was evident. Section title: Results Educational score: 3.5972745418548584 Domain: clinical Document type: Clinical case Language: en During follow-up, the tooth exhibited recurrent pathology at the fracture line. The patient was scheduled for retreatment of the tooth. Repeated RET included additional ultrasonic irrigation and calcium hydroxide dressing. One year after retreatment, the apical rarefaction resolved with continued maturogenesis of the apical fragment and approximation of the coronal and apical fragments attached by a calcified tissue and surrounded by the continuous periodontal ligament . Section title: Discussion Educational score: 4.098699569702148 Domain: biomedical Document type: Study Language: en Regenerative endodontics has become a promising alternative for treating immature teeth with apical pathology . A recent meta-analysis reported a 100% survival rate for immature teeth after RET for at least one year . Similar success rates that ranged between 93.1% and 96% were found for teeth that underwent RET due to trauma, dens evaginatus, or caries . These results are similar to the one-year survival and success rates reported in the current study (97.9% and 95.8%, respectively). Despite high survival and success rates , failures can still occur, presenting clinicians with challenging treatment decisions, particularly in immature teeth . Despite initial signs of healing and continued root development observed during short-term follow-up examinations, six cases ultimately failed. These cases were successfully managed through secondary interventions. Section title: Discussion Educational score: 3.512302875518799 Domain: biomedical Document type: Study Language: en The timeframe during which RET failures are detected varies significantly, ranging from less than six months following treatment to as long as four, eight, and even 11 years . In the literature, the average follow-up time reported was shorter: 19.9 months for the mineral trioxide aggregate plug and 16.7 months for the RET group . Section title: Discussion Educational score: 4.098474502563477 Domain: biomedical Document type: Study Language: en In the current study, one tooth was extracted immediately after the RET due to persistent infection, and one failure was observed three months after the initial follow-up visit, while the other five cases showed failures between two and seven years after the RET was completed, and another tooth was extracted due to a root fracture, despite initial signs of success. This study emphasizes that while short-term success is encouraging, it does not guarantee long-term stability in RET cases. The success rate dropped over time from 95.8% to 83.3%. The study strongly advocates for extended, diligent monitoring to ensure the best possible outcomes for patients undergoing regenerative endodontic procedures. Section title: Discussion Educational score: 4.088117599487305 Domain: biomedical Document type: Study Language: en The initial success of the regenerative treatment was observed during the follow-up in the cases described above. It was manifested in the healing of the periapical lesion and the continued development of the roots. However, over time, a new periradicular rarefaction reemerged. RET failures have been attributed to persistent infection or inadequate root canal disinfection due to a lack of mechanical debridement, which is associated with an effort to avoid further weakening of the thin and fragile dentinal walls . Indeed, histological examination after RET’s failure demonstrated bacteria and biofilm on the canal walls . Section title: Discussion Educational score: 3.992306709289551 Domain: biomedical Document type: Study Language: en It was reported that most of these failures were following a secondary dental trauma (56%), as observed in case number 6; abnormal anatomy of the dens invaginatus (25%), as observed in case number 3; and a result of persistent infection, as observed in case 5 . Failures that occur after the initiation of orthodontic treatment , as reported in case number 2, raise concerns as to the immune competency of the revitalized tissue after regenerative endodontic procedure (REP) . Teeth treated by RET may not withstand biting forces and fracture, especially if they are poorly developed and the canal walls are short and thin . Thus, they necessitate extraction, as they may not be restorable . Section title: Discussion Educational score: 2.763512134552002 Domain: biomedical Document type: Other Language: en Regenerative treatment failures can be successfully treated by several treatment options, including repeated RET , orthograde root canal treatment with or without creating an apical barrier with a bio-ceramic material, or performing a surgical intervention . Section title: Discussion Educational score: 3.8925390243530273 Domain: biomedical Document type: Other Language: en Repeated RET should be considered in an attempt to improve the low prognosis resulting from the thin root walls and the unfavorable crown-to-root ratio. RET is likely to fail without adequate root canal disinfection . Efforts are being made to improve the efficacy of the antibiotic paste placed in the canals during RET . Current disinfection protocols applied during RET cannot eliminate bacterial infection from the root canals . Supplementary agitation of sodium hypochlorite by means such as ultrasonic activation or the use of XP finisher (FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) can effectively reduce the biofilm and enhance disinfection of the root canal , especially in wide root canals . Section title: Discussion Educational score: 3.788783550262451 Domain: biomedical Document type: Other Language: en In the event of a failed regenerative endodontic procedure (REP), the procedure can be repeated, offering another opportunity for success in cases where the initial REP did not yield the desired outcomes . Modifying the protocol during repeated procedures and treatments may be necessary, allowing for variations in the procedure. The current protocol includes the use of several antimicrobial agents . Alternatives like calcium hydroxide or modified TAP can be considered, along with a different intra-coronal barrier. Another bioceramic material, such as Biodentine, which has different characteristics and setting times, may perform better . Section title: Discussion Educational score: 3.832932710647583 Domain: biomedical Document type: Clinical case Language: en RET was successfully repeated in two cases. The successful treatment outcome in case number 6 after repeated RET can be attributed both to the patient's young age and the wide diameter of the canal at the fracture line . This may imply that when a failure occurs in teeth with a large apical diameter, especially when the patient is young, the preferred treatment modality will be a repetition of the RET. However, a surgical approach may be required when a calcified barrier has formed apical to the bioceramic plug in the canal . Section title: Discussion Educational score: 3.7614917755126953 Domain: biomedical Document type: Other Language: en Surgical endodontics can be a viable option to address regenerative endodontic failures. The main advantage of this procedure in teeth with short roots is maintaining the crown-to-root ratio. It is usually recommended to resect 3 mm of the root end during apical surgery to remove the majority of ramifications and lateral canals . Since the apical canal is quite wide, it may be advisable to minimally resect the root by one mm or to smooth out any uneven edges of the root. Section title: Discussion Educational score: 3.9418230056762695 Domain: clinical Document type: Clinical case Language: en Favorable outcomes were reported after minimal root resection and extended retrograde root end preparation . This approach can also be suggested when the orthograde access to the infected tissue in the canal is blocked. Removal of the bioceramic plug from the canal may risk the tooth in fracture. When anatomical constraints prevent apical surgery, intentional replantation (IR) can be an alternative treatment option. A case report highlights the successful intentional replantation of an immature central incisor that had undergone RET and later developed a chronic apical abscess. Despite signs of external root resorption, the replanted tooth remained symptom-free throughout a four-year follow-up period . Section title: Discussion Educational score: 3.8297417163848877 Domain: biomedical Document type: Study Language: en In most cases, failure occurred after the healing of the periapical lesion and continued root development. Despite the late failure, it can be assumed that teeth with a better crown-to-root ratio and the increase in the root wall thickness are advantageous compared to more immature teeth, which are more susceptible to fracture . Additionally, the time between regenerative endodontic therapy (RET) and the decision to perform a full root canal treatment (RCT) due to reemerging pathology may allow for greater patient cooperation, particularly among young individuals. Section title: Conclusions Educational score: 3.61621356010437 Domain: biomedical Document type: Other Language: en Teeth that have undergone successful RET and demonstrated treatment failure at the follow-up appointments can undergo additional treatment that will allow the preservation of the teeth and healing of the periapical lesion. The treatment options include ReRET, orthograde root canal treatment with or without an apical plug, or surgical root canal treatment. Despite the initial treatment failure, a successful outcome was observed after a secondary intervention. | Study | biomedical | en | 0.999997 |
PMC11699719 | Section title: Background Educational score: 4.622372627258301 Domain: biomedical Document type: Study Language: en Iron administration in piglets in their first days of life is a basic zootechnical measure relevant for welfare and health. Dependent on body weight, pigs are marginally equipped with iron at birth ranging from 124 to 180 mg iron/kg dry matter in piglets from 0.8 kg to 1.6 kg body weight . For piglet growth with daily weight gains of 250 g, approximately 10 mg iron/day is needed , resulting in iron depletion within the first three weeks of the suckling period without iron supplementation. Iron concentrations of 1.7–5.4 µg/mL in sow colostrum and 1.3–4.6 µg/mL in milk are not adequate to fill this supply gap . Although study outcomes about oral iron supplementation vary with respect to efficacy oral iron uptake from external sources is assessed to be insufficient due to low intestinal iron absorption . The immaturity of the duodenal mechanisms of iron absorption was found to be related to low expression of iron transporters (duodenal divalent metal transporter (DMT) 1 protein, ferroportin) and a high expression of hepcidin as the major iron-regulatory hormone during the first days of life . A physiological tight control of iron metabolisms and regulation of iron release into extracellular body compartments is essential to prevent toxic effects of iron . It can be assumed, that breeding success with regard to high piglet growth rates and high number of piglets born alive overstrain genetically determined and evolutionary meaningful iron homeostasis control mechanisms. The naturally low foetal iron reserves are still low in litters of hyperprolific sows, while in parallel growth rate and feed efficacy of piglets have increased, and also the demand for essential nutrients as iron . While in adults iron homeostasis is dependent on absorption in the duodenum and its recycling by macrophages, iron deficiency anaemia (IDA) in piglets with the consequence of growth retardation, depression and a disturbance of cognitive development can only be prevented by iron supplementation . Section title: Background Educational score: 4.359146595001221 Domain: biomedical Document type: Study Language: en Immediately after birth a haemodilution caused by colostrum uptake and subsequent shifts in body fluid to the circulatory system leads to low haemoglobin (Hb) concentrations in piglets. The heaviest piglets are considered to be at higher risk to become anaemic due to their larger blood volume . If not treated, a latent iron deficiency in the first living days will finally result in a microcytic hypochromic anaemia characterized by Hb concentrations below 90 g/L and small erythrocytes . Cut-off Hb concentrations considered critical for piglet health differ among experts and are still under discussion. This might be due to a lack of currently valid values for Hb corrected for breed, production phase and age . Some authors suggest a Hb of 110 g/L to exclude latent iron deficiency , which corresponds to the global cut-off for children younger than 5 years defined in 1968 in a WHO technical report . In swine, either Hb concentrations below 80 g/L are considered critical for piglet health or Hb concentrations below 90 g/L . Some authors recommended for diagnostic of IDA the determination of iron-related parameters (e.g. ferritin, transferrin and transferrin saturation, iron, total iron binding capacity) next to established haematological and reticulocyte parameters [ 7 , 15 – 17 ]. Iron-related parameters are more difficult to interpret with respect to health status, because iron homeostasis is tightly regulated. Section title: Background Educational score: 4.1907057762146 Domain: biomedical Document type: Study Language: en Due to the biological duality of iron, balancing of risks and benefits of iron administration in suckling piglets is under research in different production systems with varying available iron sources from feed, water and environment. A comparison of piglets raised in intensive indoor and outdoor production systems and supplemented with 100 mg iron dextran on day three of age resulted in higher Hb concentrations on day 28 of life in outdoor pigs . Some authors conclude that no prophylactic iron treatment is necessary in outdoor pig production . A comparison of organic piglets either raised indoor with iron supplementation or outdoor without iron supplementation resulted in higher Hb concentrations in outdoor pigs, because soil contained sufficient bioavailable iron . In outdoor pigs with physiologically low Hb concentrations on their third day of life and receiving no iron treatment the majority of piglets was not anaemic at weaning . Treated pigs showed lower Hb levels at weaning, indicating reduced iron absorption from the duodenum due to downregulation of ferroportin . Other authors conclude that iron administration in general is necessary in outdoor pig production . In organic farms with prolonged suckling periods one iron injection was found to be not sufficient in spite of iron uptake from external sources as soil and piglet starter diet . Contradictory results might be explainable by differences in bioavailable iron in soil or by other factors as genotype of the sow or season . Section title: Background Educational score: 4.152715682983398 Domain: biomedical Document type: Study Language: en In intensive pig production daily iron requirements of suckling piglets are usually complied by subcutaneous or intramuscular (IM) administration of iron dextran or gleptoferron in the first days of life . Currently, combined iron products containing an active substance against coccidia are also used on farms. The advantage of these products combining two routine measures in one is that they save time and effort. The parenteral application of a coccidiostat is more comfortable than the formerly established oral application with same efficacy . However, when using a combination product targeting two diseases, an adjustment of the necessary amount of iron to prevent anaemia in the frame of a farm-specific treatment protocol is not possible. The aim of this study was to test for non-inferiority of iron dextran (Uniferon ® , Pharmacosmos A/S, Holbaek, Denmark), which can be used in flexible volumes, versus to the combinatory product Forceris ® (Forceris ® , Ceva Santé Animale, Libourne, France) containing gleptoferron and toltrazuril. In addition, different administration schemes for iron dextran with respect to time point and dosage were compared on a conventional farm. Section title: Study farm Educational score: 4.214751243591309 Domain: biomedical Document type: Study Language: en The animal experiment was performed in a farrow-to-finish farm with 230 sows (Danbred genetics) and 2550 fattening places in a swine dense region in North-Western Germany. Farrowing intervals were five weeks with a four-week suckling period and farrowing groups of approximately 57 sows. Sows of one farrowing group farrowed in three farrowing units with 20 places each. No nurse sows were used, and split suckling was not performed. The farm was known positive but stable for porcine reproductive and respiratory syndrome virus (PRRSV) according to category II-vx and for Actinobacillus ( A. ) pleuropneumoniae and Mycoplasma (M.) hyopneumoniae . No disease outbreaks were observed in the year before start of the study. All sows were vaccinated every four months with a live attenuated PRRSV-1 vaccine (Suvaxy ® PRRS MLV, Zoetis Deutschland GmbH, Berlin, Germany) and against influenza virus with a triple vaccine containing H1N1, H1N2 and H1N3 (Respiporc FLU3, Ceva, Düsseldorf, Germany) as well as a vaccine against H1panN1 (Respiporc FLUpanH1N1, Ceva). Sows were further vaccinated every six months against parvovirus and erysipelas (Parvoruvac ® , Ceva). Piglets are vaccinated routinely against edema disease on day five of life (Ecoporc SHIGA, Ceva) and on day 23 of life against PRRSV-1 (Suvaxy ® PRRS MLV), porcine circovirus 2 (PCV2) and M . hyopneumoniae in combination (Porcilis ® PCV M Hyo, MSD Tiergesundheit, Unterschleißheim, Germany). In addition, piglets were vaccinated twice on day 23 and three weeks later with an autogenous vaccine containing killed Streptococcus ( S .) suis and A. pleuropneumoniae strains. On average 16.7 live and 1.8 dead born piglets with 15% suckling piglet losses were recorded on the farm in the study year. The piglet´s average weights at weaning were 7 kg and the average daily weight gain in the fattening period 0.9 kg. Section title: Study farm Educational score: 4.166804313659668 Domain: biomedical Document type: Study Language: en Study piglets were selected randomly and stratified by weight and sex out of 17 litters of sows in different farrowing units, which had farrowed on the same day. A total number of 136 piglets out of the 17 litters were included in the study on their second day of life. Sows farrowed in conventional crates on a fully slatted floor. Farrowing was not induced medically, supervised on the main farrowing days from 6am to 10pm and assisted if necessary. The ambient temperature in the farrowing room during farrowing was 26 °C due to warm weather conditions in summer. Piglets had access to a water-heated resting area with a surface temperature of 33 °C. No cross-fostering was performed during the experiment in the 17 litters. In all farrowing pens an automatic replacement milk system was implemented to provide milk supplement from day 5 to 11 of life. Creep feeding started at day 12 of life ad libitum with a supplementary feed as training mash (wet creep feed) provided via the automatic replacement milk system until weaning. Since day 16 of life additional dry creep feed was provided in an extra trough ad libitum until three days after weaning. Since day 3 after weaning creep feeding was paralleled by feeding nursery starter diet ad libitum (Table 1 ). At weaning on day 26 of life piglets were brought litter-wise to the nursery pens with three litters stalled in one pen with place for 33 piglets on fully slatted plastic floor. Pens were equipped with automatic dry feeders with eight feeding places and four nipple drinkers. In addition temporary drinking trays were provided as well as three chains with wooden beams as enrichment objects. Lightweight piglets were collected from different litters and commingled in separate nursery pens. Ambient temperature in the nursery unit was 30 °C for two days at the beginning of the nursery period and decreased to 28 °C until end of the first week. In the following six weeks temperature was decreased by 1 °C to 22 °C until end of nursery. After a seven-week nursery period, pigs were brought to the fattening unit in a neighbouring building with place for ten pigs per pen on fully slatted floors. Water was used from a well and feed for pigs of all production stages was bought from a commercial feed company. Analysis of drinking water showed low nitrate (127 mg/L), low sulphate (107 mg/L) and low iron (< 0.1 mg/L) concentrations, and low number of colony forming units (CFU) of 33–49 CFU/mL after incubation at 22 °C and 17–39 CFU/mL after incubation at 36 °C without any detection of coliforms. Feed iron contents are recorded in Table 1 . Section title: Study farm Educational score: 1.3029855489730835 Domain: biomedical Document type: Other Language: en Husbandry conditions followed the national German regulations on pig husbandry . Section title: Study farm Educational score: 2.951953649520874 Domain: biomedical Document type: Other Language: en Table 1 Declarations of iron content in commercial feed used on the farm for different production stages Milk supplement Wet creep feed Creep feed Nursery starter diet Nursery diet Fattening Feeding period (living days) 5–11 12–28 16–32 32–46 47–80 81–240 Iron content (mg/kg) 75 175 106 140 140 not declared Substance Fe(II)sulfate-monohydrate Fe(II)sulfate-monohydrate, Fe (II)-Glycin-chelat-Hydrat Fe(II)sulfate-monohydrate Recommendation for iron content (mg/kg) 150 1 240 2 175 3 240 2 90-150 2 100–120 4 90-150 2 100-120 4 50–60 4 1 , 2 reviewed by , 3 , 4 Section title: Study design Educational score: 4.162632942199707 Domain: biomedical Document type: Study Language: en From the current farrowing group only litters from those sows were included in the study, which had farrowed on the same main farrowing day. Out of this subgroup of 34 sows 17 sows with at least ten and less than 18 live born piglets were selected. In the study group 88% of the sows were in parity 4–7, one sow had the first and one the ninth litter. Within each of the 17 litters eight healthy piglets (four males and four females if available) weighing at least 1 kg were allocated to four treatment groups by target randomization based on weight and sex strata resulting in two piglets per group in each litter, which were sampled and treated according to Table 2 . For intramuscular (IM) treatment disposable syringes with a maximum volume of 2 mL (HSW HENKE-JECT ® , Henke Sass Wolf, Tuttlingen, Germany) connected to disposable needles (20-gauge, 0.90 × 40 mm (Sterikan ® , B.Braun, Vet Care GmbH, Tuttlingen, Germany) were used. Needles were only used once. Piglets were examined, sampled and weighed at start of the study on day 2 of life, on day 11 of life, at weaning, end of nursery and end of fattening. Ear tagging was performed on day 2 of life. On day 3 of life piglets were tail-docked and on day 6 male piglets were castrated after pain treatment with 0.4 mg meloxicam (Metacam ® , Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany)/kg body weight (BW) and under general anaesthesia by IM injection of 25 mg ketamine (CP-Pharma Handelsgesellschaft mbH, Burgdorf, Germany)/kg BW and 2 mg azaperon (Stresnil ® , Elanco, Kiel Germany)/kg BW. No further zootechnical measures or treatments were performed routinely and no treatment was necessary in piglets of the study group. Section title: Study design Educational score: 2.208937644958496 Domain: biomedical Document type: Study Language: en While piglets of all four groups were treated on day 2 of life, only piglets of group 4 were treated again on day 11 of life. All pigs were weighed and sampled again two days before weaning on day 24 of life, weighed again on day 74 of life at the end of nursery and on day 160 of life at the end of fattening. Section title: Study design Educational score: 4.1369404792785645 Domain: biomedical Document type: Study Language: en Study design was elaborated to prove (i) non-inferiority of IM administration of 200 mg iron dextran (1 mL Uniferon ® ) compared to IM administration of 200 mg iron (III)-gleptoferron and 45 mg toltrazuril (1.5 mL Forceris ® , Ceva Santé Animale, Libourne, France) with respect to the major target variable Hb measured prior to weaning on day 24 of life and (ii) to test if additional amount of iron given on day 2 of life or a repeated iron administration on day 11 of life were beneficial for growth and Hb concentrations. An effect of toltrazuril on gut microflora and therefore indirect on intestinal iron absorption cannot be excluded, so that toltrazuril was given in all experimental groups . Toltrazuril as one component in the combinatory product Forceris ® was orally administered as a singular product (Cevazuril ® , Ceva Santé Animale, Libourne, France) in groups 1, 3 and 4. Similar approaches have been performed previously in other comparative studies . For oral administration a disposable syringe with a maximum volume of 2 mL (HSW HENKE-JECT ® ) were used. The syringe was placed laterally through the piglet`s mouth cleft behind the tongue ground before 0.9 ml Cevazuril ® was applied. The piglet’s mouth was closed with gentle pressure until the product was swallowed. Section title: Study design Educational score: 3.8397839069366455 Domain: biomedical Document type: Study Language: en Table 2 Experimental groups of piglets provided with different iron treatment strategies Group Treatment Sample size ( n ) 1 Single products: 200 mg iron dextran IM, oral application of 45 mg toltrazuril on day 2 34 2 Combinatory product: 200 mg gleptoferron and 45 mg toltrazuril IM on day 2 34 3 Single products: 300 mg iron dextran IM, oral application of 45 mg toltrazuril on day 2 34 4 Single products: 200 mg iron dextran IM, oral application of 45 mg toltrazuril on day 2 and 200 mg iron dextran IM on day 11 34 Within each of 17 litters eight healthy piglets were allocated to the four treatment groups by target randomization based on weight and sex strata resulting in two piglets per group in each litter Section title: Clinical examination and blood analyses Educational score: 2.623784303665161 Domain: biomedical Document type: Study Language: en During the suckling period clinical examination and scoring of skin lesions dorsal on carpal joints and head, claw lesions and navel were performed on day 2, 11 and 24 of life. At the end of nursery on day 74 and at the end of fattening on day 160 of life again a clinical examination and scoring of tail and ear lesions were performed. Clinical variables and their scores are summarized in Table 3 . Section title: Clinical examination and blood analyses Educational score: 4.08535623550415 Domain: biomedical Document type: Study Language: en Blood samples were collected from the Vena cava cranialis in volumes of 2 mL in collection tubes containing ethylenediaminetetraacetic acid (EDTA) (Kabevette ® , Kabe Labortechnik, Mümbrecht-Elsenroth, Germany) using 21-gauge, 0.80 × 40 mm needles (Sterikan ® , B.Braun). Blood samples were placed in an isolated box with cool pads until end of sampling, transported to the laboratory at the Clinic for Swine and Small Ruminants, University of Veterinary Medicine, Hannover, stored at 6 °C overnight and analysed within 24 h. Haematological variables (Hb, packed cell volume (PCV), erythrocytes, mean corpuscular haemoglobin concentration (MCHC), leucocyte count, thrombocyte count) were automatically analysed in a haematology analyser . Section title: Clinical examination and blood analyses Educational score: 3.853902578353882 Domain: biomedical Document type: Study Language: en Table 3 Clinical variables recorded on days 2, 11, 24, 74 and 160 of life Variables Clinical scores 0 1 2 3 4 5 Claws No claw lesions Pododermatitis haemorrhagica Panaritium - - - Skin lesions head No skin lesions Slight and superficial scratches Dominant red scratches Wet wounds - - Skin dorsal on carpal joints No skin lesions Slight and superficial scratches and crusts Extended crusts Wet wounds - - Navel 1 Physiological diameter and consistency Diameter 0.8–1.5 cm Diameter > 1.5 cm Wet navel Purulent secretion or abscess Hernia umbilicalis Diarrhoea No Pulpy feces Liquid feces - - - Ear Intact skin Dry scratches Bloody wound - - - Tail lesion Intact skin Dry scratches Bloody wound - - - Tail losses Length after tail docking ≥ half length of tail ≥ two third of tail Total tail loss - - Clinical scores were used in suckling piglets for skin lesions dorsal on carpal joints and head, claw lesions and navel. In nursery and fattening pigs tail and ear lesions were scored. All pigs were examined for any other deviations from physiological findings 1 only suckling piglets Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 4.159938335418701 Domain: biomedical Document type: Study Language: en A sample size calculation for a non-inferiority t-test between experimental groups 1 (200 mg iron dextran) and 2 (200 mg gleptoferron) was performed using NCSS PASS with target power (1-β) 0.8 and target significance level α of 0.05. The calculation was based on a non-inferiority assumption according to expected Hb concentrations as the major target variable deduced from results in three published comparison trials [ 32 – 34 ]. According to these studies a difference of 5 g Hb/L between treatment groups was considered equivalent and defined as a margin for non-inferiority testing between different schemes of iron administration in this study (Table 2 ). Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 2.806947946548462 Domain: biomedical Document type: Study Language: en Sample size calculation resulted in 29 piglets per group, i.e. in total 116 piglets. Due to an average suckling piglet mortality of 15% in the trial farm, the required number of piglets was adjusted by five reserve animals per group resulting in 136 piglets to be included in trial. Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 1.620261788368225 Domain: biomedical Document type: Other Language: en Data were recorded in Excel, version 2016 (Microsoft Corporation, Albuquerque, USA) and imported for statistical evaluations into SAS ® , version 9.4 TS level 1M5 (SAS Institute, NC, USA). Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 3.5659663677215576 Domain: biomedical Document type: Study Language: en Spearman`s rank correlation coefficients were calculated for all variables with a quantitative outcome. Frequencies of clinical findings were compared by chi-square testing between the groups. Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 4.071544647216797 Domain: biomedical Document type: Study Language: en For the main primary hypothesis under study non-inferiority testing was performed to prove that treatment of piglets in group 1 (200 mg iron dextran) is non-inferior to treatment of piglets in group 2 (gleptoferron) with respect to Hb at weaning. A boundary threshold of 5 g/L Hb was chosen for this test (see above). Procedure of non-inferiority testing was based on Schuirmann’s method of two one-sided tests (TOST). Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 3.445122003555298 Domain: biomedical Document type: Study Language: en Within explorative analyses for secondary endpoints group differences with respect to red blood cell measurements and weight gain were tested in an analysis of variance model followed by least square means tests for group effects. Due to the explorative nature of these analyses no multiple adjustments were conducted. Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 3.9633054733276367 Domain: biomedical Document type: Study Language: en In addition, it was explored if treatment effects were influenced by birth weights. To include the potential effects of birth weight in the model, piglets were allocated to three birth weight classes. Three weight groups were defined, i.e. “low weight piglets” from the minimum to the 25% quantile, “medium weight piglets” from the 25% to the 75% quantile, “high weight piglets” from the 75% quantile to the maximum. Allocation of animals by birth weight class and treatment to the groups are shown in Table 4 . General linear models with fixed effects group and birth weight class and the combined effects of both were analysed with respect to red blood cell variables at weaning. Section title: Sample size calculation for a non-inferiority study and statistical evaluation Educational score: 3.454331159591675 Domain: biomedical Document type: Study Language: en Table 4 Number of piglets in experimental groups and range of body weights Experimental groups Group 1 Group 2 Group 3 Group 4 Weight classes Body weight (kg) n Body weight (kg) n Body weight (kg) n Body weight (kg) n Weight class 1 (low) 1.14–1.48 9 1.12–1.48 7 1.26–1.46 9 1.10–1.48 10 Weight class 2 (medium) 1.52–1.94 16 1.50–1.94 20 1.50–1.94 14 1.56–1.94 14 Weight class 3 (high) 1.98–2.20 8 2.02–2.30 7 1.96–2.46 8 1.96–2.36 9 Number of piglets allocated in experimental groups and weight classes: sample size and range of weights at study start on day 2 of life, n: number of piglets within weight class Section title: General clinical and laboratory findings Educational score: 4.205248832702637 Domain: biomedical Document type: Study Language: en The 17 sows included in the study had in average 15.6 live born, 8.9 dead born and 14.4 weaned piglets. The suckling piglet mortality was 7.5% in the litters of the study group. During the suckling period four animals out of the study groups were euthanized and sent for necropsy. Due to chronic disease (starvation, arthritis, ascites) these pigs were considered as biological outliers and were excluded from the final statistical examination. One additional pig with severe arthritis was excluded from the study. All injection sites were inconspicuous in all animals during the whole course of the study. In the majority of suckling piglets, a slight pododermatitis haemorrhagica (reddening of the heels) was found, while only one animal showed a panaritium (swelling, reddening and pain sensitivity of the coronary skin) on day 24 of life. Head skin lesions were mainly superficial scratches on day 2 of life. About 30% of the animals showed more severe head skin lesions (score 2 and 3) on day 11 of life. Skin lesions dorsal on carpal joints were as well most severe on day 11 of life with 7.6% of pigs showing open wounds. Most dorsal carpal skin wounds were superficial. Navel inflammations decreased during the suckling period (Table 5 ). Two pigs were diagnosed with umbilical hernia. A severe purulent inflammation of the navel was found on day 11 in three animals. The incidence of diarrhoea was very low during the whole suckling period and was not treated. During nursery a severe outbreak of disease caused by S . suis led to animal losses. During the second half of the nursery period a severe tail biting outbreak occurred. In total, due to animal losses and loss of ear tags, 42 animals were excluded from further evaluation. Among the remaining 89 pigs at the end of nursery tail injuries were found in more than half of the pigs. Due to ear tag losses only 76 pigs were identified and examined at the end of fattening. Acute tail wounds were diagnosed still in 5.3% of pigs at the end of fattening and 6.6% of the pigs had a total loss of tail. A summary of clinical findings is shown in Table 5 . There were no differences in frequencies of various clinical findings between different treatment groups. Section title: General clinical and laboratory findings Educational score: 3.8292148113250732 Domain: biomedical Document type: Study Language: en Table 5 Selected clinical findings in all examined pigs (%) in the respective stage of production Proportion of pigs with clinical findings (%) Day of life 2 ( n = 131) 11 ( n = 131) 24 ( n = 131) 74 ( n = 89) 160 ( n = 76) Pododermatitis haemorrhagica 64.9 66.4 3.1 - - Head skin lesions (score 2 and 3) 0.8 30.5 5.3 - - Dorsal carpal joint skin lesions (score 2 and 3) 0.0 36.6 58.0 - - Navel inflammation 27.5 24.4 14.5 - - Diarrhoea 3.8 1.5 0.8 - - Tail injuries - - - 55.1 5.3 Tail losses - - - - 14.5 Bloody ear injuries - - - 18.0 0.0 Selected clinical findings in % from all examined pigs (n) in the respective stage of production until end of fattening irrespective of treatment group Section title: General clinical and laboratory findings Educational score: 2.9471819400787354 Domain: biomedical Document type: Study Language: en Piglets included in the study had a relatively high average birth weight of 1.7 kg and an adequate average daily weight gain of 0.460 kg during the nursery period. Growth rates during fattening were in average 0.926 kg resulting in average heavy weights at slaughter of 111 kg in the study pigs. Body weight development in the different treatment groups is shown in Table 6 . Section title: General clinical and laboratory findings Educational score: 4.187612533569336 Domain: biomedical Document type: Study Language: en Table 6 Body weight and average daily weight gain (ADWG) in different treatment groups (mean ± standard deviation) Group 1 Group 2 Group 3 Group 4 Day of life n Body weight (kg) n Body weight (kg) n Body weight (kg) n Body weight (kg) 2 33 1.71 ± 0.32 34 1.72 ± 0.3 31 1.71 ± 1.86 33 1.69 ± 0.34 24 33 6.85 ± 1.45 34 6.62 ± 1.57 31 6.61 ± 1.86 33 6.61 ± 1.81 74 23 31.17 ± 6.36 23 30.15 ± 5.57 20 29.78 ± 5.92 23 29.02 ± 7.52 160 17 116.82 ± 12.65 20 110.13 ± 20.07 19 109.29 ± 14.99 20 108.23 ± 22.65 Group 1 Group 2 Group 3 Group 4 n ADWG (kg) n ADWG (kg) n ADWG (kg) n ADWG (kg) Suckling period (day 2–24) 33 0.23 ± 0.06 34 0.22 ± 0.07 31 0.22 ± 0.07 33 0.22 ± 0.08 Nursery period (day 24-day 74) 23 0.50 ± 0.12 23 0.48 ± 0.10 20 0.47 ± 0.10 23 0.46 ± 0.13 Fattening period (day 74–160) 17 0.97 + 0.19 20 0.91 + 0.19 19 0.92 + 0.13 20 0.91 + 0.2 Suckling, nursery and fattening period (day 2-160) 17 0.73 ± 0.08 20 0.68 ± 0.19 19 0.68 ± 0.09 20 0.67 ± 0.14 Section title: General clinical and laboratory findings Educational score: 4.13072395324707 Domain: biomedical Document type: Study Language: en On day 2 of life 33 pigs (25%) and at weaning only 2 pigs showed Hb values below 90 g/L. Both anaemic piglets at weaning suffered either from purulent navel inflammation or an umbilical hernia. Blood cell parameters are visualized in Figs. 1 , 2 , 3 , 4 and 5 and summarized in Table 7 . Manifold correlations between red blood cell parameters were found (data not shown). Hb at weaning showed a significant correlation with body weight at weaning ( P < 0.01) and the ADWG in the suckling period ( P = 0.002). The PCV at weaning was significantly correlated with weight at weaning, at end of nursery, at end of fattening and ADWG ( P < 0.01). MCHC at weaning was significantly negatively correlated with weight at weaning, at end of nursery, at end of fattening and ADWG ( P < 0.01). Section title: General clinical and laboratory findings Educational score: 2.7870309352874756 Domain: biomedical Document type: Study Language: en Fig. 1 Haemoglobin concentrations (g/L) in piglets belonging to different treatment groups and day of life Blue boxes: 2nd day of life, study start, red boxes: 24th day of life at weaning; different letters right to the boxes indicate significant differences between respective groups 1 and 4 ( P = 0.04) in a least square means comparison Section title: General clinical and laboratory findings Educational score: 4.191370010375977 Domain: biomedical Document type: Study Language: en Table 7 Mean (±standard deviation) haematological measures in different groups on day 2 and 24 of life Day of life Group 1 ( n = 33) Group 2 ( n = 34) Group 3 ( n = 31) Group 4 ( n = 33) Haemoglobin (g/L) 2 95.4 (±11.1) 95.2 (±11.9) 98.4 (±8.5) 97.7 (±11.5) 24 120.6 (±8.5) 121.8 (±11.6) 123.4 (±13.3) 126.3 (±11.1) Packed cell volume (L/L) 2 0.28 (±0.03) 0.30 (±0.11) 0.29 (±0.03) 0.29 (±0.04) 24 0.38 (±0.03) 0.38 (±0.04) 0.38 (±0.04) 0.39 (±0.04) Mean corpuscular haemoglobin concentration (g/L) 2 335 (±11) 331 (±42) 339 (±15) 338 (±23) 24 321 (±11) 323 (±12) 326 (±11) 325 (±10) Red blood cell count (10 12 /L) 2 4.53 (±0.65) 4.42 (±0.52) 4.61 (±0.47) 4.55 (±0.661) 24 5.92 (±0.45) 5.78 (±0.51) 5.88 (±0.52) 5.93 (±0.44) Thrombocyte count (10 9 /L) 2 199 (±68) 221 (±61) 214 (±65) 206 (±50) 24 466 (±85) 442 (±72) 464 (±87) 434 (±107) Leucocyte count (10 9 /L) 2 8.05 (±1.77) 8.23 (±2.17) 8.09 (±1.83) 8.02 (±1.75) 24 13.93 (±4.47) 14.11 (±4.67) 13.88 (±3.55) 14.33 (±5.17) Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 4.046794891357422 Domain: biomedical Document type: Study Language: en To underline the equivalence of treatments a non-inferiority test was conducted (TOST) as described. Based on statistical results shown in Table 8 the non-inferiority testing resulted in a P-value of 0.008, so that the first hypothesis, that iron dextran is non inferior to gleptoferron with respect to Hb at weaning was confirmed. Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 4.090306282043457 Domain: biomedical Document type: Study Language: en Table 8 Descriptive outcome of non-inferiority testing (TOST) with regard to haemoglobin concentrations (g/L) at weaning Group N Mean (±standard deviation) Mean 95% confidence limits 1 33 120.6 (±8.5) 117.6 123.7 2 34 121.8 (±11.6) 117.8 125.8 Difference group 1–2 (Satterthwaite) -1.16 -6.12 3.79 Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 4.1238298416137695 Domain: biomedical Document type: Study Language: en In a second explorative analysis the four treatment groups were compared with respect to red blood cell variables and weight gain. The analysis of variance models with fixed effect group did not result in any statistical significant group effect on variables listed in Tables 6 and 7 . A group wise least square means comparison resulted in a significantly higher Hb concentration at weaning ( P = 0.04) in piglets of group 4 (twice administration of 200 mg iron dextran on day 2 and day 11) compared to piglets of group 1 (one administration of iron dextran on day 2) as shown in Fig. 1 . No significant group effect was found for PCV , erythrocytes , leucocytes or thrombocytes . Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 2.2088816165924072 Domain: biomedical Document type: Study Language: en Fig. 2 Packed cell volume (L/L) in piglets belonging to different treatment groups and day of life. Blue boxes: 2nd day of life, study start, red boxes: 24th day of life at weaning Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 2.1975362300872803 Domain: biomedical Document type: Study Language: en Fig. 3 Erythrocytes (T/L) in piglets belonging to different treatment groups and day of life. Blue boxes: 2nd day of life, study start, red boxes: 24th day of life at weaning Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 2.2308945655822754 Domain: biomedical Document type: Study Language: en Fig. 4 Leucocytes (G/L) in piglets belonging to different treatment groups and day of life. Blue boxes: 2nd day of life, study start, red boxes: 24th day of life at weaning Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 2.234049081802368 Domain: biomedical Document type: Study Language: en Fig. 5 Thrombocytes (G/L) in piglets belonging to different treatment groups and day of life. Blue boxes: 2nd day of life, study start, red boxes: 24th day of life at weaning Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 4.131636619567871 Domain: biomedical Document type: Study Language: en Finally, data exploration targeted to reveal different treatment effects in different birth weight classes of piglets. The two-way analysis of variance for the dependent variable Hb at weaning with fixed effects treatment group and weight class revealed no statistically significant effects. Visualization in an interaction plot hints towards countervailing effects as of Hb was affected in different ways by different treatments within the different weight classes . While lowest and highest birth weight classes represent 25% of data each including extreme values, the median birth weight group represents the interquartile range and therefore 50% of the data without extreme values. In this subgroup a treatment response becomes visible with higher Hb concentrations in pigs treated on day 2 and day 11 of life with a full dose of iron dextran . In these medium weight piglets (interquartile weights at study start) significant differences in treatment effects on Hb at weaning were found between group 1 and 4 ( P = 0.01) as well as between group 2 and 4 ( P = 0.03). A significant difference in PCV at weaning was found between group 1 and 4 ( P = 0.02). These findings reflect an effect of the repeated iron treatment on day 11 . Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 2.8779618740081787 Domain: biomedical Document type: Study Language: en Fig. 6 Interaction plot of different birth weight classes and treatment groups with effect on Hb concentrations. Haemoglobin (Hb) concentrations of individual piglets at weaning are depicted by symbols of the respective birth weight classes. Coloured lines indicate the mean Hb concentrations of the different birth weight classes in the different treatment groups at weaning Section title: Comparing treatment groups – equivalence and explorative comparisons Educational score: 3.3531882762908936 Domain: biomedical Document type: Study Language: en Fig. 7 (a) Haemoglobin concentration (g/L) and (b) Packed cell volume (L/L) at weaning in medium weight piglets Medium weight piglets (1.48–1.94 kg body weight at study start) of group 1 and 4 differed in Hb concentration and PCV. Different letters right to the boxes indicate significant differences between respective groups Section title: Discussion Educational score: 4.587457180023193 Domain: biomedical Document type: Review Language: en Iron (III)-hydroxide-dextran complex (Uniferon ® , Pharmacosmos A/S, Holbaek, Denmark) is an injectable 20% iron dextran for prevention and treatment of anaemia in swine. In spite of the standard dose of 200 mg iron administered routinely to piglets within the first days of life, IDA can develop due to the high growth rates of modern crossbred pigs and especially in heavy pigs . Next to anaemia, iron deficiency can negatively impact organ functions as especially the intestinal absorption and digestion capacities, due to e.g. destruction of tight junctions and decrease in villus height . The benefit of a second iron dose was therefore examined in several studies, mostly resulting in increased Hb concentrations at weaning, while contradictory results were found for an effect on ADWG . This might be due to known detrimental effects of routine iron bolus administrations, which can be of significance for piglet performance under certain factor constellations. Some authors therefore see no advantage in an additional iron dosage later in the suckling period . Several studies have focused on toxic effects of iron and the beneficial effects of split iron dosages to maintain the tightly balanced physiological iron metabolism . Hepcidin increased significantly after intramuscular administration of 150 mg Fe/kg BW on day three of life, which was not shown with lower doses of 35.5 mg Fe/kg BW given twice on day three and 14 of life . High hepcidin concentrations can block ferroportin with the consequence of lower iron utilization from external sources as creep feed or soil . In one study, the administration of less iron in the first days of life (40 mg) was adequate to improve iron status of piglets without severely increasing the hepcidin expression. A split iron supplementation in piglets was therefore recommended to minimize DNA damage and oxidative stress . These manifold different study results indicate that different protocols in iron administration might be suitable. Next to genetically defined growth intensity real iron requirements for piglets are dependent on several other farm-specific factors as e.g. blood loss during zootechnical measures, iron sources in the environment and health status of piglets. Therefor suitable protocols in iron administration might differ between farms, groups and individuals . The exact need for iron supplementation is considered to be farm specific due to various factors influencing iron availability and requirements, so that diagnosis of IDA should be based on laboratory diagnostical findings . Any parenteral iron supplementation should always target at a balance between the benefit of anaemia prevention and risk related to oxidative damage. These two sides of iron supplementation in piglets are still controversially discussed . Section title: Discussion Educational score: 4.227662086486816 Domain: biomedical Document type: Study Language: en In this study, flexible iron dosages and two iron sources (iron dextran and gleptoferron) were compared. Iron dextran was non-inferior to gleptoferron in a combinatory product with respect to Hb concentrations at weaning. Following the non-inferiority concept, if there are benefits in dosage flexibility and costs - while the efficacy is not worse (non-inferior) than gleptoferron in a combinatory product - iron dextran is a reasonable and effective treatment. In the past, comparative studies between both iron sources came to different results. In a study with piglets out of 26 litters no differences in red blood parameters or ADWG were found between pigs treated with iron dextran or gleptoferron . In two recent comparative studies with piglets from only four litters each, differences in various parameters were recorded, which were difficult to interpret due to a lack of sample size justification. In the first study, a comparison between the administration of 200 mg gleptoferron and 200 mg iron dextran in 25 piglets resulted in significant differences in mean Hb concentrations at weaning of 113 g/L in piglets treated with gleptoferron and 101 g/L in piglets treated with iron dextran . Comparable to our study on day 2 of life 28% of the piglets had Hb concentrations < 90 g/L. On day 18 Hb values were all above 90 g/L and higher than at weaning on day 31, which was 7 days later than in our study. In the second study with 32 piglets mean Hb concentrations of 114 g/L at weaning did not differ between piglets treated with 200 mg gleptoferron or iron dextran, while mean concentration-time profiles of iron in serum were significantly increased in the gleptoferron group . From serum iron levels no beneficial effect of iron administrations for the piglets can be deduced, because any bolus iron injection is reflected by increased tissue hepcidin expression as the systemic iron-regulatory hormone . In case that more iron is supplemented than can be bound by transferrin in serum, free toxic iron can catalyze reactive oxygen species . The intracellular iron storage protein ferritin in serum was also compared between the treatment groups but can also be interpreted as an inflammation marker due to its function as an acute phase protein . For this reason, in our study Hb was defined as the target variable. Study design and number of pigs examined in our study differed from those in the cited comparative studies. No differences in ADWG were observed in the cited comparative studies, which is in accordance with our study. A higher dosage of iron on day 2 of life or a second full dosage of iron on day 11 of life had unexpectedly minor effects in our study. A second iron dose led to higher Hb concentrations but not ADWG at weaning. This outcome was similar to other studies, as e.g. in a comparison of gleptoferron injections at different points of time and oral iron supplementation, which led to different Hb concentrations but not ADWG between pigs with one or two iron injections . A second iron dosage of 100 mg on day 11 of life did neither improve red blood cell parameters nor ADWG at weaning on day 21 in a study testing different iron dosages . In contrast to that, in a recent study in organic pig production piglets profited clearly from a second dose of iron on day 14 of life due to the longer suckling period and subsequently later iron intake from feed . Section title: Discussion Educational score: 4.165380477905273 Domain: biomedical Document type: Study Language: en If ADWG can be increased by additional administration of iron is still a matter of debate and might depend on several factors, as availability of iron from the sows, blood loss during zootechnical measures, start and iron content of creep feed. Different studies come to different results as reviewed previously . A dose-dependent effect of iron dosage on ADWG in the suckling period was found up to a dosage of 100 mg iron on day 3 after birth and for up to 200 mg in the nursery period . In most studies, no effect of an additional iron administration next to a primary dose of 200 mg on ADWG in the suckling period was found [ 43 – 45 ]. In some studies, the effect of a second iron dosage in the suckling period was only significant on ADWG in the post-weaning period . This is not in accordance with findings in our study. In the post-weaning period several pigs suffered from S.-suis -related disease and in a later time period pig health was impacted by a tail-biting outbreak. Both diseases lead to inflammatory responses with influence on growth rates . Piglets with different birth weights and treated with the same dose of iron responded differently to iron provided in the nursery diet and the effect of a second iron injection was also influenced by red blood cell parameters at the time point of second iron injection . It can be hypothesized, that the significant effect of the second iron administration in medium weight piglets in our study might be due to a different Hb concentration at the respective time point in this group. Although pigs of different weight classes did not differ in their red blood cell parameters on their second day of life, differences can be expected on day 11 of life. Iron supply by only one injection in low weight piglets with lower ADWG might had been adequate, while high weight piglets might have profited from a second dose of iron resulting in higher Hb concentrations in the post-weaning period not examined in this study. For the medium weight piglets the beneficial effect of a second iron dose on Hb concentration was already detectable at weaning. In general, a positive association between ADWG in the post-weaning period and the Hb concentration at weaning exists . Also in our study several red blood cell parameters were positively correlated with the ADWG. Section title: Discussion Educational score: 4.1094136238098145 Domain: biomedical Document type: Study Language: en In a recent study, a second iron injection resulted in a 4% increase in ADWG from weaning to slaughter . In that study, piglets had no access to creep feed during the suckling period and diets contained less iron (100 mg/kg) than diets in our study (Table 1 ). Mean Hb concentrations at weaning in pigs with only one iron injection (107 g/L) were lower than in our study (group 1 and 2: 121 ± 10 g/L), where piglets had access to external iron sources in milk supplement and creep feed. In our study, iron content in creep feed was below 240 mg/kg as a published recommendation , but was within the range of iron concentrations in published creep feed studies . Creep feed containing 175 mg iron/kg and fed from day 7 after birth was found to be adequate for good growth rates irrespective of parenteral iron administration . Section title: Discussion Educational score: 4.2321343421936035 Domain: biomedical Document type: Study Language: en Voluntary iron intake by creep feed with appropriate iron content is considered an important iron source during the suckling period allowing iron absorption precisely regulated according to the needs of the piglets . On the other hand, the regulation of intestinal iron absorption is not fully functional before 5–6 weeks of age . Physiologically restricted iron absorption in the gut in young piglets is a protective mechanism to avoid injury of the intestinal barrier and negative influence on the gut microbiota favouring growth of pathogenic bacteria . Any disturbance of gut development and gut flora, which can be due to a lack of colostrum especially in large litters, can have severe consequences for piglets’ health, especially in presence of pathogenic microorganisms. As pathogens require iron for their metabolism, any iron supplementation can interfere with the iron regulation of the host based on internalization of iron in intracellular compartments during inflammation . In our study group the average numbers of live born and weaned piglets were in the upper range of piglet producing farms with high herd productivity in Europe . Large litters need intensive care in the suckling period, usually including cross-fostering, which was not performed in this study . The high proportion of pigs with head and carpal joint lesions in our study indicated increased teat fighting although milk supplement was provided. This finally did not negatively impact the suckling piglet mortality in the litters of the study group, so that management of these large litters can be assessed as successful in this farm. Nevertheless, the ADWG of 226 ± 69 g in the study pigs during the suckling period was relatively low . Next to birth weight as the most important factor various factors related to sow, pen, litter and piglet have impact on ADWG in suckling piglets . It was reported that foreleg lesions in piglets can affect weight gain in piglets. In our study carpal joint lesions were 37% in the second week of life, which is due to intense teat stimulation in the large litters with high competition. It can be assumed, that more cross fostering resulting in no more than one piglet per functional teat in a litter would have led to less carpal joint lesions and a higher ADWG. In three herds with ADWG in piglets ranging from 204 to 210 g sow-related factors had a significant impact on ADWG, as e.g. poorly milking sows three weeks after farrowing . These sow-related factors were not analysed in our study in detail, but all sows were always in a good health status. S. suis -related disease was responsible for 7.5% piglet mortality in the nursery period, so that an inadequate colostrum supply due to the large litter sizes might have been a potential cofactor for the detrimental course of disease. Skin lesions at the head and carpal joints in weaned piglets were additional risk factors for entry of S. suis and subsequent bacteriaemia. A post-weaning ADWG of 475 ± 132 g in surviving pigs was within a physiological range . Section title: Discussion Educational score: 3.809727191925049 Domain: biomedical Document type: Review Language: en To find a compromise between benefits and drawbacks of additional iron supplementation, new and flexible iron supply strategies are necessary taking different pig breeds, husbandry and management conditions into account. The advantage of combinatory products addressing anaemia and coccidiosis by one single shot can be counteracted by the lack of adjustment of the necessary amount of iron to prevent anaemia as long as possible. Additional scientific data are necessary to reassess iron supplementation strategies in pigs and other livestock animals with respect to the double-edged character of this element. Section title: Conclusions Educational score: 4.160741329193115 Domain: biomedical Document type: Study Language: en In large litters of hyperprolific sows, a high heterogeneity of piglet birth weights is accompanied by a high growth potential resulting in varying demands for iron between individuals. While some individuals might benefit from 200 mg Fe, heavier piglets might be undersupplied, and smaller piglets might be burdened by this amount of iron due to its toxic effects. In our study iron dextran as a traditional substance was non-inferior to gleptoferron in a combinatory product. Iron dextran can be used in flexible dosage and treatment schemes according to varying requirements on different farms as an alternative product in case that no treatment against coccidiosis is necessary. In the study farm, Hb was increased at weaning by a second full dose of iron on day 11 of life, but without improvement of ADWG. Differences in published study outcomes about the effects of additional iron dosages might in part depend on piglet`s access to creep feed containing iron already in the suckling period superimposing the effect of additional parenteral iron administration. The consequences of metabolic changes by iron administration balancing its benefits should be addressed in further clinical studies comparing different protocols of iron administration. | Study | biomedical | en | 0.999999 |
PMC11699721 | Section title: Introduction Educational score: 2.5075201988220215 Domain: biomedical Document type: Other Language: en Beta-blockers are widely used in the treatment of various conditions, including hypertension, 1 cardiac arrhythmias, 2 migraines, 3 and certain cardiovascular diseases such as Marfan syndrome. 4 Their prescription may also be necessary during pregnancy, primarily for pre-existing chronic conditions. Section title: Introduction Educational score: 3.9696948528289795 Domain: biomedical Document type: Review Language: en Prescribing beta-blocker treatments during pregnancy raises concerns due to their potential effects on both the mother and the fetus. Indeed, beta-blockers can interfere with the physiological adjustments necessary for pregnancy: increased cardiac output, heart rate, and blood volume, and decreased peripheral vascular resistance. 5 Previous studies have suggested several potential adverse effects associated with the use of beta-blockers during pregnancy. These adverse effects include increased risks of intrauterine growth restriction, 6 neonatal hypoglycemia, 7 neonatal bradycardia, 7 hypotension, 8 and prolonged neonatal hospitalization. 9 Section title: Introduction Educational score: 4.106557846069336 Domain: biomedical Document type: Study Language: en Due to their inhibitory action on beta-adrenergic receptors, beta-blocker treatment can lead to a decrease in maternal heart rate. This hemodynamic change in the mother raises questions about the treatment's impact on fetal heart rate. 10 An in vivo study in mice showed that administration of propranolol resulted in a decrease in fetal heart rate associated with an increase in heart rate variability, whereas saline had no effect on either mothers or fetuses. 11 These results, which seem contradictory at first glance, testify to the lack of knowledge about beta-blocker pharmacology, particularly during pregnancy. Any potential alteration in fetal heart rate in humans could have significant implications for obstetric monitoring and particularly for the interpretation of fetal heart rate monitoring. Indeed, a significant variation in fetal heart rate can be an indicator of fetal distress, requiring rapid medical intervention to prevent serious complications such as fetal asphyxia and hypoxia. 12 Due to the underlying maternal conditions for which beta-blockers are prescribed, there is an increased risk of fetal distress. In this context, accurate interpretation of fetal heart rate monitoring becomes even more crucial. Section title: Introduction Educational score: 4.04840087890625 Domain: biomedical Document type: Study Language: en This study aims to determine whether the use of beta-blockers during pregnancy alters the mean fetal heart rate among patients treated with beta-blockers, compared to fetuses whose mothers receive no treatment. Section title: Methods Educational score: 3.4325692653656006 Domain: biomedical Document type: Study Language: en For this study, we conducted a retrospective case-control study involving 90 patients, divided into two groups of 45 patients each. The first group consisted of pregnant patients treated with beta-blockers, while the second group was comprised of pregnant women receiving no treatment. Section title: Methods Educational score: 2.640706777572632 Domain: biomedical Document type: Study Language: en The data collected included all patients on beta-blockers who gave birth after 24 weeks of gestation to a singleton pregnancy in two hospital-university maternity units in Ile de France, specialized in the management of maternal pathologies, particularly cardiac, between 2009 and 2021. Data were collected from the patients’ computerized medical records and anonymized. Section title: Methods Educational score: 2.332965612411499 Domain: biomedical Document type: Study Language: en Between the two groups, patients were matched based on age, parity, and gestational age at delivery to minimize potential biases associated with these factors. The control patient was the first patient corresponding to these matching criteria in our database and not already included in the study. Section title: Methods Educational score: 3.128669023513794 Domain: biomedical Document type: Study Language: en Patients on Labetalol only and those treated with Propranolol for Graves’ disease were excluded from the analysis, as the mechanism of action of Labetalol (a noncardio-selective alpha/beta-blocker) and the impact of Graves’ disease on maternal and fetal heart rates 13 could introduce biases into our results. Section title: Methods Educational score: 4.129446506500244 Domain: biomedical Document type: Study Language: en The demographic characteristics (age, parity, and body mass index) of the patients are presented in Table 1 . Information regarding the type of beta-blocker treatment, its indication, and dosage were extracted from medical records and presented in Table 2 . In case of changes in dosage during pregnancy, the highest dosage was retained. Table 1 Main characteristics of untreated patients and patients with beta-blockers Table 1 Untreated patients n =45 (%) Patients with beta-blockers n =45 (%) P Age, median (IQR) 31 (27–34) 31 (27–34) 1 Parity I parous 25 (56) 25 (56) 1 II parous 18 (40) 18 (40) III parous 2 (4.4) 2 (4.4) BMI (kg/m 2 ) <18.5 2 (4.4) 9 (20) 0.11 18.5–24.9 23 (51) 24 (53) 25–29.9 11 (24) 5 (11) 30–34.9 5 (11) 2 (4.4) 35–39.9 0 (0) 1 (2.2) >40 2 (4.4) 1 (2.2) Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. Table 2 Details of treatment received by patients Table 2 Treatment (INN) n (%) Daily dose mg Indication Atenolol 14 (31) 50–100 mg Marfan syndrome 14 Bisoprolol 21 (46) 2.5–10 Marfan syndrome 18 Others 3 Labetalol 1 (2) 200 Marfan syndrome Nebivolol 1 (2) 10 Marfan syndrome Propranolol 8 (17) 40–120 mg Marfan syndrome 2 Others 6 Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. Section title: Methods Educational score: 4.07817268371582 Domain: biomedical Document type: Study Language: en The evaluation of mean fetal heart rate was performed by reading the fetal heart rate monitoring and manually assessing the baseline heart rate on the day of delivery, during labor, or before scheduled C-section, depending on the obstetric context. Situations of suspected intrauterine infection or maternal fever were excluded due to their potential influence on fetal heart rate. It was classified into three categories: less than 110 bpm, between 110 and 150 bpm, and greater than 150 bpm, following the usual definition of normal fetal heart rate. 14 Section title: Methods Educational score: 3.37520694732666 Domain: biomedical Document type: Study Language: en Maternal heart rate on the day of delivery was also recorded from the fetal monitoring recordings or surveillance sheets in the delivery room or operating room. This rate was categorized into three categories for statistical comparison: less than 60 bpm, between 60 and 100 bpm, and greater than 100 bpm. Section title: Methods Educational score: 2.9550609588623047 Domain: biomedical Document type: Study Language: en Regarding obstetric data, gestational age and mode of delivery were recorded. For neonatal data, fetal weight, and birth percentile according to Audipog curves 15 were noted. Section title: Methods Educational score: 3.937929391860962 Domain: biomedical Document type: Study Language: en A descriptive analysis was conducted to describe the characteristics of the study population. Quantitative variables are expressed as mean with confidence interval or median with interquartile range, while qualitative variables are expressed as number and percentage. Comparison of qualitative variables was performed using a Chi-squared test or Fisher's exact test when assumptions were not met, and means were compared using Student's t test. An alpha risk of 5% and a significance level of 0.05 were considered in all analyses. Data were processed using R software via the P value interface. 16 Section title: Methods Educational score: 1.0311405658721924 Domain: biomedical Document type: Other Language: en The study was approved by an ethics committee . 17 Section title: Results Educational score: 1.9957431554794312 Domain: biomedical Document type: Study Language: en In terms of demographic characteristics, there was no significant difference in age between the two groups, with a median age of 31 years. As shown in Table 1 , the two groups were also similar in terms of parity and body mass index. Section title: Results Educational score: 3.9253110885620117 Domain: biomedical Document type: Study Language: en For the treatment received by patients on beta-blockers, as depicted in Table 2 , a large majority were treated for Marfan syndrome, accounting for 80% of the cohort ( n =36/45). Other indications for treatment included sinus tachycardia ( n =2), migraine pathology ( n =2), and cardiomyopathy ( n =2). One patient was treated due to dilated cardiomegaly, another for rheumatic mitral insufficiency, and the last for Bouveret-Hoffmann syndrome. Section title: Results Educational score: 3.9960720539093018 Domain: biomedical Document type: Study Language: en Bisoprolol was the most frequently prescribed treatment ( n =21, 46%), at doses ranging from 2.5 to 10 mg per day, followed by atenolol, mainly for Marfan syndrome. Notably, the majority of patients, 37/45 (82%), were already on beta-blocker treatment before pregnancy. Two patients (4.4%) started beta-blockers during the first trimester of pregnancy, two (4.4%) during the second trimester, and 4 (8.8%) during the third trimester. One patient received bisoprolol followed by labetalol due to poor tolerance. Section title: Results Educational score: 4.140109062194824 Domain: biomedical Document type: Study Language: en The main analysis of the study, presented in Table 3 , revealed no significant difference in the distribution of mean fetal heart rate between the two groups across the three categories mentioned earlier. Most fetuses, regardless of maternal beta-blocker use, had heart rates between 110 and 150 beats per minute, with similar percentages in both groups (87% vs 93%, respectively), P =.71. These findings suggest that beta-blocker use does not significantly impact the average fetal heart rate. Table 3 Maternal and mean fetal heart rate the day of delivery among untreated patients and patients with beta-blockers Table 3 Colonne1 Untreated patients n =45 (%) Patients with beta-blockers n =45 (%) P Maternal heart rate <60/min 1 (2.2) 7 (18) <.01 60–100/min 37 (82) 30 (79) >100/min 7 (16) 1 (2.6) Mean fetal heart rate <110/min 2 (4.4) 0 (0) .71 110–150/min 39 (87) 27 (93) >150/min 4 (8.9) 2 (6.9) Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. Section title: Results Educational score: 4.080177307128906 Domain: biomedical Document type: Study Language: en However, a significant difference was observed in maternal heart rates between the two groups. There was a notably higher prevalence of maternal heart rates above 100 beats per minute in untreated patients compared to those on beta-blockers (16% vs 2.6%, respectively), P <.01. This result aligns with the known bradycardic effect of beta-blockers. Section title: Results Educational score: 4.04542350769043 Domain: biomedical Document type: Study Language: en When the analysis focused on the subgroup of patients treated with bisoprolol, fetal heart rates were not significantly different between the groups. Specifically, 100% of fetuses from patients on bisoprolol had heart rates between 110 and 150 bpm, compared to 93% of fetuses from untreated patients ( P =1). Section title: Results Educational score: 4.189011096954346 Domain: biomedical Document type: Study Language: en The secondary analysis on obstetric and neonatal outcomes revealed several significant differences between the groups, as shown in Table 4 . Significant differences were noted in the onset of labor and mode of delivery. Specifically, cesarean sections were more frequent in patients on beta-blockers (53%) compared to untreated patients (8.9%), P <.01. This difference predominantly concerns cesarean deliveries before labor, linked to the underlying chronic pathology of these patients, leading to specific labor management and a risk of cesarean section due to cardiac decompensation. Table 4 Secondary Analysis. Obstetric and neonatal outcome among untreated patients and patients with beta-blockers Table 4 Colonne1 Untreated patients n =45 (%) Patients with beta-blockers n =45 (%) P Gestational age SA, median (IQR) 38 (36.6–38.4) 38 (36.6–38.4) 1 Onset of labor Spontaneous 27 (60) 7 (16) <.01 Induction 14 (31) 14 (31) C-section 4 (8.9) 24 (53) Mode of delivery Spontaneous vaginal 28 (62) 13 (29) <.01 Instrumental vaginal 6 (13) 5 (11) C-section 11 (24) 27 (60) Birth weight g, median (IQR) 2890 2625 <.01 Birth percentile <10th 6 (13) 16 (36) .014 Hautier. Effect of maternal beta-blocker treatment. AJOG Glob Rep 2024. Section title: Results Educational score: 3.6603076457977295 Domain: biomedical Document type: Study Language: en Regarding birth weight, the results suggest that newborns from mothers on beta-blockers tend to have a lower birth weight than those from untreated mothers, P <.01. These findings suggest a potential association between beta-blocker use during pregnancy and an increased risk of neonatal hypotrophy. Section title: Principal findings Educational score: 3.9849441051483154 Domain: biomedical Document type: Study Language: en Our study revealed a differential effect of beta-blockers on maternal and fetal heart rates in patients treated with beta-blockers during pregnancy compared to untreated pregnant women. Section title: Results in the context of what is known Educational score: 2.968319892883301 Domain: biomedical Document type: Study Language: en The literature is limited concerning the hemodynamic effects of beta-blockers on fetuses. Our work contributes uniquely to this area by focusing specifically on the impact of beta-blockers on mean fetal heart rate. Section title: Results in the context of what is known Educational score: 3.9652273654937744 Domain: biomedical Document type: Study Language: en Our findings complement existing research on labetalol (alpha-beta-blocker treatment), which, due to its use in hypertensive disorders of pregnancy, has a richer literature. For instance, a systematic review published in 2004 gathered studies on the impact of major antihypertensive drugs on fetal heart rate during pregnancy. 18 The review included studies on labetalol, methyldopa, nifedipine, and hydralazine primarily. The authors did not find adverse effects of these treatments on fetal heart rate. In the neonatal period, the effects of Labetalol have been described, by Thewissen et al 19 who found no differences in newborns of mothers treated with Labetalol compared to the control group concerning heart rate, blood pressure, and the need for vasopressor support. Section title: Results in the context of what is known Educational score: 4.108003616333008 Domain: biomedical Document type: Study Language: en Regarding the molecules studied in our research, Montan et al's 20 1984 trial compared the average fetal heart rate in 40 full-term pregnant women before and after initiating atenolol treatment for hypertension. They found a statistically significant decrease in the average fetal heart rate: 143 vs 133 ( P <.001) after atenolol initiation. However, the clinical impact of such a decrease appears minimal, as the fetal heart rate remains within normal limits, not raising concerns during monitoring. Section title: Results in the context of what is known Educational score: 4.0664567947387695 Domain: biomedical Document type: Study Language: en Montan et al also observed a decrease in variability of 5 to 10 bpm for at least 20 minutes in 13.1% of fetuses after atenolol introduction compared to 2.3% before treatment ( P <.01). This parameter could falsely alert during monitoring, especially among fetuses suspected of being hypotrophic in patients treated with beta-blockers. Our study did not investigate this parameter, limiting the applicability of our findings. A more comprehensive or automated approach to interpreting fetal heart rate in patients on beta-blockers could offer more precise information for better monitoring interpretation in these high-risk obstetric patients. Section title: Results in the context of what is known Educational score: 3.8078620433807373 Domain: biomedical Document type: Study Language: en Among patients with Marfan syndrome, two were treated with nebivolol. There is no data on the specific effect of this molecule on fetal hemodynamic parameters. A study in rats published in 2016 by Altoama et al 21 compared the effects of bisoprolol and nebivolol on fetuses and found a decrease in average fetal weight gain when the mother was treated with nebivolol ( P <.01). The effects on heart rate were not studied. Section title: Clinical implication Educational score: 2.873819351196289 Domain: biomedical Document type: Other Language: en This result provides guidance to clinicians in interpreting fetal cardiac monitoring in these patients with chronic conditions, often at high obstetric risk. Section title: Clinical implication Educational score: 3.195218086242676 Domain: biomedical Document type: Other Language: en More broadly, patients on beta-blocker treatment during pregnancy require close monitoring and appropriate clinical management to minimize risks to both the mother and the fetus. Further studies are needed to validate our findings and understand the underlying pathophysiological mechanisms. Section title: Research implications Educational score: 4.006204128265381 Domain: biomedical Document type: Study Language: en From a pathophysiological perspective, it is established that so-called “cardio-selective” beta-blockers inhibiting beta-1 receptors like bisoprolol and atenolol cross the placental barrier. 22 These molecules could, therefore, have a potential negative chronotropic or inotropic effect on the fetus. One hypothesis we can propose to explain the absence of effects of these molecules is the immaturity of the fetus's beta-adrenergic receptors. Further research is needed to validate this hypothesis. Section title: Research implications Educational score: 3.982653856277466 Domain: biomedical Document type: Study Language: en Contradictory results exist in animals, notably in the 2022 article by Khandoker et al. 11 In this work, conducted in mice, subcutaneous injection of propranolol in pregnant females led to a decrease in fetal heart rate compared to saline injection, with no effect on maternal heart rate. Given that the transplacental passage of beta-blockers in mice has not been demonstrated, 23 the extrapolation of animal models to human pregnancy remains limited. Section title: Strengths and limitations Educational score: 3.0726516246795654 Domain: biomedical Document type: Study Language: en It is important to note that this study has certain inherent limitations due to its design. It is a retrospective, bicentric case-control study, which potentially limits the external validity of our results. Furthermore, the retrospective nature of the study could introduce selection and data collection biases. However, thanks to the computerized collection of our data, the comprehensive nature of the data is ensured. Section title: Strengths and limitations Educational score: 2.9172260761260986 Domain: biomedical Document type: Study Language: en Additionally, the sample sizes per treatment were small due to the rarity of the involved pathologies, which may limit the statistical power of the analysis and make it difficult to detect significant differences. Larger sample sizes could allow subgroup analyses and more detailed results per molecule. A potential dose-dependent effect could also be studied in a larger cohort. Section title: Strengths and limitations Educational score: 4.053598403930664 Domain: biomedical Document type: Study Language: en Despite these limitations, this study has several strengths worth noting. The specificity of recruitment of maternal pathologies from the two maternity involved in this study provides genuine originality to our results. The matching between groups on important variables such as age, parity, and gestational age strengthens the robustness of the obtained results. The observed difference in heart rate in mothers on beta-blockers compared to the control group supports good treatment adherence by patients, reinforcing our findings. Similarly, the significant increase in neonatal hypotrophy in treated patients, a known effect of beta-blockers, 6 aligns with fetal exposure to these treatments. Section title: Strengths and limitations Educational score: 3.81868839263916 Domain: biomedical Document type: Study Language: en Finally, excluding patients with pathologies that could interfere with fetal heart rate constitutes a rigorous methodological approach, allowing for better isolation of the beta-blockers’ effect on fetal heart rate. Section title: Conclusion Educational score: 3.461277723312378 Domain: biomedical Document type: Study Language: en Our study did not find a significant effect of beta-blockers on fetal heart rate in mothers treated during pregnancy compared to the control group. These women require specialized care management, and the interpretation of fetal monitoring should not be biased by maternal treatment. Further research is needed to understand the underlying physiological mechanisms. Section title: CRediT authorship contribution statement Educational score: 0.9950365424156189 Domain: other Document type: Other Language: en Sarah Hautier: Writing – original draft, Investigation, Funding acquisition, Formal analysis, Data curation. Thi Minh Thu Nguyen: Resources. Arane Kim: Resources. Tiphaine Barral: Resources. Dominique Luton: Validation, Supervision, Conceptualization. | Other | biomedical | en | 0.999996 |
PMC11699727 | Section title: Introduction Educational score: 4.0419921875 Domain: biomedical Document type: Review Language: en Aging is an independent risk factor for cardiovascular diseases, especially for heart failure (HF). Cardiac diastolic dysfunction (CDD) underlies HF with preserved ejection fraction (HFpEF) and is generally considered an aging-related condition. CDD is characterized by impaired relaxation with normal systolic function, however, it impacts approximately 3 million people in the United States and up to 32 million people worldwide. 1 , 2 As reported, CDD has been one of the leading causes of cardiovascular mortality in the elderly. 3 , 4 Patients with CDD have a very poor prognosis with more than 50% deaths 5 years after diagnosis. 5 Nevertheless, effective treatments to improve clinical outcomes and long-term survival in elderly patients with CDD are scarce. The underlying mechanisms are still being explored. 6 , 7 , 8 Section title: Introduction Educational score: 4.202032566070557 Domain: biomedical Document type: Study Language: en Cardiac troponin I (cTnI) plays a key role in regulating diastolic function. Changes including deficiency and mutations in cTnI are associated with CDD. 9 , 10 In elderly human hearts, the cytoplasmic concentration of cTnI is decreased in the left ventricular (LV) myocardium. 11 Aging-related CDD is also associated with decreased cTnI expression caused by abnormal histone acetylation modification. 10 Epigallocatechin gallate (EGCG), the main green tea catechin extracted from Camellia sinensis , has emerged as a mediator of cardiovascular health. 12 Cardioprotective effects of EGCG have been documented in several clinical studies. 13 , 14 In our recent clinical trial, cardiac functions and HF symptoms were significantly improved in cardiomyopathy patients who received EGCG capsules daily for 12 months. 15 Green tea intake was also related to enhanced blood vessel function and lower heart disease risk. 12 Several studies have shown that EGCG, as a histone acetylation modification regulator, could alter gene expression via inhibiting histone deacetylase 1 (HDAC1) and regulate organ functions. 16 , 17 So far, little attention has been paid to whether specific long-term intervention with EGCG could prevent the onset and progression of CDD and cTnI expression decline. In this study, we investigated the effects of three doses of EGCG on CDD and the underlying mechanisms. Section title: Animals Educational score: 4.1690263748168945 Domain: biomedical Document type: Study Language: en Female C57BL/6 mice were purchased from the Animal Center of Chongqing Medical University. All studies were carried out in strict accordance with the recommendations approved by Chongqing Medical University (Chongqing, China). All mice were housed in five per cage in the controlled environment with a 12 h strict light/dark cycle at 23 ± 1 °C and fed a standard diet from 12 to 18 months of age for 6-month administrations. Mice had free access to drinking water and food. As summarized in Figure 1 A, forty 12-month-old mice were randomized into four groups: aged, 50 mg EGCG (50 mg/kg/day), 100 mg EGCG (100 mg/kg/day), and 200 mg EGCG groups (200 mg/kg/day), containing 10 animals each. EGCG-treated mice were orally administered EGCG (purity: 99%, Selleck, USA) mixed with drinking water. Moreover, the aged mice were orally administered ordinary drinking water without an intervention agent. Mice aged 3 months were assigned as young control group ( n = 10). Drinking water containing EGCG was freshly prepared at about 5:00 p.m. every day at room temperature for EGCG-treated groups, and water left in the bottle was measured to determine the amounts consumed and then replaced. All mice were subjected to transthoracic echocardiography for assessment of cardiac functions at the age of 12 and 18 months, as well as a treadmill test. Heart tissues were harvested for histological analysis and determination of cardiac proteins, histone acetylation modification, and myocardial apoptosis and fibrosis. Figure 1 Long-term administration with EGCG prevents diastolic dysfunction later in life in the aged mice. (A) Diagram of the study design: mice were subjected to transthoracic echocardiography for assessment of cardiac functions, to treadmill test for evaluation of exercise ability, and to mechanistic studies. (B, C) Changes in transcription and protein expression of cardiac troponin I (cTnI) at different ages. (D) Representative M-mode echocardiographic images acquired from LV short-axis views. (E) Representative Doppler echocardiographic tracings. (F, G) Quantitative measurements of cardiac functions including ejection fraction (EF), fractional shortening (FS), the ratio of early ventricular filling to late ventricular filling caused by atrial contraction (E/A), and isovolumetric relaxation time (IVRT) ( n = 8–10). All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 1 Section title: Echocardiography Educational score: 4.131434917449951 Domain: biomedical Document type: Study Language: en Mice were subjected to conventional transthoracic echocardiography and Doppler analyses using GE Vivid E9 color Doppler ultrasound system (USA) equipped with a high-frequency probe (11 MHz) before (12 months old) and after (18 months old) interventions. Experimental animals were anesthetized with 2% isoflurane for 3–5 min and then maintained with 1%–1.5% during data collection. Ventricular dimension, septal and ventricular wall thickness at end-diastole and end-systole were measured to calculate systolic function using standard M-mode images under a short axis view. The mitral blood inflow and diastolic function were evaluated using the pulse-wave Doppler. Additionally, the following parameters were measured as well: early ventricular filling (E) to late (A) ventricular filling caused by atrial contraction (E/A), isovolumetric relaxation time (IVRT), and isovolumetric contraction time (IVCT). All data were analyzed using in vivo echocardiography report software. Both the echocardiography operator and data analyzer were blinded to the groups. Section title: Treadmill test Educational score: 4.070170879364014 Domain: biomedical Document type: Study Language: en A treadmill test with a six-lane was used to assess exercise capacity, as previously described. 18 After one-week training, the exhaustion test was performed, which comprised 3 exercise tests on the treadmill for consecutive days. All mice received a 1.2-mA shock if they stopped running and slid onto an electric grid placed at the rear end of the treadmill. An exercise test was started at a speed of 10 m/min on a 5° incline. The speed was increased by 1 m/min every 3 min until the mice fatigued, that is, the animals could no longer keep pace with the belt, and at this point, the running distance was recorded. Section title: Heart tissue harvesting Educational score: 3.81354022026062 Domain: biomedical Document type: Study Language: en Mice at 3, 10, 12, 14, 16, and 18 months of age were anesthetized with carbon dioxide and sacrificed. Heart tissues were washed with phosphate-buffered saline and stored at −80 °C. After administrations, heart tissues from young (3 months old), aged (18 months old), and EGCG groups were harvested. Section title: Morphological observations Educational score: 4.140385627746582 Domain: biomedical Document type: Study Language: en LV samples were prepared for the transmission electron microscope (TEM), cut into small pieces about 1 mm 3 , fixed immediately with 3% glutaraldehyde, and sent to the TEM center of Chongqing Medical University. All samples were sliced by ultramicrotome (Leica, German) and observed using a Transmission Electron Microscope (Hitachi, Japan). Formalin-fixed and paraffin-embedded tissues were cut into sections and stained with hematoxylin-eosin (HE). Cardiac fibrosis was observed using Masson's trichrome and collagen volume fraction (CVF) was calculated by NIH ImageJ Software as the ratio of fibrosis to total tissue area. Section title: Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) Educational score: 4.028187274932861 Domain: biomedical Document type: Study Language: en TUNEL assay was carried out according to the manufacturer's instructions (Roche, Germany) for observing cardiomyocyte apoptosis. The nuclei were marked in blue, while TUNEL-positive cells were stained in green. The fluorescence intensity of apoptotic cardiomyocytes was documented by a fluorescence microscope (Nikon, Japan). The apoptotic index was estimated as the ratio of the number of apoptotic cells to the total number counted. Section title: Mouse primary cardiomyocyte culture, transfection and treatment Educational score: 4.048986434936523 Domain: biomedical Document type: Study Language: en Primary cardiomyocyte culture from neonatal C57BL/6 mice was performed as previously described. 19 The collected cells were divided into Blank, DMSO, NC, HDAC1 overexpression, and HDAC1 overexpression + EGCG groups. Cardiomyocytes were transfected with empty vector or overexpression HDAC1 adenovirus. In HDAC1 overexpression + EGCG group, cardiomyocytes were treated with EGCG (40 μM) right after transfection with HDAC1 overexpression adenovirus. Cells were collected 24 h after administrations for subsequent analysis. Section title: Quantitative real-time PCR Educational score: 4.123401641845703 Domain: biomedical Document type: Study Language: en Total RNA was isolated from heart tissues using an RNA Extraction kit (Bioteck, China). Single-strand cDNA synthesis was performed using oligo dT-Adaptor primers and an AMV reverse transcriptase kit (TaKaRa, Japan). cDNA amplification was performed according to a quantitative real-time PCR assay employing a SYBR Green RealMasterMix kit (Tiangen, China). β-actin was used as a housekeeping gene to normalize transcription levels of the genes of interest. Genes of interest, including cTnI, A/B-type natriuretic peptide (ANP and BNP), HDAC1/2/3, sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a), sarcalumenin (SAR), and control were detected with the corresponding gene-specific pairs of primers, as detailed in Table S1 . Section title: Western blotting Educational score: 4.08452033996582 Domain: biomedical Document type: Study Language: en Western blotting was performed in accordance with previous protocol. 20 The homogenized samples were obtained using ice-cold RIPA buffer (Beyotime, China) containing protease and phosphatase inhibitors, and the protein concentration was determined using the Bradford Assay (Thermo Fisher, USA). Proteins of interest and control transferred to the PDVF membrane were analyzed using primary antibodies against cTnI (Abcam, USA), HDAC1 (CST, USA), or GAPDH (Arigo, China). The membranes with proteins were incubated with corresponding HRP-labeled secondary antibodies (Beyotime, China). Immunoblots were visualized by enhanced chemiluminescence luminal reagent (Mishushengwu, China) through Imaging System (Bio-Rad, USA) and quantitative analyses were acquired using Quantity One software (Bio-Rad, USA). Section title: Chromatin immunoprecipitation Educational score: 4.102316856384277 Domain: biomedical Document type: Study Language: en Chromatin was collected using the Chromatin Extraction Kit (Abcam, England) and immunoprecipitated in accordance with the instructions of the chromatin immunoprecipitation (ChIP) one-step kit (Abcam, England). The chromatin was fragmented into 200–1000 bp DNA fragments by ultrasonication. Protein-DNA complexes were incubated using antibodies against acetylated histone 3 (AcH3, Abcam, England), acetylated lysine 9 on histone H3 (AcH3K9, Abcam, England), acetylated lysine 27 on histone H3 (AcH3K27, Abcam, England), HDAC1 (Cell Signaling Technology, USA), histone deacetylase 2 (HDAC2, Cell Signaling Technology, USA), histone deacetylase 3 (HDAC3, Cell Signaling Technology, USA), GATA4 (Abcam, England) or MEF2c (Abcam, England), alongside non-immune IgG (as a negative control) and RNA polymerase II (as a positive control). The primer sequences targeting the −1000 bp upstream regions of the cTnI promoter were listed in Table S2 . Section title: HDAC1 activity assay Educational score: 4.142897605895996 Domain: biomedical Document type: Study Language: en HDAC1 activity was detected using an HDAC1 Activity Assay Kit (Biovision, USA). Proteins were extracted as per the manufacturer's instructions and the protein concentrations were tested using the Bradford Assay (Thermo Fisher, USA). A total of 50 μg protein for each immunoprecipitation was added with 6 μL HDAC1 antibody in sample groups or 6 μL IgG in control groups, followed by incubation at 4 °C overnight. After incubation, the protein-A/G bead slurry of 25 μL was added and incubated at 4 °C for 1 h. The beads were collected by centrifugation and wash. For each reaction, sample and control groups were mixed and incubated with Reaction Mix containing HDAC Assay Buffer and HDAC Substrate at 37 °C for 2 h. The Positive Control, Developer, and Standard Curve were prepared according to the manufacturer's instructions. Reaction supernatant of 100 μL was transferred to individual wells and the fluorescence value was read at Ex/Em = 380/500 nm. Then the AFC standard curve was gained. Sample HDAC Activity = (2 × B)/(T × S) = pmol/min/mg = mU. B was AFC amount from the Standard Curve (pmol). Sample dilution factor was 2. T represented reaction time (120 min) and S represented sample amount (50 × 10 −3 mg). Section title: Statistical analysis Educational score: 3.2604804039001465 Domain: biomedical Document type: Study Language: en All data were presented and analyzed using SPSS 23.0 software (USA) and GraphPad Prism 8.0 (USA). The results were shown as mean ± standard deviation (SD) and analyzed by one-way ANOVA followed by Tukey's or Bonferoni's or Sidak's multiple comparison post hoc. At least three independent triplicated experiments were performed for each experimental set-up. P values less than 0.05 were considered statistically significant, indicated as ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Section title: The expression of cTnI decreased gradually during cardiac aging Educational score: 4.060004234313965 Domain: biomedical Document type: Study Language: en To identify the key temporal point of cTnI expression decline during cardiac aging, the transcript and protein levels were measured in mice at 3, 10, 12, 14, 16, and 18 months of age. As shown in Figure 1 B and C, cTnI mRNA expression declined markedly at 14 months of age (14 m, 16 m, or 18 m vs . 12 m, P < 0.01, respectively), and its protein expression was decreased significantly at 16 months of age (16 m or 18 m vs . 12 m, P < 0.05, respectively). This study was aimed to investigate the preventive effects of EGCG on cardiac aging. Therefore, adult mice at 12 months of age were given EGCG for 6 months. As previously described, cTnI expression had not yet decreased at 12 months. Section title: Long-term administration of EGCG prevented CDD in the aged mice Educational score: 4.2623677253723145 Domain: biomedical Document type: Study Language: en Echocardiographic data showed that diastolic function was preserved at the age of 12 months ( Table S3 ), along with equivalent IVRT and E/A ratio to the young mice, which also suggested EGCG interventions should be initiated at this age. We then assessed heart functions in the young and aged mice treated with or without EGCG ( Table S3 ; Table 1 ). In the present study, the LV systolic function, as shown by pEF and fractional shortening, remained unchanged in the aged mice (18 months old), the same as in the young mice . However, a deteriorative and noticeable reduction of LV diastolic function was observed in the aged mice, as reflected by a significant increase of the E/A ratio ( P < 0.001) and a prolongation of IVRT ( P < 0.05) compared to the young mice . Fortunately, long-term administration of 100 and 200 mg EGCG effectively prevented CDD: the E/A ratio was 0.74-fold and 0.48-fold lower, and IVRT was 0.79-fold and 0.65-fold lower in 100 and 200 mg EGCG-treated mice, respectively, as compared with the aged mice, but no significant improvement of both was observed after 50 mg EGCG administration . Table 1 Cardiac functions after 6-month EGCG administration (18 months). Table 1 Parameters Aged Young Aged+50 mg EGCG Aged+100 mg EGCG Aged+200 mg EGCG IVSd (cm) 0.089 ± 0.020 0.086 ± 0.017 0.091 ± 0.010 0.089 ± 0.014 0.085 ± 0.008 LVIDd (cm) 0.305 ± 0.062 0.223 ± 0.046 0.271 ± 0.021 0.270 ± 0.030 0.269 ± 0.029 LVPWd (cm) 0.072 ± 0.025 0.075 ± 0.010 0.081 ± 0.010 0.080 ± 0.018 0.068 ± 0.005 LVIDs (cm) 0.153 ± 0.066 0.100 ± 0.025 0.119 ± 0.014 0.104 ± 0.023 0.116 ± 0.034 EDV (ml) 0.082 ± 0.050 0.030 ± 0.020∗∗ 0.054 ± 0.013 0.055 ± 0.017 0.051 ± 0.014∗ ESV (ml) 0.012 ± 0.016 0.002 ± 0.004 0.004 ± 0.005 0.004 ± 0.005 0.003 ± 0.005 EF (%) 86.52 ± 8.97 90.09 ± 3.90 90.01 ± 4.69 92.06 ± 2.21 90.11 ± 7.62 FS (%) 51.76 ± 11.39 55.03 ± 5.75 55.59 ± 7.94 58.21 ± 4.28 57.09 ± 11.19 SV (ml) 0.066 ± 0.031 0.030 ± 0.020∗∗ 0.049 ± 0.012 0.046 ± 0.015 0.047 ± 0.015 E/A 2.17 ± 0.24 1.19 ± 0.53∗∗∗ 1.89 ± 0.24 1.61 ± 0.42∗ 1.05 ± 0.39∗∗∗ IVRT (ms) 16.38 ± 3.68 11.07 ± 3.79∗ 13.02 ± 5.36 10.24 ± 2.72∗∗ 10.64 ± 3.25∗∗ A, late ventricular filling caused by atrial contraction; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; FS, fractional shortening; IVSd, intraventricular septum at end-diastole; LVIDd, left ventricular internal dimensions at end-diastole; LVIDs, left ventricular internal dimensions at end-systole; LVPWd, left ventricular posterior wall thickness at end-diastole; All values are presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001; another four groups compared to 3 m, respectively. Section title: Long-term administration of EGCG prevented CDD in the aged mice Educational score: 4.078634262084961 Domain: biomedical Document type: Study Language: en Furthermore, ANP and BNP in LV myocardium, classical molecular markers of HF, were significantly enhanced in the aged mice, along with a marked reduction of heart aging markers SERCA2a and SAR compared to the young subjects . Long-term EGCG intakes at all doses effectively improved cardiac aging phenotype as shown by a conspicuous reduction of HF markers and relieved the aging-related decreases of SERCA2a and SAR in the aged heart, compared with the aged mice . Figure 2 Long-term EGCG supplementation alleviates age-associated heart failure. (A, B) Gene expression of A-type natriuretic protein (ANP) and B-type natriuretic peptide (BNP). (C, D) Gene expression of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) and sarcalumenin (SAR). (E) Treadmill test for assessment of exercise capacity ( n = 8–10). All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 2 Section title: Long-term administration of EGCG enhanced exercise performance in aged mice Educational score: 4.0838093757629395 Domain: biomedical Document type: Study Language: en A running exhaustion test was conducted to evaluate exercise capacity by treadmill test . Our findings showed that the average running fatigue distance of the aged mice was 54.52% lower than that of the young mice ( P < 0.001). Concurrently, the aged mice supplemented with 50, 100, and 200 mg EGCG, respectively, exhibited increases in running fatigue distance by 37.01%, 31.36%, and 58.45% (each of EGCG groups vs . the aged group, P < 0.001), indicating EGCG intakes augmented exercise performance in the aged mice, with the most pronounced therapeutic effect observed in those treated with 200 mg EGCG. Section title: Long-term administration of EGCG repressed cardiomyocyte apoptosis and alleviated mitochondrial destruction in aged mice Educational score: 4.2069807052612305 Domain: biomedical Document type: Study Language: en As the heart aging, cardiac structural and morphological changes became evident . Heart tissue sections were stained with HE, Masson, and TUNEL to detect the degrees of myocardial fibrosis and cardiomyocyte apoptosis, and evaluate the effects of EGCG on the aged mice. And ultrastructure of the myocardium was also observed under TEM. Our data disclosed that heart aging was accompanied by cardiac fibrosis with a markedly increased interstitial fibrotic area , however, no significant inhibitory effect of all EGCG doses on myocardial fibrosis was observed in the aged mice . There was a slight decrease in CVF in the aged mice treated with 100 and 200 mg EGCG, but the difference was not statistically significant. Intriguingly, a remarkable increase of cardiomyocyte apoptosis rate in the aged mice was revealed by TUNEL staining, as opposed to the young mice , but it was normally reduced to the level of youth in the aged mice treated at all EGCG doses , suggesting EGCG could prevent myocardial apoptosis in the aged heart. Meanwhile, the TEM showed that the sarcomeres were arranged in complex patterns and partially dissolved in the aged mice, accompanied by disrupted Z lines and an increased mitochondrial flameng score compared to the young mice . Nevertheless, destruction of myofilaments and swollen mitochondria were alleviated in the aged mice administered with EGCG supplementations at all doses . Figure 3 Long-term EGCG intake alleviates aging-related cardiomyocyte apoptosis and mitochondrial damage. (A, B) Representative histology of heart tissue sections stained with hematoxylin-eosin stain and Masson's trichrome stain (yellow arrows collagen fibrosis; scale bar: 100 μm). (C) TUNEL stain for evaluation of cardiomyocyte apoptosis (red arrows apoptotic cells; 400 × ). (D) Transmission electron microscope (TEM) for the display of myocardial ultrastructure . (E–G) Quantitative analysis of myocardial fibrosis, cardiomyocyte apoptosis, and mitochondrial destruction ( n = 3–4). All results are represented as mean ± standard deviation. ∗∗∗ P < 0.001. Figure 3 Section title: Long-term administration of EGCG promoted cTnI expression via enhancing AcH3K9 in the aged mice Educational score: 4.247036933898926 Domain: biomedical Document type: Study Language: en Abnormal cTnI expression, as reflected by its deficiency, low levels, and mutations has been associated with CDD. 9 , 10 Emerging evidence revealed gene expression is consistent with histone acetylation modification. 10 , 16 In this study, we detected the levels of cTnI expression and histone acetylation modification near its promoter both in young and aged mice with or without EGCG, to identify the impacts of EGCG on cTnI regulation. The cTnI mRNA and protein levels were significantly lower in the aged mice compared to the young mice . Supplementation with 100 and 200 mg EGCG increased these reduced levels to those observed in the youth, however, 50 mg EGCG supplementation did not alter the protein decline . Furthermore, a significantly declined AcH3 near cTnI's promoter ( P < 0.001) was found in the aged group, along with decreased AcH3K9 ( P < 0.001) and unchanged AcH3K27 ( P > 0.05) compared with the young mice . Supplementations with 100 and 200 mg EGCG notably increased the downregulated AcH3 and AcH3K9 near cTnI's promoter (100 or 200 mg EGCG vs . the aged group: P < 0.01 for both; 200 mg EGCG vs . the young group: P > 0.05) and did not affect AcH3K27 (each of EGCG groups vs . the aged group: P > 0.05). However, no significant improvement of AcH3 and AcH3K9 was found after 50 mg EGCG intake . In addition, the lower transcription factor binding levels of MEF2c and GATA4 near cTnI's promoter were confirmed in the aged mice and improved to the levels as seen in the young mice by 100 and 200 mg EGCG administrations . The results indicated that EGCG could increase cTnI expression via enhancing AcH3 and AcH3K9 near cTnI's promoter and increasing the recruitment of MEF2c and GATA4 in the aged mice. Figure 4 Long-term administration of EGCG promoted cTnI expression via inhibiting HDAC1 and enhancing AcH3K9. (A, B) Quantitative analysis of cTnI transcription and protein expression by quantitative PCR and Western blot, respectively. (C) The enrichment levels of AcH3K4, AcH3K9, and AcH3K27 near cTnI's promoter by chromatin immunoprecipitation (ChIP) assays. (D) The enrichment levels of MEF2c and GATA4 near cTnI's promoter by ChIP assays. (E) Quantitative analysis of class I HDACs by quantitative PCR. (F) The binding levels of class I HDACs near cTnI's promoter by ChIP assays ( n = 8–10). All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 4 Section title: Long-term administration of EGCG inhibited HDAC1 in the aged mice Educational score: 4.139965057373047 Domain: biomedical Document type: Study Language: en To better understand the target of EGCG, we detected Class I HDACs, the upstream modifier of AcH3K9 in the heart tissues. The data showed the transcript level of HDAC1 was significantly higher in the aged mice than in the young mice ( P < 0.001), and there was no difference in HDAC2 and HDAC3 between the two groups . The elevated protein level of HDAC1 was consistent with its transcription level . But the boosted transcript and protein levels of HDAC1 were markedly inhibited by all doses of EGCG . Furthermore, the HDAC1 binding level near cTnI's promoter was significantly higher in the aged mice than in the young mice ( P < 0.001), but 100 and 200 mg EGCG supplementations significantly inhibited the elevated HDAC1 binding level and had no effects on HDAC2 and HDAC3 . These results suggested that EGCG might enhance AcH3 and AcH3K9 near cTnI's promoter by inhibiting HDAC1. Section title: EGCG enhanced cTnI expression via inhibiting HDAC1 Educational score: 4.101084232330322 Domain: biomedical Document type: Study Language: en To directly confirm the regulatory effects of HDAC1 on cTnI expression, primary cardiomyocytes transfected with HDAC1 overexpression adenovirus were cultured in vitro . As shown in Figure 5 A and B, cTnI mRNA and protein expression levels were significantly reduced in the HDAC1 overexpression group compared with the Blank group ( P < 0.001 for transcript and P < 0.01 for protein). The transcription and protein levels for HDAC1, the enrichment of HDAC1 near cTnI's promoter, and its activity in cardiomyocytes were elevated in the HDAC1 overexpression group compared with the Blank group, along with decreased AcH3K9 . These data indicated that HDAC1 induced-histone deacetylation modification played a key role in low cTnI expression regulation. Figure 5 EGCG elevates cTnI expression via suppressing HDAC1 and increasing AcH3K9. (A) Quantitative analysis of cTnI and HDAC1 transcription expression by quantitative PCR. (B) Quantitative analysis of cTnI and HDAC1 protein expression by Western blot. (C) The enrichment levels of AcH3K9 and HDAC1 near cTnI's promoter by chromatin immunoprecipitation assays. (D) HDAC1 activity. All results are represented as mean ± standard deviation. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Figure 5 Section title: EGCG enhanced cTnI expression via inhibiting HDAC1 Educational score: 4.312540054321289 Domain: biomedical Document type: Study Language: en To better understand the effects of EGCG, primary cardiomyocytes were treated with 40 μM EGCG in the HDAC1 overexpression group . The results showed the transcription and protein levels for cTnI were increased by EGCG treatment in cardiomyocytes with HDAC1 overexpression ( P < 0.05 for transcript and P < 0.01 for protein). We then detected the histone acetylation modification levels. The results revealed the protein for HDAC1 and its enrichment near cTnI's promoter were significantly reduced ( P < 0.001 for both), and the subsequent improvement of AcH3K9 was detected in HDAC1 overexpression group with EGCG administration ( P < 0.001), accompanied by depressed HDAC1 activity ( P < 0.01). Nevertheless, EGCG had no inhibitory effects on the mRNA expression of HDAC1 in primary cardiomyocytes with overexpressed HDAC1 ( P > 0.05). These data confirmed that EGCG could enhance cTnI expression via inhibiting the HDAC1 protein expression, its enrichment near cTnI's promoter and the activity of HDAC1, and then elevating AcH3 and AcH3K9, as shown in Figure 6 . Figure 6 The schematic illustrating that long-term EGCG administration prevents CDD and cTnI decline in aging hearts. The scheme depicts the role of physiological aging in the pathophysiology of CDD. The hallmarks of HFpEF such as CDD, reduced exercise performance, increased heart failure markers, myocardial fibrosis, cardiomyocyte apoptosis, and mitochondrial destruction, which might be associated with cTnI decline induced by HDAC1. According to our results, long-term EGCG administration counteracts aging-associated cardiomyocyte apoptosis and mitochondrial destruction, through which it improves the physiological cardiac function and structure. In the aging mice, EGCG regulates cTnI gene expression via inhibiting the expression of HDAC1, reducing its binding level near cTnI's promoter region and lowering the activity, as well as enhancing AcH3 and AcH3K9. The black blue arrow indicates lower and the red arrow indicates higher. See text for details. AcH3, acetylated histone 3; AcH3K9, acetylated lysine 9 on histone H3; CDD, cardiac diastolic dysfunction; cTnI, cardiac troponin I; EGCG, epigallocatechin gallate; HDAC1, histone deacetylase 1; HFpEF, heart failure with preserved ejection fraction. Figure 6 Section title: Discussion Educational score: 4.1440110206604 Domain: biomedical Document type: Study Language: en In recent years, EGCG, the major polyphenol component of green tea extract, has been widely reported to have extensive biological activities in various organs. 21 , 22 , 23 In our study, we found that cTnI expression and diastolic function were decreased gradually during cardiac aging, therefore, EGCG was administered in drinking water before their declines occurred. Then the effects of different concentrations of EGCG were observed. The results showed that 100 and 200 mg EGCG supplementations in drinking water for 6 months prevented the development of aging-related CDD and cTnI expression decline. This effect was due to long-term intervention with 100 and 200 mg EGCG up-regulating cTnI expression via inhibiting HDAC1 and increasing AcH3K9 near cTnI's promoter. However, there was no significant difference in the performance between the 50 mg EGCG group and the aged group. We speculated that the concentrations of EGCG or its derivatives in the blood circulation of mice administered with 50 mg EGCG were too low to generate the cardioprotective effects. In our previous study, intraperitoneal injection of 50 mg EGCG could improve CDD in restrictive cardiomyopathy (RCM) mice, indicating that a higher dose of oral EGCG may be necessary to avoid first-pass elimination. Section title: Discussion Educational score: 4.287960052490234 Domain: biomedical Document type: Study Language: en CDD, a clinical syndrome associated with aging, is becoming increasingly prevalent. Recent studies have indicated that abnormal expression of cTnI contributes to the pathophysiology and development of CDD. 9 , 10 , 11 , 24 Homozygous knock-in mice with cTnI R21C mutation developed hypertrophy after 12 months of age and exhibited abnormal diastolic function that is characterized by longer filling times and impaired relaxation. 24 Mice carrying cTnI R193H mutation manifested bi-enlarged atria and severe CDD. 25 A clinical study found that the myocardial cytoplasmic cTnI concentration was decreased in the elderly human hearts, which was consistent with the occurrence of CDD in the elderly population. 11 In hemodialysis patients with pEF, serum cTnI levels were significantly associated with CDD and risk of mortality independent of echocardiographic variables and other biomarkers. 26 Our data showed that cTnI mRNA and protein expression declined markedly at 14 and 16 months of age, respectively, with the lowest expression occurring at 18 months. In fact, histone acetylation is a dynamic and reversible process that regulates gene expression and is mediated by histone acetylase and HDACs. 27 , 28 Our data demonstrated that the expression of HDAC1 and its enrichment near cTnI's promoter were increased in the aged mice, and the level of AcH3K9, a marker for gene activation, was reduced, indicating aging-related low cTnI expression was associated with HDAC1 induced deacetylation of H3K9 near cTnI's promoter. We wonder whether blocking cTnI decline could prevent the occurrence and development of CDD. Section title: Discussion Educational score: 3.908491849899292 Domain: biomedical Document type: Study Language: en In Asia, especially in China, there is a tradition of drinking tea for thousands of years. EGCG has been reported to have multiple effects on cardiovascular diseases and could improve CDD in RCM mice and patients with abnormal cTnI. 15 , 25 EGCG dissolved in drinking water was used in the present study. This feeding regimen is noninvasive and well tolerated by animals and mimics the daily consumption of green tea by an average adult human. 29 Based on the daily water consumption and weight of mice, we calculated the mean dose to be about 50, 100, and 200 mg/kg/day. Section title: Discussion Educational score: 4.362284183502197 Domain: biomedical Document type: Study Language: en Our data showed that long-term EGCG administration blunted aging-related CDD and exercise performance decline, accompanied by significant reductions of HF markers and increases of SERCA2a and SAR in the EGCG-treated aged mice, suggesting EGCG could prevent cardiac aging and improve HF. SAR, a luminal Ca 2+ buffer protein, is located in the longitudinal sarcoplasmic reticulum of muscle and heart and regulates Ca 2+ reuptake by interacting with SERCA and maintaining its stabilization. 30 , 31 Senescent SAR knock-out mice exhibited typical systolic dysfunction due to the decreases in SERCA2a expression and activity. 30 AAV/SERCA2a gene therapy has been performed in patients with advanced heart failure due to systolic dysfunction. 32 However, in our study, no difference was found in myocardial contractility between the young and aged mice. Systolic dysfunction may not be a major issue in older mice and the population. 2 , 3 , 6 With increasing age, hearts present undesirable structural and morphologic phenotypes, characterized by elevated collagen fiber deposition, myocardial apoptosis, and mitochondrial destruction. Here, we found that EGCG may attenuate myocardial apoptosis and mitochondrial destruction in the aged mice, but had no effects on cardiac fibrosis. EGCG could maintain the baseline mitochondrial function and integrity, which is essential for preventing myocardial apoptosis during aging. 33 Taken together, long-term EGCG supplementation is associated with ameliorative diastolic function and a repressible aging process. Section title: Discussion Educational score: 4.268246173858643 Domain: biomedical Document type: Study Language: en Epigenetically, EGCG has been classified as an HDAC inhibitor, which can repress Class I HDACs to enhance histone acetylation and increase the transcription of target genes. 34 , 35 In this study, long-term 100 and 200 mg EGCG treatment could alleviate the decreases in cTnI transcript and protein levels. Enrichment of HDAC1 near cTnI's promoter was reduced, and AcH3 and AcH3K9 were elevated after EGCG treatment in the aged mice, along with elevated recruitments of transcription factors. Although 50 mg EGCG inhibited HDAC1 expression, it did not increase cTnI mRNA expression nor improve histone acetylation and enrichment of transcription factors near cTnI's promoter. To better understand the effect of EGCG on HDAC1, primary cardiomyocytes with HDAC1 overexpression were treated with EGCG. We found both cTnI mRNA and protein were decreased in cardiomyocytes with HDAC1 overexpression, however, EGCG could reverse cTnI expression. The level of AcH3K9 was increased, and protein expression of HDAC1 and its enrichment near cTnI's promoter were significantly reduced by EGCG treatment. Additionally, the activity of HDAC1 was also markedly suppressed. These data strongly suggested that EGCG could increase cTnI expression via inhibiting HDAC1. Section title: Discussion Educational score: 3.4475300312042236 Domain: biomedical Document type: Study Language: en Unfortunately, there were some limitations in the present study. EGCG blood concentrations were not measured due to a lack of corresponding devices. Water containing EGCG was freshly prepared daily, its blood concentration might be maintained at a certain level to ensure the effectiveness of EGCG. Here, we did not detect the methylation levels near cTnI's promoter. Our previous study revealed no significant changes in methylation levels in mice of different ages, and histone acetylation might be more important as an epigenetic regulator than gene methylation in regulating cTnI gene expression. 10 Section title: Conclusion Educational score: 4.0891432762146 Domain: biomedical Document type: Study Language: en The primary finding of this study is that EGCG may be an effective agent for preventing aging-related CDD and cTnI expression decline by inhibiting HDAC1 and increasing AcH3 and AcH3K9 near cTnI's promoter in aged mice . We provide a novel insight into the fact that long-term administration of EGCG is an effective strategy in preventing the onset and development of aging-related CDD and cTnI expression decline. Drinking green tea daily may improve diastolic function in elderly individuals and enhance their quality of life. Section title: Ethics approval Educational score: 0.9915826916694641 Domain: biomedical Document type: Other Language: en This study was approved by the Institutional Review Board of Children's Hospital of Chongqing Medical University. All procedures were performed in accordance with the Helsinki Declaration. Section title: Author contributions Educational score: 0.9353786706924438 Domain: other Document type: Other Language: en JT designed the study. JJQ and ZLJ experimented and analyzed the data. JT and JJQ interpreted the results of the experiments. JJQ and LJL prepared the figures. JJQ and ZLJ drafted the manuscript. All authors read and approved the final manuscript. Section title: Conflict of interests Educational score: 0.8620539307594299 Domain: other Document type: Other Language: en The authors declared no conflict of interests. Section title: Funding Educational score: 0.9768024682998657 Domain: other Document type: Other Language: en This study was supported by grants from the Science and Technology Foundation of Chongqing , China , the Key Grant from the National Clinical Research Center for Child Health and Disorders (China) , the National Natural Science Foundation of China , and the National Key Clinical Specialty (China). | Review | biomedical | en | 0.999996 |
PMC11699737 | Section title: Background Educational score: 3.6605095863342285 Domain: biomedical Document type: Review Language: en Chronic respiratory diseases are a leading cause of disability and mortality globally . They include a range of infectious and non-infectious health conditions such as tuberculosis, chronic obstructive pulmonary disease (COPD), asthma, occupational lung diseases and pulmonary hypertension. As with many other chronic conditions, their prevalence is increasing . Despite being a heterogeneous group, chronic respiratory diseases share similar risk factors, such as smoking, air pollution, dust and occupational chemicals . Early diagnosis, close monitoring, appropriate therapy, and easy access to health care can improve outcomes . Section title: Background Educational score: 3.915191888809204 Domain: biomedical Document type: Review Language: en Rurality may be a determinant of risk and outcome of chronic respiratory disease. For example, COPD incidence is higher in populations living in rural and remote areas, who are, on average, also more exposed to risk factors such as indoor pollution and the use of biomass fuel . More than half of all rural residents lack access to critical healthcare and many need to travel long distances to reach facilities. Patients may be reluctant to seek care due to travel costs, time expenditure and loss of potential income, resulting in healthcare-seeking delays and limited access to specialist services and pulmonary rehabilitation . Lower health literacy and socioeconomic status may also influence the underutilisation of care and the persistence of unhealthy habits such as smoking . The urban-rural divide ultimately results in a higher disease burden in remote communities, characterized by delayed diagnoses and higher hospitalization and mortality rates . Section title: Background Educational score: 2.767766237258911 Domain: biomedical Document type: Other Language: en E-health refers to the use of electronic technologies in health care and has greatly expanded due to technological advances and the spread of mobile phones and other digital tools. Additionally, the COVID-19 pandemic introduced an urgent need to replace physical appointments, increasing the use of e-health solutions . Importantly, e-health can be a cost-effective resource for diagnosis, monitoring, and health education , and telemonitoring has been shown to improve health utilization in rural settings . Section title: Background Educational score: 3.9623525142669678 Domain: biomedical Document type: Study Language: en In patients with chronic respiratory diseases, e-health interventions have been shown to reduce emergency department attendance [ 17 – 19 ], hospitalisation rates , the number of exacerbations and to increase pharmacological treatment compliance and physical activity rates . The use of e-health interventions may, therefore, help to reduce health disparities in remote populations living with these diseases [ 20 – 22 ]. Indeed, there is evidence that telehealth interventions are non-inferior for improving COPD self-management in rural areas . However, beyond the effectiveness of e-health interventions, whether implementation is successful is likely context-specific, and a comprehensive understanding of factors influencing this process is paramount. The aim of this qualitative evidence synthesis (QES) was, therefore, to analyse the qualitative and mixed-methods literature on e-health interventions in people with chronic respiratory diseases in remote settings and thereby identify barriers and facilitators for implementation. Section title: Study design and research question Educational score: 3.743532180786133 Domain: biomedical Document type: Study Language: en The QES was structured according to Cochrane’s EPOC guidance and the ENTREQ reporting guidelines (checklist: Supplementary Table 1) . The PerSPE(c)TiF framework was used to define the research question (Supplementary Table 2) . In brief, the aim of this QES was to explore any factors related to the successful implementation of e-health technologies in remote settings worldwide, for diagnosis and follow-up of chronic respiratory diseases from the perspective of patients, healthcare workers and other stakeholders. Section title: Search strategy Educational score: 2.342708110809326 Domain: biomedical Document type: Study Language: en Scoping searches on PubMed were used to refine the search strategy. Three key domains (‘e-health’, ‘rural’ or ‘remote’ setting, and ‘chronic lung diseases’) were identified. With the help of a librarian, search terms and variations were generated and combined. The search was piloted in MEDLINE (Ovid). Section title: Search strategy Educational score: 1.9920445680618286 Domain: biomedical Document type: Study Language: en Five databases were searched in April 2023: MEDLINE, CINAHL, Embase, Web of Science and PsycINFO. The full search strategy is shown in Supplementary Table 3. Additionally, relevant reviews were screened for additional articles. Grey literature was not searched. Section title: Study screening and selection Educational score: 4.046089172363281 Domain: biomedical Document type: Study Language: en Citations were imported to Endnote version 20.3 on 21.4.2023, and duplicates were removed, followed by title/abstract screening and full-text review based on predefined inclusion and exclusion criteria (Table 1 ). Articles were screened by two authors (ES and EB) independently. In case of disagreement, the article was included in the full-text review. All qualitative and mixed-methods studies published in English describing diagnosis, monitoring, or treatment of patients with chronic respiratory diseases, defined according to the criteria of the American Lung Association , in remote and rural settings using e-health were included. We chose to include abstracts as well as full-text articles in order to obtain the most comprehensive data, as the chosen methodology of thematic synthesis is appropriate for relatively thin data, keeping the level of confidence in the data in mind when interpreting these. With regard to COVID-19, studies on diagnosis, monitoring or long-COVID were included, while studies on short-term treatment were not, as findings from the former were expected to be relevant for this QES. Section title: Study screening and selection Educational score: 4.000218868255615 Domain: biomedical Document type: Study Language: en Table 1 Inclusion and exclusion criteria Criteria for selection Inclusion criteria Exclusion criteria Type of article Full-text articles or abstracts of primary research Literature review, systematic review, QES Methodology Any qualitative or mixed-methods approach Quantitative research without qualitative components Study population Patients of any age suffering from or suspected of chronic respiratory diseases including multi-morbidity patients, and/or their families; any stakeholder working on chronic respiratory disease e-health interventions including but not limited to healthcare workers, administrative personnel, policy makers, technology manufacturers, researchers. Patients with conditions other than chronic respiratory conditions; stakeholders not working on chronic respiratory disease e-health interventions Type of setting Remote or rural settings (no specific geographic restrictions) Non-remote settings Type of Intervention Any e-health intervention directed at the management of chronic respiratory disease patients (diagnostic process, follow up, monitoring and treatment) Interventions without e-health components; e-health interventions not directed at chronic respiratory disease patients; multi-disease e-health interventions where findings for subgroup of chronic respiratory disease patients are not described Timing No time limit Language English Non-English language Type of outcomes Any qualitative outcomes related to factors influencing the use of e-health, including but not limited to: - User attitudes and satisfaction with the technologies - Factors for prolonged engagement - Factors hindering e-health implementation Quantitative outcomes not accompanied by qualitative findings Section title: Quality assessment Educational score: 4.095580101013184 Domain: biomedical Document type: Study Language: en The assessment of the methodological limitations was done according to the Critical Appraisal Skills Programme (CASP) tool for qualitative research to evaluate the methodological rigour of selected studies . The CASP assessment is presented in Supplementary Table 4. The findings were also assessed for confidence according to GRADE CERQual method (Supplementary Table 5). CERQual takes into account four different factors: methodological limitations, relating to the study design or conduct , coherence, assessing the fit between the primary study findings and review findings , adequacy, describing the overall richness and quantity of the data and relevance, describing the extent to which the data is applicable and relevant to the review question . After assessing each individual component, the overall confidence was judged by one author (ES) as high, moderate, or low. Section title: Data analysis and thematic synthesis Educational score: 4.167338848114014 Domain: biomedical Document type: Study Language: en The RETREAT framework , taking into account the review question, epistemology, timeframe, resources, expertise, audience and types of data, guided the choice of a thematic synthesis approach (Supplementary Table 6). The selected articles were uploaded to Atlas.ti software . Articles were coded sentence-by-sentence based on units of meaning, applied to both first and second-order constructs present in the results and discussion sections of papers, to capture both primary opinions and authors’ interpretations . First-order constructs (quotes from participants in the original study) are presented in italics, and second-order constructs are presented in normal font. Descriptive themes were generated grouping similar codes into a hierarchical tree structure. Thematic synthesis was combined with an a priori ‘best fit’ framework approach . The themes were discussed between the authors (ES, FB, and EB) and existing frameworks were sought to guide the analysis and presentation of the data, ultimately identifying the Digital Health Equity Framework (DHEF) . The DHEF draws on the leading health disparities framework. It includes key determinants of health at different levels and incorporates a digital environment domain which enabled us to classify and interpret factors related to implementation of e-health in remote locations. In order to identify themes, deductive coding was used applying the DHEF and an inductive coding process was utilised to capture novel insights. Aligning with the DHEF, themes were arranged according to their relevance to determinants of health at different levels: individual (e.g., patient or healthcare provider attitude towards technology), interpersonal (e.g., patient-tech-healthcare provider relationship), community (e.g., healthcare infrastructure), society (e.g., tech policies and data standards). An additional level was added relating to the intervention itself (e.g., technology characteristics, design quality and product presentation). Within the data, barriers and facilitators were identified, highlighting those unique to remote populations. Section title: Search results Educational score: 2.4345791339874268 Domain: biomedical Document type: Study Language: en After removing duplicates, 1562 articles were identified, with 134 studies sought for retrieval. Based on citation searching from relevant reviews, four additional studies were sought for retrieval. After full-text assessment, 41 studies were included . Section title: Search results Educational score: 2.3725192546844482 Domain: biomedical Document type: Other Language: de Fig. 1 PRISMA Flowchart Section title: Search results Educational score: 4.102588653564453 Domain: biomedical Document type: Study Language: en Table 2 – overview of selected articles First author, year Country, location Setting Type of publica-tion Participants Medical condition E-health intervention Study population CERQual assess-ment of confidence in the evidence Age % Female Languages spoken Alexander, 2021 USA, North Carolina McDowell, Mitchell, and Yancey counties, designated as Health Professional Shortage Areas for primary care and predominately rural Qualitative research Patients ( n = 17) COPD Telehealth Mean age 61 years +-SD 9.1 41 English High Alwashmi, 2019 Canada Newfoundland and Labrador – area with substantial remote and rural population Qualitative research Nurses, physicians, pharmacists ( n = 30) COPD Mobile health interventions Mean age 39 years NR NR High Anticona, 2015 Peru Rural areas in the Highlands and the Amazon basin Qualitative research Physicians ( n = 30) Multiple, including chronic respiratory diseases Technological innovations, including telehealth and mobile health Age 26–30 years 43 Spanish Low Boyd, 2007 Australia, Victoria Five different rural Victorian towns Mixed methods Patients, developers, community members (8 focus groups of 4–10 participants) Asthma Multimedia education program NR NR NR Moderate Brown, 2017 USA, North Dakota Oakes, located in rural Dickey County Mixed methods Patients ( n = 18) Asthma Telehealth for asthma education NR NR NR Moderate Chaiyachati, 2013 South Africa, KwaZulu-Natal Umzinyathi, a rural district of KwaZulu-Natal Mixed methods Mobile healthcare workers ( n = 5) Multidrug resistant TB Mobile phone application Median age 32 years (range 27–46) 20 English Moderate Chen, 2012 USA, West Virginia Rural Appalachia Abstract Patients ( n = 5) Lung cancer Nurse-interpreted telementoring intervention with patient ducation Mean age 66 years +- 6.4 60 NR Moderate Concotelli, 2012 USA, New Mexico NR Abstract Patients CF Telehealth adolescent support group NR NR NR Low Cox, 2022 Australia, Victoria 33% of participants “from a rural location” Abstract Patients ( n = 20) Chronic respiratory diseases Telerehabilitation NR NR NR Low de Batlle, 2021 Spain Lleida, a large rural area of over 4300 km 2 Mixed methods Patients ( n = 76) COPD Mobile health-enabled integrated care model Mean age 82 years 46 NR Moderate De San Miguel, 2013 Australia, Western Australia Metropolitan area in Western Australia Mixed methods Patients ( n = 80) COPD Home-based telehealth monitoring Mean age 73 years, range 54–88) 52 English Moderate Demchenko, 2015 Canada, Saskatchewan People living more than 100 km from Saskatoon Abstract Patients ( n = 46) Lung cancer Nurse-clinical administered telehealth clinic NR NR NR Low Douglas, 2013 Canada, Alberta First Nations communities in Alberta Mixed methods Education instructors, phycisians, academics Asthma Self-management education curriculum NR NR NR Moderate Ellington, 2021 Uganda, Jinja district Two federally funded Ugandan primary care health facilities, one peri-urban and one rural Qualitative research Health care workers ( n = 31) Lower respiratory disease Mobile health tool N = 10 < 30 years; n = 11 30–40 years; n = 4 > 40 years 68 English and local languages Moderate Godden, 2011 UK, Scotland Five participants “worked exclusively in remote and rural areas, while the others provided services across the region for urban, rural and remote patients” Mixed methods Phycisians, nurses, respiratory care professionals ( n = 20) COPD, Asthma, Lung cancer, OSAS Telehealth NR 65 NR High Goodridge, 2011 Canada, Alberta “This health region encompasses an area of more than 40,000 km2, has population densities ranging from 1.1–2.0 persons per km2 and is designated as having no metropolitan influence by Statistics Canada.” Qualitative research Patients ( n = 7) COPD, Bronchiectasis Not specified Mean age 75 years (range 57–88) 71 English Low Guthrie, 2020 USA, Texas “Northeast Texas, a 35-county area with a population close to 1.5 million, over half living in a rural area” Abstract Patients CF Telemedicine NR 55 NR Low Hatem, 2022 USA, Massachusetts NR Abstract Patients COPD, COVID, interstitial lung disease Telehealth-supported home based pulmonary rehabilitation program Mean age 71 years 5 NR Low Johnson, 2021 USA, South Carolina Four of nine schools were classified as rural Mixed methods Nurses, phycisians, respiratory therapist, program coordinator ( n = 26) Asthma Mobile health application 25-34y n = 4; 35-44y n = 7; 45-54y n = 10; 55-64y n = 4; 65 + n = 1 96 NR Moderate Khan, 2017 Canada Northern communities in Saskatchewan Mixed methods Phycisians TB, various conditions Multiple NR NR NR Low Kok, 2023 Uganda, various districts Rural communities, with a distance of at least 7 km to a health facility Mixed methods Community health workers ( n = 24) COVID Telehealth Mean age 38 years 90 Luganda, Lusoga, Runyankole Moderate Latycheva, 2013 Canada Five First Nations and Inuit communities from across Canada Mixed methods Patients, parents, grandparents, community members, teachers Asthma Web-based asthma education materials N = 14 6 to 12 years; n = 18 12 to 18 years; age of adults participants NR NR English High Locke, 2019 USA, Rural patients, as defined by the US Census Bureau Mixed methods Patients COPD, Asthma Video telehealth inhaler training program Mean age 69.2 years 0 NR Moderate Lundell, 2020 Sweden, Västerbotten county A large and sparsely populated area with long distances to health care facilities Qualitative research Patients ( n = 13) COPD Home telemonitoring system NR 62 Swedish Moderate MacGeorge, 2021 USA, South Carolina NR Abstract Nurses, teachers, administrators Asthma Telehealth-delivered school-based program NR NR NR Low Mair, 1999 UK NR Mixed methods Phycisians, Patients COPD Telecommunications technology Mean age 62 years (range 52–72) 67 NR Low Mathur, 2019 India, Assam state Five rural blocks from Darrang and Kamrup district of the Assam state Qualitative research Patients ( n = 40) TB Outbound automated calls Mean age 34 years, range 18–60 20 Assamese Moderate Mc Veigh, 2019 USA, Idaho Large veteran hospital in Boise, Idaho, providing services to veterans located in mountainous regions of the county Qualitative research Nurses, phycisians, social workers ( n = 16) Non-malignant respiratory disease Telemedicine/digital platform for palliative care NR NR English Moderate McGee, 2020 USA, various states Patients residing in rural/geographically isolated areas Mixed methods Patients ( n = 49), informal caregivers ( n = 49) Chronic lower respiratory disease Telehealth services Patients: mean age 706 years 6.1 English Moderate Mendez, 2021 Chile, Santiago Participants from any Chilean region Mixed methods Patients, physiotherapists ( n = 22) COPD Mobile phone application Median 37 (IQR 33–39 y) 50 Spanish High Musiimenta, 2020 Uganda, Southwestern TB clinic within Mbarara Regional Referral Hospital in rural, southwestern Uganda Mixed methods Patients ( n = 35), social supporters ( n = 15) TB Mobile health intervention Median 32 (patients); 37 (social supporters) 43 (patients); 60 (social supporters) Runyankole Moderate Ng, 2014 Australia, South Australia Port Augusta and Whyalla (300 and 381 km from Adelaide respectively) and Tennant Creek (500 km from Alice Springs) Abstract Patients COPD, CF, OSAS Telehealth consultations NR NR NR Low Otty, 2023 Australia, Queensland Regional tertiary public hospital in northern Australia, with a significant number of geographically dispersed rural and remote communities Qualitative research Patients, informal caregivers ( n = 19) Lung cancer Telehealth consultations Median 64 (IQR 52–82 y) 48 NR Moderate Petitte, 2014 USA, West Virginia Patients’ homes in rural Appalachia Mixed methods Patients ( n = 10) Lung cancer Home telemonitoring intervention Median 66 (IQR 58–73) 50 NR Low Ratchakit, 2020 Thailand, Chiang Rai TB clinic at Chiangrai Prachanukroh Hospital, a tertiary-care hospital located in Thailand’s northernmost province Mixed methods Patients ( n = 80) TB Mobile health intervention Mean 50 ± 16 (intervention arm) 40 Thai Moderate Raza, 2009 USA, Michigan/Wisconsin Tertiary care hospital in Milwaukee and small rural hospitals in Iron Mountain (MI) and Appleton (WI) for veterans located in northern Wisconsin and the Upper Peninsula of Michigan Mixed methods Patients ( n = 314) Pulmonary specialist diagnostic consult Telehealth consultations NR NR NR Moderate Roberts, 2012 UK, Scotland Sheltered housing in Oban and in a community hall on the Isle of Luing Qualitative research Patients, phycisians, nurses, nurse specialists, housing warden, project managers (n = NR) COPD Home and community telehealth monitors NR NR NR High Ruseckaite, 2021 Australia, Victoria NR Abstract Patients, informal caregivers ( n = 15) CF Telehealth consultations Mean 40.2 (patients); 41.8 (caregivers) NR NR Low Venter, 2012 New Zealand Rural communities in Turangi and Taupo areas Mixed methods Patients ( n = 20) COPD Home telemonitoring intervention NR NR NR Moderate Wilson, 2022 USA, Midwest states Rural areas across five Midwest states Abstract Phycisians, nurse practicioners, phycisian assistants ( n = 10) COPD Telemedicine NR NR NR Low Young, 2012 USA, Wisconsin Family Health Center of Marshfield, serving 254 rural municipalities in north-central Wisconsin Mixed methods Patients ( n = 98) Asthma Telepharmacy intervention Mean 44.6 (SD 15.8) 75 English High CF cystic fibrosis, COPD chronic obstructive pulmonary disease, NR not reported, OSAS obstructive sleep apnea syndrome, TB tuberculosis Section title: Study characteristics and quality assessment Educational score: 4.029427528381348 Domain: biomedical Document type: Study Language: en We included 11 primary qualitative studies, 21 mixed-methods studies, and 9 abstracts. The studies were published between 1999 and 2023. The geographical location varied; fifteen studies took place in the USA, six in Canada, six in Australia, three in Uganda, three in the UK, two in India and one in Peru, South Africa, Spain, Sweden, New Zealand, Chile and Thailand each (see Table 2 for details on the setting). Sixteen studies (39%) reported the language spoken by the participants, which was English in nine studies, Spanish in two studies, and Assamese, Swedish, Runyankole and Thai in one study each. One study reported that interviews and focus groups were done in English and ‘local languages’, and one study was done in Luganda, Lusoga and Runyankole. None of the studies reported the proportion of participants whose mother tongue was not English. Five studies explicitly mentioned the inclusion of Indigenous, First Nations or Aboriginal participants , two of which reported involvement of these groups in the study design itself, either in the form of an advisory group or by partnering of the researchers with First Nations leaders . Twelve studies included a mix of different chronic respiratory diseases, ten of the studies focused on COPD, seven on asthma, four on tuberculosis, four on lung cancer, three on cystic fibrosis and one on COVID-19 (Table 2 ). Section title: Study characteristics and quality assessment Educational score: 3.9925992488861084 Domain: biomedical Document type: Study Language: en The results of the CASP assessment of methodological limitations of the included studies are shown in Supplementary Table 4. According to the CERQual assessment of the findings, there were seven studies that inspired high confidence in their findings, while twenty instilled moderate confidence. The remaining fourteen studies elicited low confidence in their results (Table 2 and Supplementary Table 5). The reasons for low confidence ratings were diverse; notably, for several studies only abstracts were published without corresponding full articles, impacting the ability to assess the methodology and the richness and thickness of the findings. Section title: Description of themes Educational score: 2.901259183883667 Domain: biomedical Document type: Study Language: en Our synthesis, guided by the DHEF, yielded 19 themes across five levels, each with interrelated facilitators and barriers for the implementation of e-health interventions in remote locations. A graphical representation of the themes is shown in Fig. 2 . The key facilitators and barriers identified through this QES are illustrated in Table 3 . Fig. 2 Graphical representation of the relevant themes at five different levels Section title: Description of themes Educational score: 4.044046401977539 Domain: biomedical Document type: Study Language: en Table 3 Overview of facilitators and barriers for e-health interventions associated with each level Level Facilitators Barriers Individual • Patient willingness to become more autonomous, increase health literacy and self-management quality • Usability as educational tool • Patient familiarity with technology • Patient and HCP interest in learning new technologies • Scheduling flexibility and reduction of in-person appointments • No need for companion • Sense of reassurance with constant monitoring/access • Personalized care • Reduced travel costs, overall affordability • Cost-effectiveness • May not be helpful unless integrated with behavioral change interventions • Patient age and poor digital literacy • Patient and HCP lack of knowledge about existing resources • Patient low literacy levels • Patient and HCP low interest in new technologies, technology perceived as challenging, skepticism • Preference for in-person consultation and fear of loss of human contact • Sense of hyperconnectivity • Increased workload and fear of not being adequately compensated (HCP) • Negative attitude towards tech devices (e.g., smartphones) • Steep set-up phase • Cost of device, lack of insurance Interpersonal • HCP can manage more patients • Reduced risk of infectious diseases transmission • Improved constant monitoring • Clinical team building and creation of multidisciplinary teams • Greater family involvement • Reduced non-verbal communication • HCP feeling constantly available “on demand” • Sense of false security in the patient • Deterioration of relationships within pre-existing teams due to increased responsibilities Community • Community engagement • Use of role models • Cultural appropriateness • Availability of the translation in local languages • Focus on multiple conditions • Poor networks • Lack of pre-existing health infrastructure • Focus on one disease at a time only Society • Lack of concern about personal health data privacy • Use of HIPAA compliant technologies • Trust in tech developer • Governmental and local leaders’ involvement • Private-public partnerships • Perceived as cost-saving • COVID-19 experience • General availability and diffusion of tech devices • Risk of health data leakage • Lack of global privacy standards • Concerns around cost-effectiveness • Maintenance of the infrastructure • High training demand Technology and device • Ease of set-up and use • Easy interventions and advice • Translation in local language • Availability of tech support • Large fonts, audio support (e.g., for visually impaired) and recorded materials • Appealing graphic features, fun approach, customization • Portable, light devices • Content and language complexity • Excessive length of interactive sessions • Unavailability in local language • Non-communicability between different software • Bulky, heavy devices • Difficult to clean HCP healthcare provider, HIPAA Health Insurance Portability and Accountability Act Section title: Health literacy and self-efficacy Educational score: 3.6328165531158447 Domain: biomedical Document type: Other Language: en E-health requires a different form of patient participation than traditional consults. This participatory nature could positively influence self-efficacy and health literacy. Many healthcare providers (HCPs) and patients felt that e-health facilitated autonomy and increased the quality of self-management. E-health was often used as a tool for education and was seen to have advantages compared to traditional in-person education, allowing for more detailed instruction over an extended period . For patients, being able to review information at their convenience was important. Additionally, using e-health, they gained a better comprehension of their own physiological reactions and general health status , leading to improved self-management of disease . Section title: Health literacy and self-efficacy Educational score: 0.9750158190727234 Domain: other Document type: Other Language: en “She was so proud of herself that she contacted the research team the next day to describe her successful self-management.” Oakes, North-Dakota . Section title: Health literacy and self-efficacy Educational score: 2.1697771549224854 Domain: biomedical Document type: Study Language: en Nonetheless, the view of e-health increasing self-efficacy was not universal. Whereas many patients found e-health helpful in interpreting their symptoms, some did not experience this benefit . Some participants felt that e-health was taking responsibility away from them, as the HCP would monitor their parameters . Furthermore, some HCPs noted that in certain socio-economic contexts, a diagnostic/therapeutic e-health intervention was not sufficient as a standalone measure but should rather be integrated with behavioural change interventions . Section title: Health seeking behaviours Educational score: 2.006091833114624 Domain: biomedical Document type: Other Language: en Despite the differing opinions on the ultimate impact on self-efficacy, one clear beneficial aspect of e-health interventions in remote locations was the positive impact these had on health-seeking behaviours among patients. HCPs recognized the value of e-health in reaching patients who might otherwise avoid traditional consults for various reasons, or who would have ignored early warning symptoms . Section title: Health seeking behaviours Educational score: 1.721598744392395 Domain: biomedical Document type: Other Language: en Similarly, e-health facilitated communication with patients who typically only seek medical attention during exacerbations or when prompted by family members: Section title: Health seeking behaviours Educational score: 1.082553744316101 Domain: other Document type: Other Language: en “some young asthmatic patients who don’t ever come and see me unless they’re exacerbated or that , you know , or their mothers phone me and say they’re not very well. for the youngsters that’s good. Even my asthmatic guy , who I hardly ever see , whenever I send him a text I always get a reply”. Scotland . Section title: Technology access and digital literacy Educational score: 2.986948251724243 Domain: biomedical Document type: Study Language: en While e-health had the potential to positively impact health-seeking behaviour, the benefits could be hampered by problems with training. Adequate training for both patients and HCPs emerged as a key factor to enable technology access and the implementation of e-health interventions . Familiarity with technology, rather than age, was thought to be a key positive determinant of e-health adoption, suggesting that future generations of elderly individuals might be better equipped to utilize e-health solutions : Section title: Technology access and digital literacy Educational score: 1.0282315015792847 Domain: other Document type: Other Language: en “I had patients who are older than 90 who never owned a computer in their life and managed to do their sessions on their iPads and send it to me with no trouble. So , I think it depends on maybe education level and understanding , and maybe how things are explained to them.” Canada, Newfoundland and Labrador . Section title: Technology access and digital literacy Educational score: 1.9593344926834106 Domain: biomedical Document type: Study Language: en Previous experiences with technology also influenced acceptance, with nurses familiar with smartphones showing greater comfort and likelihood of using new interventions compared to those less technologically savvy . Section title: Technology access and digital literacy Educational score: 1.8064028024673462 Domain: biomedical Document type: Other Language: en Nonetheless, the influence of advanced age on e-health adoption was widely recognized, with several studies noting that elderly patients tended to use e-health less frequently . Some elderly patients reported struggling to learn how to use the devices , while HCPs expressed scepticism about their ability to do so . Section title: Technology access and digital literacy Educational score: 1.793267011642456 Domain: biomedical Document type: Other Language: en Another obstacle to the adoption of e-health within health systems was the lack of knowledge about existing e-health resources . Section title: Technology access and digital literacy Educational score: 1.6687335968017578 Domain: other Document type: Study Language: en Additionally, lower levels of education were linked to a preference for audio over text materials due to low literacy levels, identified as a barrier to use in multiple studies . Section title: Attitudes towards technology Educational score: 1.9273598194122314 Domain: biomedical Document type: Study Language: en Related to the quality of training at the time of implementation, we noted that pre-existing attitudes towards technology also played a role. Both patients and HCPs were more likely to use e-health when they had positive attitudes towards learning new technologies: Section title: Attitudes towards technology Educational score: 1.0760740041732788 Domain: other Document type: Other Language: en “[Providers] usually like new technology , I think they will be excited to use it and therefore they are likely to download [the app]. In addition , people prefer digital information than opening and reading what is in the […] book.“ Uganda, Jinja district . Section title: Attitudes towards technology Educational score: 2.615053653717041 Domain: biomedical Document type: Study Language: en Conversely, negative attitudes towards technology acted as a barrier. In patients, the following were specific barriers for the acceptability of new technologies: lack of interest in technology , lack of motivation of the patient or the caregiver , negative attitudes towards mobile phones and technology in general or the “technologization” of society , not feeling comfortable with e-health or technology , perceiving it difficult to learn a technology , and a strong preference for in-person consults . Some HCPs were also sceptical towards technology, expressing concerns that experienced physicians may be reluctant to change their way of practicing medicine . Section title: Attitudes towards the use of e-health interventions Educational score: 1.946457028388977 Domain: biomedical Document type: Study Language: en The personal attitudes of patients and HCPs regarding e-health interventions varied greatly across settings and countries. Generally, patients were more likely to engage if they felt comfortable with e-health prior to use , saw it as convenient , wanted to know more about their condition or health information or had a baseline interest in e-health . Section title: Attitudes towards the use of e-health interventions Educational score: 1.7588374614715576 Domain: biomedical Document type: Other Language: en Overall, e-health was perceived to be associated with several benefits. With some interventions, physical appointments were not needed, which made scheduling for patients easier and convenient . “to go into a facility […] sometimes that’s really onerous for people, especially people who are suffering from COPD, so they’re going to have shortness of breath and exertion and find it even harder to get from the parking lot into the hospital” Canada, Newfoundland and Labrador . Section title: Attitudes towards the use of e-health interventions Educational score: 1.9025309085845947 Domain: clinical Document type: Other Language: en Non-live interventions were especially acceptable, allowing patients to access care on a flexible schedule, accommodating those unable to take time off work during business hours . This encouraged patients to seek help earlier (especially when experiencing debilitating symptoms ), reduced travel costs , increased independence due to lack of need for a companion and allowed provision of care during times when a trip was not possible, e.g. due to weather conditions . Section title: Attitudes towards the use of e-health interventions Educational score: 1.865710973739624 Domain: biomedical Document type: Other Language: en Some HCPs felt encouraged that the interventions gave patients more responsibility and made them more accountable, as their actions were tracked. This led to higher therapy adherence and better outcomes . Some patients felt reassured by being monitored 24 h a day and receiving personalized care . Section title: Attitudes towards the use of e-health interventions Educational score: 3.0824320316314697 Domain: biomedical Document type: Study Language: en Nonetheless, numerous sources also reported negative attitudes towards the use of e-health technologies. Patients’ and HCPs’ negative feelings arose primarily from a sense of hyperconnectivity, increased workload , and scepticism about the reliability of devices and efficacy of the interventions . Many patients and HCPs reported a sense of threat over the risk of losing human contact and face-to-face clinical interactions [ 35 – 37 , 49 , 67 ]. In one study, this was described as feeling “chained” to the digital environment: Section title: Attitudes towards the use of e-health interventions Educational score: 1.1092056035995483 Domain: other Document type: Other Language: en “[…] since thoughts about the system and remembering to perform the tests were always present. This could lead to a feeling of being “fed up” and a wish for “a break” from the system” Sweden, Västerbotten county . Section title: Attitudes towards the use of e-health interventions Educational score: 2.514256715774536 Domain: biomedical Document type: Other Language: en Some HCPs thought that their reliance on their mobile phones would affect the way patients view them, believing it is rather a distraction or a tool to compensate for lack of knowledge, and could act as a barrier to use . Some HCPs feared that they would lose their current autonomy by being tracked and followed through standardised interventions . Devices could also display false abnormalities, such as low oxygen saturation from cold fingers, causing unnecessary anxiety and doubt— issues typically avoided when an HCP would interpret results in traditional consultations . Section title: Attitudes towards the use of e-health interventions Educational score: 1.5949398279190063 Domain: clinical Document type: Other Language: en Many HCPs complained that their workload for the same number of patients increased due to extra tasks and information to review and retain , especially in the set-up phases of a new intervention . Section title: Attitudes towards the use of e-health interventions Educational score: 1.9305338859558105 Domain: biomedical Document type: Study Language: en Individual patient characteristics also contributed to shaping attitudes in a negative way. For example, the severity of health conditions could affect the feasibility: pulmonary rehabilitation and e-health interventions requiring physical exercise were viewed by some patients as unacceptably demanding . Section title: Attitudes towards the use of e-health interventions Educational score: 1.6606911420822144 Domain: biomedical Document type: Other Language: en Evidence of the efficacy of e-health for improving care was seen as important for both patients as well as for HCPs: Section title: Attitudes towards the use of e-health interventions Educational score: 1.0745748281478882 Domain: other Document type: Other Language: en “ […] I think that type of evidence is what’s going to change my mind as a practitioner about whether it’s worth using it or not.” Canada, Newfoundland and Labrador . Section title: Attitudes towards the use of e-health interventions Educational score: 1.2530267238616943 Domain: other Document type: Other Language: en “more information justifying the use of the telemonitoring system was needed, since the purpose of using it was unclear” Sweden, Västerbotten county . Section title: Financial considerations and incentives Educational score: 2.1507294178009033 Domain: biomedical Document type: Other Language: en The use of e-health was facilitated for patients when the equipment was free or at least affordable , for example by saving on commutes . For HCPs, the use of e-health was facilitated if the intervention was perceived as cost-effective for the healthcare facilities they were working in . Conversely, high costs of electronic devices or use of personal devices and mobile data , the need to share devices, such as a mobile phone, due to the high cost of acquisition of own device , and lack of insurance represented barriers to the use of e-health interventions: Section title: Financial considerations and incentives Educational score: 1.4384204149246216 Domain: other Document type: Other Language: en “A nurse with experience conducting mHealth programs endorsed this concern by adding that only about 10% of participants may remain in the mHealth intervention if the insurance company stopped paying for the service.” Canada, Newfoundland and Labrador . Section title: Financial considerations and incentives Educational score: 1.7253707647323608 Domain: biomedical Document type: Other Language: en Uncertainty about costs caused scepticism and reluctance to engage in e-health. When the participants had little information about actual costs, they feared that new interventions would be expensive . Section title: Financial considerations and incentives Educational score: 1.6337950229644775 Domain: biomedical Document type: Other Language: en A significant disincentive was the lack of financial rewards, where the HCP would feel not adequately compensated for using e-health , even though it brought clinical benefits: Section title: Financial considerations and incentives Educational score: 1.0951505899429321 Domain: other Document type: Other Language: en “a physician insisted: ‘ I mean we’re all so busy that nobody wants to do anything for free because why would I do that for free if I get paid for it.’ ” Canada, Newfoundland and Labrador . Section title: Interpersonal level factors Educational score: 2.2410457134246826 Domain: biomedical Document type: Study Language: en The following level of our framework captures the internal dynamics of the healthcare teams and health facilities, and relationships with and between patients and their families, influencing the feasibility and acceptability of the interventions. Section title: Patient-technology-provider relationship Educational score: 2.6985225677490234 Domain: biomedical Document type: Study Language: en E-health significantly impacted patient-HCP relationships and communication patterns, with reduced face-to-face interaction emerging as a key sub-theme. This allowed HCPs to manage more patients , decreased the need for physical infrastructure , and, during the COVID-19 pandemic, reduced the risk of transmission . However, this also resulted in negative feelings among both patients and HCPs, as remote visits were felt to affect nonverbal communication and decrease interpersonal connection . Section title: Patient-technology-provider relationship Educational score: 0.921660304069519 Domain: other Document type: Other Language: en “I like to have a bit of actual contact and eye contact , and hear the tone of someone’s voice , and a gentle touch sometimes can be so reassuring , you know. I think it’s going to be lost with this type of technology.” Canada, Newfoundland and Labrador . Section title: Patient-technology-provider relationship Educational score: 1.7949588298797607 Domain: biomedical Document type: Other Language: en Nonetheless, some noted that e-health allowed for the maintenance of good communication if the patient and HCP already knew each other . Indeed, many participants expressed the need or preference to continue face-to-face contact in some form, even when e-health was used . Section title: Patient-technology-provider relationship Educational score: 1.3816859722137451 Domain: other Document type: Other Language: en The idea of being connected 24/7 created expectations in some cases, and some HCPs expressed concerns about the constant need to be always on demand: Section title: Patient-technology-provider relationship Educational score: 1.2723039388656616 Domain: clinical Document type: Other Language: en “[a patient] would often e-mail about his condition […] with questions that required careful consideration and reference to the notes , so it was actually pretty labour intensive without really much or any associated clinical benefit” Scotland . Section title: Patient-technology-provider relationship Educational score: 1.666908860206604 Domain: biomedical Document type: Other Language: en Frequent contact also resulted in a feeling that patients were being better monitored by HCPs , but on the other hand, was criticized by HCPs for potentially giving a false sense of security . Section title: Clinical team dynamics Educational score: 1.8848166465759277 Domain: biomedical Document type: Other Language: en Within organisations, e-health could facilitate contact between HCPs. For example, pharmacists could connect with physicians and physicians could connect with specialists not normally easily reachable such as pulmonologists, pulmonary rehabilitation experts and palliative care physicians . This seemed to be the case in remote areas specifically, as often only a primary care physician was available to offer care, and the patient was unable or unwilling to move to a hospital with specialised care: Section title: Clinical team dynamics Educational score: 1.122227430343628 Domain: other Document type: Other Language: en “I dream of a system where the primary care docs (doctors in the community) get to phone a friend and the palliative care physician does the consultation with the primary care provider” Rural America . Section title: Clinical team dynamics Educational score: 1.3898714780807495 Domain: other Document type: Other Language: en Team members were also able to communicate with each other, building cohesion and optimising care for patients: Section title: Clinical team dynamics Educational score: 1.0595436096191406 Domain: other Document type: Other Language: en “With [this phone] we are able to work as a team , […] it has made communication between the team quite good and also for the patients because if you don’t communicate as a team.they are getting less care than they should.’’ KwaZulu-Natal, Umzinyathi district . Section title: Clinical team dynamics Educational score: 1.864368200302124 Domain: biomedical Document type: Study Language: en However, in one study, the increased workload and shifting of care needs associated with the e-health intervention resulted in the deterioration of staff cohesion as new skills and extra responsibilities became necessary . Section title: Family engagement Educational score: 1.8284236192703247 Domain: biomedical Document type: Other Language: en E-health did not only affect communication inside HCP-teams and between HCPs and patients. The possibility to view information at home through a device allowed for greater family involvement in education and therapy . Section title: Family engagement Educational score: 1.7319086790084839 Domain: biomedical Document type: Other Language: en Engaging with the family was important, especially with paediatric patients, as parent involvement was required for participation . Section title: Community level factors Educational score: 1.6949763298034668 Domain: biomedical Document type: Other Language: en The third level is represented by community factors. Aspects of the local settings and community health facilities played an important role, as did local culture. Section title: Community resources Educational score: 1.7126492261886597 Domain: biomedical Document type: Other Language: en The availability of a reliable internet or phone connection represented an important enabler to the use of e-health. Local network issues resulting in poor audio connection , or ‘freezing’ of the app and upload issues led to underuse and delays in patient care. Section title: Community resources Educational score: 1.8678317070007324 Domain: biomedical Document type: Study Language: en Some studies noted that the greatest trigger for introducing e-health was the absence of pre-established infrastructure or services. Examples included places without access to basic health resources , and locations where specialist services were unavailable . Section title: Community engagement Educational score: 1.9660433530807495 Domain: biomedical Document type: Other Language: en Proper design and planning of the e-health intervention, including community engagement, was seen as essential. Engaging patients and HCPs during the development and planning process improved the usability of technologies. Section title: Community engagement Educational score: 1.2125988006591797 Domain: other Document type: Other Language: en “The feedback [from human-computer interface testing] resulted in language changes, directional aids, greater use of graphics and age-specific content.” Australia, Victoria . Section title: Community engagement Educational score: 1.8135457038879395 Domain: biomedical Document type: Study Language: en Community engagement was equally important in the implementation phases. One study noted that once the programme was functioning, additional patients should be recruited by a local familiar HCP or another trustworthy stakeholder . Section title: Community engagement Educational score: 1.924874186515808 Domain: biomedical Document type: Study Language: en “study participants […] conveyed that uptake of tele-COPD programs may be greater if tele-health visits were described and demonstrated to patients by their own health care provider or other trusted individual(s).” USA, North Carolina . Section title: Community environment and local culture Educational score: 2.75166392326355 Domain: biomedical Document type: Review Language: en Adaptation to local cultures and conditions was important for intervention acceptability. One study commented on the cultural significance of tobacco use within the community, highlighting the challenge of addressing health-related habits when these are embedded in local culture . Another example involves the perception of symptoms, which may differ across cultures and populations . Because this review included papers from various locations, specific aspects varied. However, some common themes emerged. Two studies discussed the importance of including aspects from local culture, including local (non-western) remedies and spiritual aspects : Section title: Community environment and local culture Educational score: 1.63327157497406 Domain: biomedical Document type: Other Language: en “one participant of this study described his own uncommon approach to treat patients in his […] community by integrating traditional and Western medical practices. He felt this approach helped him to enhance his relationship with the community and increase the overall compliance with medical procedures.” Peru, Highlands and Amazon basin . Section title: Community environment and local culture Educational score: 1.5048656463623047 Domain: other Document type: Other Language: en To make the interventions more appropriate for local communities, two studies discussed the need to include some examples that are relevant or meaningful for the local cultural context, highlighting the importance of cultural appropriateness especially in indigenous communities: Section title: Community environment and local culture Educational score: 1.2620065212249756 Domain: other Document type: Other Language: en “examples of more relevant content included stories of Aboriginal people living with asthma, and information on housing issues (eg mould), as well as using different languages and visual learning formats (eg appropriate coloring, characters, images, and pictures).” First Nations and Inuit communities in Canada . Section title: Community environment and local culture Educational score: 1.0688575506210327 Domain: other Document type: Other Language: en “Specific adaptations to [the intervention] included incorporating […] the imagery of the circle; the seven sacred teachings; and inclusion of elders, family members and the community. […] The wolf was recommended as a culturally relevant mascot based on its symbolism as a teacher and member of a larger community, and for its capacity to howl, which was symbolic of strong lungs.” Canada, Alberta . Section title: Community environment and local culture Educational score: 1.4653807878494263 Domain: other Document type: Other Language: en “[patients] recommended adding environmental factors common to Aboriginal communities (eg road dust, forest fire smoke) as relevant information to newly developed materials” First Nations and Inuit communities in Canada . Section title: Community environment and local culture Educational score: 1.4581717252731323 Domain: other Document type: Other Language: en One crucial part of adapting interventions was translation into local languages : Section title: Community environment and local culture Educational score: 1.1037778854370117 Domain: other Document type: Other Language: en “A lot of kids are adopted by a grandparent and their education is very low to none and they often don’t speak English . educational materials need to be in different languages for people who do not speak or read English and French.” Community health worker; First Nations and Inuit communities in Canada . Section title: Community environment and local culture Educational score: 1.9225369691848755 Domain: biomedical Document type: Other Language: en Beyond cultural considerations, opinions varied on the best adaptation approach for different patient populations and conditions. Some favoured single-condition interventions, while others, especially caregivers of multimorbid patients, preferred interventions addressing multiple conditions . Section title: Community environment and local culture Educational score: 1.2034841775894165 Domain: other Document type: Other Language: en ‘‘I think the phones would be helpful if not only looks at TB. It should also look at other diseases or problems.like immunizations and ante-natal care.’’ KwaZulu-Natal, Umzinyathi district . Section title: Societal level factors Educational score: 1.879440188407898 Domain: biomedical Document type: Other Language: en The fourth level of the framework is represented by societal factors influencing the implementability of e-Health interventions. Section title: Data protection standards Educational score: 1.845822811126709 Domain: biomedical Document type: Other Language: en Universally for various communities, health information privacy played a large role. Depending on the setting, health data privacy and transparency of data were not seen as barriers for the utilization of e-health interventions: Section title: Data protection standards Educational score: 1.2920492887496948 Domain: other Document type: Other Language: en “Sending data regarding health parameters electronically was considered quite harmless: ‘There’s information that is more sensitive.But I mean this , so what? I’ve got COPD , okay , lots of people know that.’ ” Sweden, Västerbotten county . Section title: Data protection standards Educational score: 1.5482195615768433 Domain: biomedical Document type: Other Language: en Some noted that e-health even offered increased privacy compared to paper-based practice . Section title: Data protection standards Educational score: 1.035827398300171 Domain: other Document type: Other Language: en ‘‘we lose [papers] and we lost them under the car seats or on the floor and you find them everywhere.[the phone] is always in your pocket.’’ KwaZulu-Natal, Umzinyathi district . Section title: Data protection standards Educational score: 2.2478368282318115 Domain: biomedical Document type: Study Language: en One study cited the credibility of the technology developer to play a significant role in the privacy concerns . On the other hand, several studies reported on participants’ fears about private health information unwanted disclosure , as electronic communication was seen at risk of cyberattacks and data leakage . It should also be noted that privacy standards are not universal and may be associated with cultural values, representing potential barriers to the use of e-health interventions: Section title: Data protection standards Educational score: 1.2403568029403687 Domain: other Document type: Other Language: en “A cultural belief referred to as “Appalachian pride,” could be a barrier for tele-health adoption. Appalachian pride is comprised of multiple cultural values (e.g., privacy) that are upheld in tight-knit communities. Appalachian pride may impact tele-health adoption and preferences.” USA, North Carolina . Section title: Healthcare systems involvement Educational score: 1.5256792306900024 Domain: other Document type: Other Language: en Governmental health system involvement was seen as necessary to improve trust and coordination. Coordination through government could facilitate the implementation and scaling-up of the intervention. Section title: Healthcare systems involvement Educational score: 1.1571853160858154 Domain: other Document type: Other Language: en “. the one thing that will make it (tele-health) have more chance of success is if NHS Highland do set up infrastructure to support the delivery of that” Scotland . Section title: Healthcare systems involvement Educational score: 1.1454521417617798 Domain: other Document type: Other Language: en It was also acknowledged that the government and local policy leaders had the power to block the wider use of the intervention, and their engagement was crucial: Section title: Healthcare systems involvement Educational score: 1.298509120941162 Domain: other Document type: Other Language: en “The administrator explained that the Ministry of Health in Uganda and local district health officials would need to approve the app prior to large scale distribution across public and private facilities. Additionally, support at these leadership levels will be critical for widespread uptake and implementation of [the intervention].” Uganda, Jinja district . Section title: Healthcare systems involvement Educational score: 1.6792011260986328 Domain: other Document type: Other Language: en Regardless of cost-effectiveness, it was thought that the scaling up of any intervention involving multiple stakeholders needed to be coordinated at the governmental level . Other providers were sceptical that an e-health program would be implementable without a total overhaul of the local healthcare systems . Section title: Societal costs Educational score: 1.6506881713867188 Domain: other Document type: Other Language: en If implemented correctly, e-health was seen as potentially saving costs at the societal level through decreased use of other healthcare resources . Nonetheless, some users were concerned about the cost of implementation and maintenance of infrastructure : Section title: Societal costs Educational score: 1.0617029666900635 Domain: other Document type: Other Language: en “there is the initial cost of establishing the infrastructure, including costs related to storing data in the cloud. In addition, costs related to the maintenance and replacement of outdated technology were discussed[.] ” Canada, Newfoundland and Labrador . Section title: Societal costs Educational score: 1.0882359743118286 Domain: other Document type: Other Language: en It was suggested that the partnership of private entities with public ones could bring down costs : Section title: Societal costs Educational score: 1.075027585029602 Domain: other Document type: Other Language: en “some of this could be outsourced to private entities that are already doing this type of work, thereby reducing expenses to taxpayers.” Canada, Newfoundland and Labrador . Section title: Influence of larger scale societal developments Educational score: 1.816611886024475 Domain: biomedical Document type: Other Language: en The COVID-19 pandemic proved to be an effective facilitator for the use of e-health. Some studies cited that because of the pandemic, the number of clinic visits was reduced and replaced by e-health . This was attributed to the desire to decrease contagion , as well as the belief that e-health was more accessible than in-person consults . Section title: Influence of larger scale societal developments Educational score: 1.8603461980819702 Domain: biomedical Document type: Other Language: en General technological development was also noted as an external factor affecting e-health. As technology advanced and became more widespread in society, its use in medicine in the form of e-health also became more acceptable, reducing barriers such as confusion about compensation , complexity and privacy : Section title: Influence of larger scale societal developments Educational score: 1.343767523765564 Domain: other Document type: Other Language: en “With current advances in secure, HIPAA-compliant, web- based systems with billing capabilities, it is now possible for more health professionals to consider expansion of their practice to include this type of care for patients with asthma and other disease states.” USA, North Dakota . Section title: Influence of larger scale societal developments Educational score: 1.6757175922393799 Domain: other Document type: Other Language: en Several points relating to the future of e-health were discussed. It was expected that as the use of e-health becomes more widespread, the costs would come down due to economies of scale. While some doubted that local health systems would be ready to implement e-health , and demands for training and infrastructure may be too high , others thought scaling up e-health may also improve patient outcomes as care becomes more standardised . Section title: Influence of larger scale societal developments Educational score: 1.2700194120407104 Domain: other Document type: Other Language: en “Large-scale implementation may realize benefits in standardization of practice and economies of scale” Scotland . Section title: Technology and device level factors Educational score: 2.1871390342712402 Domain: biomedical Document type: Study Language: en Finally, the fifth level corresponds to intrinsic technology and device characteristics reported to increase or decrease the implementability of the interventions. Section title: Complexity Educational score: 1.6689304113388062 Domain: biomedical Document type: Other Language: en It was seen as important that the devices and technologies were not too complicated , easy to set up , to learn , and to use , as well as reliable . Section title: Complexity Educational score: 1.4296241998672485 Domain: biomedical Document type: Other Language: en Many studies highlighted a preference for simple and clear language . Section title: Complexity Educational score: 1.076029896736145 Domain: other Document type: Other Language: en “One nurse […] thought the language should be “set at a grade six reading level , so there’s no issues with comprehension of what they’re being asked or told.” Canada, Newfoundland and Labrador . Section title: Complexity Educational score: 1.5679256916046143 Domain: clinical Document type: Other Language: en Some patients felt that the advice was particularly useful when it pertained to achievable and easy interventions: Section title: Complexity Educational score: 1.4719690084457397 Domain: other Document type: Other Language: en “Majority of the participants preferred information that was immediate and easy to implement, i.e., nutrition, effects of tobacco and alcohol, and government schemes. ” India, Darrang and Kamrup districts . Section title: Complexity Educational score: 1.5667084455490112 Domain: biomedical Document type: Other Language: en Preference for simple, relevant, easy-to-follow advice, such as for example interactive maps for home visits, was not only limited to patients but also echoed by some HCPs . Section title: Complexity Educational score: 1.9155718088150024 Domain: biomedical Document type: Study Language: en On the other hand, overly complex systems were seen as a barrier to continued use, both for patients as well as for HCPs . Several studies noted that also contents complexity, as well as excessive session duration, led to the underuse of the interventions: Section title: Complexity Educational score: 1.0348063707351685 Domain: other Document type: Other Language: en “A participant said , “If the call comes while I am busy working , I will not listen to it if it is long. So I want the call to be shorter than 5 min.” India, Darrang and Kamrup districts . Section title: Complexity Educational score: 1.0643631219863892 Domain: other Document type: Other Language: en “most participants identified materials that were ‘too busy’ with ‘too much writing’.” First Nations and Inuit communities in Canada . Section title: Complexity Educational score: 1.3142547607421875 Domain: other Document type: Other Language: en “Interpreting the test results was also perceived as difficult, including both the numbers and the charts, which made it difficult to notice if there had been any changes” Sweden, Västerbotten county . Section title: Complexity Educational score: 1.6533799171447754 Domain: biomedical Document type: Other Language: en Related to complexity was the ability to solve problems when they emerge. Two studies recognised that there would inevitably be technical problems with e-health. The provision of technical support was recommended . Section title: Accessibility and integration into existing systems Educational score: 1.7521597146987915 Domain: biomedical Document type: Other Language: en Integration of the e-health technology into existing systems was found to be important. Some found that e-health was underused because different setups prevented different technologies from communicating, and it was, therefore, challenging to integrate new products into daily practice : Section title: Accessibility and integration into existing systems Educational score: 1.4559353590011597 Domain: other Document type: Other Language: en “not achieving a full integration with legacy EMR systems and the coexistence of 2 management systems (usual care and IC) at the same time (which implied some duplicity of tasks) were the main barriers to adoption.” Spain, Lleida . Section title: Accessibility and integration into existing systems Educational score: 3.1456727981567383 Domain: biomedical Document type: Study Language: en The importance of accessibility features, such as large fonts loud voices , audio components for the visually impaired and text for the hearing impaired , was emphasized as their absence hindered utilization for patients with corresponding disabilities. For e-health equipment utilizing visual displays like computer or mobile applications, a visually appealing graphic user interface was favoured across several studies . Graphics were found to engage users and bolster long-term adherence . Reminders and notifications were explicitly preferred and linked to improved medication adherence . Some studies highlighted the preference for recording and reflecting on the given advice . Section title: Accessibility and integration into existing systems Educational score: 0.9869788289070129 Domain: other Document type: Other Language: en “I would have liked the option to record the call. I would have recorded the call and listened to it again whenever I wanted to.” India, Darrang and Kamrup districts . Section title: Device quality Educational score: 2.139014482498169 Domain: biomedical Document type: Study Language: en Device portability and cleanliness were key factors affecting feasibility and acceptability. Bulky, heavy devices discouraged mobility , and difficulty in cleaning after shared use was cited as a deterrent . Section title: Device quality Educational score: 2.185263156890869 Domain: biomedical Document type: Study Language: en Device quality mattered significantly; poor-quality devices, particularly older ones, were deemed useless . While many studies indicated a preference for clinic-supplied devices , one study reported that using personal smartphones over separate devices increased the likelihood of utilizing interventions such as mobile apps . Section title: Device quality Educational score: 1.7576583623886108 Domain: biomedical Document type: Study Language: en There was also little consensus on how the information should be presented, as the studies were concerned with different populations and conditions, not comparing approaches. One study covering educational resources for paediatric populations indicated that a fun approach was preferred: Section title: Device quality Educational score: 1.0980788469314575 Domain: other Document type: Other Language: en “Our results are consistent, revealing the need for children to ‘be taught in a fun way by doing activities’ and the need for personal interaction with parents.” First Nations and Inuit communities in Canada . Section title: Device quality Educational score: 1.3963894844055176 Domain: other Document type: Other Language: en Customization based on user preferences and available resources emerged as the key consideration. Section title: Discussion Educational score: 3.292262077331543 Domain: biomedical Document type: Study Language: en This QES identified various factors relating to the effective implementation of e-health interventions for chronic respiratory diseases in remote locations. In general, e-health interventions were well received but also carried a number of concerns and limitations that could reduce their effectiveness. Moreover, e-health was deemed insufficient as a standalone intervention and should be integrated into existing care practices [ 35 – 37 , 58 , 64 , 72 ]. Section title: Discussion Educational score: 3.4042296409606934 Domain: biomedical Document type: Study Language: en E-health was identified by many patients living in remote communities and HCPs as a tool with the potential to overcome the obstacle of distance to health facilities, reducing commutes and related costs, and expanding access to care. Increased self-efficacy and health literacy were additional benefits at the individual level. Concerns and barriers were noted across all levels. At the community level, concerns related to lack of telecommunications and healthcare infrastructure— hence, while e-health was perceived as a solution to certain resource limitations, other resource limitations undermined its potential. The main concerns at the interpersonal level related to the reduction of in-person interactions and fear of losing personal connections. Additionally, HCPs identified additional burdens for the staff, negative attitudes towards technology, and poorly planned implementation processes as barriers to successful adoption. Section title: Discussion Educational score: 3.2254624366760254 Domain: biomedical Document type: Review Language: en Our findings are in line with previously published reviews , but some specifically relevant issues are worth highlighting. Adapting e-health interventions to the local setting was seen as especially relevant in remote and rural environments . Some studies suggested that integrating aspects important to the community, such as symbolism , traditional medical practices and habits specific to the location with ‘Western’ medical information was appreciated, leading to higher adoption. By contrast to cultural factors, considerations about the availability of e-health or applications in the preferred language of the patient/community were discussed in very few papers, highlighting an important gap in the evidence base. Section title: Discussion Educational score: 4.016016483306885 Domain: biomedical Document type: Study Language: en In addition to the setting, the target population is also key to consider at an early stage since the feasibility of e-health interventions may vary between patient groups. For example, patients in poor health with advanced disease might be physically unable to self-measure or undergo pulmonary rehabilitation without in-person assistance . Another aspect of this was described in a meta-ethnography on telemedicine in COPD: while patients with high disease burden experienced an increased need for telemedicine, patients in a stable period or at an early phase of the illness perceived the disease as becoming too prominent with telemedicine, and a need to be more detached . Familiarity with technology in general and training needs may also differ between groups, as we identified longer learning times for patients with lower education levels and advanced age . Section title: Discussion Educational score: 2.3248167037963867 Domain: biomedical Document type: Review Language: en Financial concerns were identified as significant barriers across various levels in the majority of studies. A clear understanding of financial aspects is crucial for the successful implementation of e-health initiatives. For instance, patients should be assured that e-health services are covered through insurance or government support. The importance of financial aspects was mirrored by a systematic review of patient satisfaction with telehealth demonstrating the need to provide additional means of reimbursement from the government in order to make telehealth available . Section title: Discussion Educational score: 3.471264123916626 Domain: biomedical Document type: Study Language: en It is worth noting that the term ‘e-health’ includes a wide variety of interventions, and although the choice for a specific e-health modality was not the focus of this QES, many papers discussed their reasoning behind this. Individual preferences varied: some users preferred mobile-phone-based interventions and apps , others preferred video interaction over phone calls , some wanted live interactions with immediate feedback , others preferred non-live communication done at the convenience of the patient , some liked group-based interventions , others wanted graphical aspects rather than text or audio and yet others expressed the desire to combine different modalities . While some studies described the preference for combining modalities, such as video consults with devices for measuring physical parameters, others cautioned against it, warning that multiple electronic systems from different manufacturers may not communicate . However, while individual preferences emerged, none of the studies focused on comparing different types of e-health interventions, and assessing the advantages and disadvantages of different modalities is beyond the scope of this paper. Section title: Discussion Educational score: 4.078383922576904 Domain: biomedical Document type: Study Language: en This study has a number of limitations. First, there is a risk of over- or underestimating e-health acceptability and feasibility barriers due to selection bias in the primary research, preferentially including individuals with explicit views or motivations towards e-health. Second, it is challenging to generalize from studies undertaken from a wide range of contexts—across countries, populations, languages, continents etc—given the impact of setting- and population-specific factors; nevertheless, we found themes common across settings. Third, many studies focused on the initial phases of the implementation, potentially magnifying perceptions related to technology novelty while overlooking factors influencing longer-term use and adherence. Moreover, geographical representation poses a limitation, with an overrepresentation of Global North viewpoints. Certain populations (e.g. non-English speakers) were also underrepresented in the included papers, which may further limit generalizability of our findings. Additionally, while we aimed to include various patient populations, there was a higher number of studies on COPD and asthma compared to cystic fibrosis and other chronic diseases, potentially limiting the generalizability of findings. Lastly, there’s the possibility of publication bias, wherein less impactful qualitative research might not have been published , potentially leading to an incomplete representation of viewpoints. We recognize that e-health is a rapidly evolving field, and our findings will need ongoing validation against emerging evidence, technological advances, and changes in digital policy as they become available. Section title: Discussion Educational score: 3.9656593799591064 Domain: biomedical Document type: Review Language: en Despite these limitations, this synthesis may have some implications for both practice and research. Our findings suggest that a functional infrastructure that can respond to changing demands is a prerequisite for the effective use of e-health. When considering a new intervention, all stakeholders should be involved in the design and implementation process. Informing patients about the content and goal, benefits and limitations of the intervention is essential to avoid miscommunication and to support willingness to engage . Even though customization may be more labour- and cost-intensive when compared to the use of generic interventions, attention should be paid to local and cultural aspects relating to language, beliefs and attitudes, as failure to do so may result in a lack of use in the longer term. General digital literacy was found to be an important factor relating to the use of specific e-health interventions and providing education to increase this could increase the adoption and use of e-health. Some level of governmental involvement is required to ensure compatibility of systems, adequate resources and equitable distribution of devices. Interesting areas for future research, beyond the scope of this synthesis, include comparing various e-health modalities and focusing on a specific disease under the umbrella of chronic respiratory disease, as well as comparing our findings with the use of e-health in non-respiratory diseases. Further research should include underrepresented populations and consider new technological and societal developments. Section title: Conclusions Educational score: 3.892029285430908 Domain: biomedical Document type: Review Language: en This synthesis identified several factors that can affect the successful implementation of e-health in remote and rural locations for chronic respiratory disease patients. These factors can be used to inform the design and implementation of future e-health interventions. Intervention objectives, target population, geographical location, telecommunication and health care infrastructure capacity, local culture, language, and norms, and available human resources should be carefully considered to optimise the interventions for the best outcomes. Relevant stakeholders must be consulted throughout the process to produce an appropriate and feasible intervention. Section title: Supplementary Information Educational score: 1.4485907554626465 Domain: biomedical Document type: Other Language: en Supplementary Material 1. | Review | biomedical | en | 0.999997 |
PMC11699755 | Section title: Introduction Educational score: 4.014252185821533 Domain: biomedical Document type: Review Language: en Schistosomiasis, one of the most devastating neglected tropical diseases, poses a persistent public health and economic challenge in the developing world. 1 , 2 , 3 It has recently spread from developing countries to European nations due to human migration and climate change. 4 , 5 The disease causes significant illness and death, 6 promotes the transmission of human immunodeficiency virus, 7 and can lead to bladder cancer. 8 Currently, there is no effective vaccine against schistosomes. The only available treatment is praziquantel (PZQ), a chemotherapy that has been in use for over 40 years. 9 However, relying solely on PZQ-based control programs is unlikely to achieve disease control goals, as PZQ-treated patients, especially children, quickly become reinfected. 10 , 11 Additionally, concerns about drug resistance in schistosomes, particularly in mass drug administration programs, are growing. 12 , 13 Section title: Introduction Educational score: 4.213442802429199 Domain: biomedical Document type: Study Language: en Freshwater snails serve as obligate intermediate hosts for the digenetic trematodes Schistosoma spp., the causative agents of schistosomiasis. This is because the life cycle of schistosomes involves asexual and sexual developmental stages within a snail intermediate host and a mammalian definitive host, respectively. Snail control, alone or in combination with other strategies, has proven to be the most effective means of reducing schistosomiasis prevalence in endemic areas. 14 , 15 However, the widely used molluscicide niclosamide has harmful effects on the aquatic ecosystem, as it is toxic to other aquatic animals. 16 , 17 Given the critical role of snails in the aquatic ecosystem, an ideal biocontrol strategy should aim to disrupt parasite life cycles without eliminating the intermediate snail hosts. Field evidence supports this strategy, as the introduction of schistosome-resistant Biomphalaria tenagophila snails to endemic areas in Brazil has resulted in reduced disease transmission. 18 Section title: Introduction Educational score: 4.232619762420654 Domain: biomedical Document type: Study Language: en Biomphalaria glabrata — Schistosoma mansoni has been used as a model system for studying the compatibility between snails and schistosomes, particularly snail resistance to schistosomes, since the mid-20th century. 19 These studies have primarily focused on immunological responses, with significant progress made in recent omic-based research. 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 It is well established that immunological responses have genetic bases. 30 , 31 Previous studies have shown that snail resistance or susceptibility to schistosomes has a strong genetic component. 32 , 33 , 34 , 35 , 36 Therefore, genetic mapping of schistosome resistance or susceptibility should offer valuable insights into these mechanisms and help elucidate the underlying immunological responses. This knowledge could potentially aid in developing biocontrol programs, which have shown promise in controlling vector-borne diseases through clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene drive technologies. 37 , 38 , 39 , 40 Section title: Introduction Educational score: 4.10983943939209 Domain: biomedical Document type: Study Language: en A significant study on the genetic analysis of compatibility between snails and schistosomes using the B. glabrata — S. mansoni model was published by Charles Richards in 1970. 32 Richards laid the foundation for understanding snail resistance to schistosomes through extensive classical crosses between snails with different resistance phenotypes. Genetic mapping using various mapping populations (most of which are pre-existing laboratory strains) and genotyping assays led to the identification of multiple resistant loci, located on different chromosomes, 41 , 42 , 43 , 44 , 45 , 46 yielding valuable insights into the resistance mechanisms while also leaving unanswered questions (see details in the discussion section). Section title: Introduction Educational score: 4.167630195617676 Domain: biomedical Document type: Study Language: en The success of genetic mapping relies heavily on the strategic design of mapping populations and genotyping technologies. Typically, classic mapping populations involve F2 offspring and backcrosses derived from crossing two inbred parents. Recombinant inbred (RI) lines or RILs, derived from the F2 population, offer distinct advantages for genetic mapping. RILs are created by crossing two parental strains with contrasting phenotypes followed by successive generations of inbreeding (selfing or full-sib mating). Meiotic crossover events result in a mosaic parental genome in each RI line, and subsequent inbreeding increases recombination events and leads to a rapid reduction in heterozygosity. 47 , 48 , 49 , 50 , 51 RILs have commonly been used in plant genetics and breeding but have seldom been utilized in animal genetics, particularly for non-model organisms. This is mainly due to the labor-intensive, expensive, and time-consuming process of generating animal RILs, as well as the significant challenges associated with their maintenance. Section title: Introduction Educational score: 4.13350248336792 Domain: biomedical Document type: Study Language: en We produced RILs from a cross between two well-defined homozygous lines of B. glabrata , the iM line and iBS90. 45 To dissect this genetic resource, we employed whole-genome sequencing (WGS), a high-throughput genotyping assay, to scan every single nucleotide across the genomes of 46 individual RIL snails, representing 46 phenotyped RILs. This approach, designated as RIL-WGS, allowed us to reveal a significant number of single-nucleotide polymorphisms (SNPs) and bin markers for subsequent genome-wide association study (GWAS) and quantitative trait loci (QTLs) analysis. As a result, we identified a small genomic region and genes within that region involved in anti-schistosome defense. Section title: Development and phenotyping of RIL snails Educational score: 4.272219657897949 Domain: biomedical Document type: Study Language: en Two important biological characteristics of the B. glabrata – S. mansoni system contributed to the successful genetic design of the snail RILs. Firstly, the wild-type pigmentation follows Mendelian inheritance patterns, allowing us to confirm successful crosses. Secondly, B. glabrata is hermaphroditic, enabling both crossing and selfing. The breeding of snail RILs originated from an effort that spanned over 20 years at The University of New Mexico. During this period, the iM line and iBS90 were developed through 81 and 41 generations of selfing from a single M line and BS90 snail, respectively. 45 Both lines were confirmed to be homozygous with contrasting resistance phenotypes and were used as parental snails to generate RILs. A total of 338 pairs, randomly generated from different F2 intercrosses, served as founders for subsequent selfing . Over more than 3.5 years, 137 RIL lines were obtained and tested for their phenotype. Among them, 118 RILs displayed a clear phenotype (42 resistant and 76 susceptible), while 19 had ambiguous phenotypes as only a portion of the snails in each line shed few cercariae. From the 118 RILs with clear phenotypes, we randomly selected 46 to represent 46 RI lines, with half ( n = 23) being either resistant or susceptible to schistosomes. Figure 1 Breeding scheme of RIL snails and testing of their resistant or susceptible phenotype Homozygous iM line (albino and susceptible to schistosomes) and homozygous iBS90 (pigmented and resistant to schistosomes) snails were used as parent snails. Single iM line and iBS90 snails were placed in a 1 L plastic cup and allowed to produce F1 progeny. Since pigmentation is a dominant Mendelian trait, three possible outcomes were expected in F1 snails: albino F1 snails produced by selfing the iM line snail and pigmented F1 snails generated either from selfing the iBS90 snail or from a cross between the iM line and iBS90 snails. Albino F1 snails were discarded, while pigmented F1 snails were retained. These pigmented F1 snails were placed individually in plastic cups to produce F2 snails. To distinguish the two types of pigmented F1 snails (from selfing or crossing), we examined the colors of the F2 snails. If all F2 snails were pigmented, it suggested they originated from a single F1 snail through selfing of the parental iBS90, and they were discarded. If the F2 snails showed a mix of albino and pigmented individuals, it indicated that their parental F1 snail was produced from a cross between the iM line and iBS90 snails. The F2 snails were then retained for subsequent breeding. An albino F2 snail and a pigmented F2 snail from these F2 snails were randomly paired and placed in a plastic cup to produce offspring (an F1 population). From this F1 population, individual F1 snails were kept in plastic cups and allowed to self for 10 generations; in each generation, one snail was selected to produce the next generation through selfing. As a result, the RILs were obtained. Each RI line was tested for the resistance phenotype after miracidia exposure and cercarial shedding, as described in the STAR Methods section. All photographs presented in this figure and in the graphical abstract were prepared by S.-M.Z. Section title: Quality control and evaluation of illumina reads Educational score: 4.117700576782227 Domain: biomedical Document type: Study Language: en Since the susceptible iM line and the resistant iBS90 were used as the parental snails to generate the RIL population, the Illumina reads used to assemble the genomes of the two lines 45 were retrieved for the current study. To ensure data comparability, the same quality control criteria described in the STAR Methods section were applied to all raw reads generated by 150 × 2 paired-end Illumina sequencing from the iM line, iBS90, and RILs. This resulted in 109.86, 118.34, and 664.4 Gb of clean reads from the iM line, iBS90, and RIL snails, respectively. Using our recently published chromosome-level assembly of B. glabrata as the reference genome, 52 the mapping rates of the iM line, iBS90, and RIL snails to the reference genome were 99%, 97%, and 96%, respectively. The sequence coverages for the iM line, iBS90, and the RIL population were 125X, 132X, and 16X, respectively ( Table S1 ). Section title: Identification of genome-wide SNPs Educational score: 4.157703399658203 Domain: biomedical Document type: Study Language: en A total of 9,079,154 SNPs were identified between the iM line and iBS90 parental snails. Among these, 273,307 SNPs were found in coding regions (including upstream −5kb and downstream +2kb), with 119,241 SNPs resulting in nonsynonymous substitutions. These SNPs were used to genotype the RIL population using CLC genomics workbench. The resulting sequence variation data were exported as variant call format files, which were then combined using the Bcftools software package. The mean depth of SNP coverage for the iM line, iBS90, and RIL snails was 125X, 111X, and 14.20 ± 0.38X, respectively ( Table S2 ). The distribution of SNP coverages is shown in Figure 2 A. After filtering out markers with high missing genotype (>20%), low coverage (<5), and low minor allele frequency (<10%), a total of 7,330,259 SNP markers were retained for downstream analysis of the association between phenotype and genome-wide SNPs. The distribution of these high-quality SNPs across the 18 chromosomes shows a general correlation with genomic sizes (r = 0.71) . The longest chromosome (chr 1) has the highest number of SNPs, while chromosome 6 has the lowest. The average SNP density across the 18 chromosomes is 9.11 ± 0.47/kb, with the highest density on chromosome 18 (11.97/kb) and the lowest on chromosome 3 (5.04/kb) . Figure 2 SNP analysis (A) Distribution of mean coverage (depth) for the 9,079,154 SNPs identified in the 46 RIL snails. (B) Number of SNPs across the 18 chromosomes. (C) Density of SNPs across the 18 chromosomes. Data are represented as mean ± SEM. Section title: GWAS and sliding-window analysis Educational score: 4.19385290145874 Domain: biomedical Document type: Study Language: en GWAS was performed on the RIL population using 7,330,259 SNP markers. A total of 120,698 SNPs were identified at a significant level of p ≤ 1 × 10 −5 . Among these, 837 SNPs were found to have significant associations ( p ≤ 5 × 10 −8 ) and were distributed across six chromosomes (chr 4, 5, 12, 15, 16, and 18). However, most of the SNPs (99.28%, 830/836) were located on chromosome 5 . Figure 3 GWAS (A) Manhattan plot showing genomic regions associated with snail resistance in the RIL population. Fisher’s exact test (−log10( p value)) was used to investigate the association between the resistant phenotype and SNPs ( n = 7,330,259). The bold dark dashed line and gray dashed line indicate the genome-wide significance levels at p = 5 × 10 −8 and p = 1 × 10 −5 , respectively. (B) Genetic divergence test (F ST ) between resistant and susceptible snails in 10-kb windows ( n = 51,284) for variants across the 18 chromosomes. The bold dark dashed line indicates significant genome-wide F ST at p ≤ 0.05 for each chromosome. (C) Sliding window-based F ST analysis on chromosome 5. Individual variants are represented by gray circles (displaying only those with F ST > 0.01, n = 74,499), while mean values are shown using sliding windows of 10-kb, marked by red lines. The blue bracket indicates a ∼3 Mb genomic region (positions: 39,634,500 nt–42,686,436 nt) with the highest divergence. Data are represented as mean ± SEM. Section title: GWAS and sliding-window analysis Educational score: 4.120139122009277 Domain: biomedical Document type: Study Language: en Fixation index F ST analysis of the 7,330,259 SNP markers was performed using a 10-kb window analysis on susceptible and resistant RIL groups. This approach allowed us to identify regions that showed differences between the two groups. A jackknife procedure was used to test whether F ST values were statistically different from zero. 53 We used a significance level of p ≤ 0.05, with a weighted F ST value of 0.3061 from the genome-wide distribution, to define high F ST outliers. Out of the 51,284 10-kb windows (F ST > 0), a total of 2,568 10-kb windows showed significant divergence between the two phenotypes. Among these, 2,563 10-kb windows were located on chromosome 5, two on chromosome 12, and three on chromosome 16 . Section title: GWAS and sliding-window analysis Educational score: 4.212114334106445 Domain: biomedical Document type: Study Language: en From the genome-wide 7,330,259 SNP markers, 470,229 SNPs on chromosome 5 were extracted and filtered to exclude SNPs with a significant deviation from the 1:1 segregation ratio ( p < 0.01) and low homozygous genotypes (≤40). This resulted in 74,499 SNPs at F ST > 0.01, including 42,473 SNPs at F ST > 0.3061 and 373 SNPs with the highest F ST value (>0.433) in a small genomic region (39.6–42.6 Mb) ( Table S5 ). Similar filtering criteria were applied to chromosomes 12 and 16, resulting in 16 significant SNPs on chromosome 12 and 5 significant SNPs on chromosome 16 ( Table S6 ). Overall, this analysis revealed a ∼3 Mb region (position: 39,634,500 nucleotides [nt]–42,686,436 nt) on chromosome 5 that exhibited the highest divergence in 10-kb windows between the susceptible and resistant RIL groups . The average F ST value of the specific 3 Mb region (0.41 ± 0.004) was significantly greater than that of the neighboring 3 Mb region to the left (0.29 ± 0.004), as determined by a pooled t test . We designated this genomic region or QTL as the B. glabrata schistosome resistance region 1 (BgSRR1). Section title: Bin marker identification and recombination pattern analysis Educational score: 4.15346622467041 Domain: biomedical Document type: Study Language: en Further analysis was conducted using bin marker-based genetic mapping. 54 , 55 To identify genomic intervals without recombination events in RIL populations, a 10-kb sliding window with Binmarker v.2.3 was used to generate a total of 2,190 bin markers from the 7,330,259 SNPs across the 18 chromosomes ( Table S7 ). The length of the bins was found to be correlated with the number of SNPs per bin (r = 0.91) . On average, the length of a bin was 353,427 bp, and each bin contained an average of 3,347 SNPs . Analysis of the distribution of the genome-wide recombination pattern revealed that the centromeric regions of most chromosomes had significantly fewer recombination events . Figure 4 Bin marker analysis (A) Scatterplot of bin markers, with the x axis indicating the length of the bin markers and the y axis indicating the number of SNPs per bin. Red dashed lines represent the linear trend line. Subplots on the top and right display histograms of bin length and the number of SNPs in each bin marker. Data are represented as mean ± SEM. (B) Distribution of bin markers across the 18 chromosomes. (C) Distribution of genome-wide recombination breakpoints on each of the 18 chromosomes. The red bar shows the relative number of observed recombination crossover sites in the RIL population. Section title: QTL analysis of snail resistance to schistosomes Educational score: 4.14360237121582 Domain: biomedical Document type: Study Language: en To further refine our analysis, we filtered 2,190 bin markers by excluding those with a significant deviation ( p < 0.001) from the 1:1 segregation ratio. The remaining markers were then used to construct a linkage map and conduct QTL analysis. A total of 2,121 bin markers were used to construct the genetic map, resulting in a map distance of 1,311.4 cM, with an average distance of 0.62 cM between adjacent markers . The number of bin markers varied across chromosomes, ranging from 81 on chromosome 8 to 182 on chromosome 11. The largest marker gap was observed on chromosome 1 with a length of 15.47 cM, followed by chromosomes 4, 8, and 12, each with gaps of approximately 13 cM ( Table S8 ). Figure 5 Recombination bin map of the 46 RILs The colors red, green, and blue represent the genotypes of the iM line (AA), the iBS90 (BB), and the heterozygous genotype (AB) and missing data, respectively. P1 and P2 refer to the iM line and iBS90, respectively. Section title: QTL analysis of snail resistance to schistosomes Educational score: 4.2609686851501465 Domain: biomedical Document type: Study Language: en QTL analysis indicated that snail resistance is controlled by a major QTL on chromosome 5, located between bin marker BgChr5_39634500:1:1 and BgChr5_39675885:1157240:1117, with a significant logarithm of odds (LOD) score of 6.04 . The bin marker on the left contains only one SNP at position 39,634,500 nt on chromosome 5, while the bin marker on the right encompasses 1,117 SNPs and spans a physical length of 1,157,240 bp. This QTL has an additive effect of 0.35 and accounts for 46.2% of the phenotypic variance. The 95% confidence interval for this QTL ranges from 37.5 cM to 39.5 cM, corresponding to a physical position from 39.6 to 41.9 Mb on chromosome 5, further confirming the 3 Mb region identified as the region of highest F ST divergence . Figure 6 QTL profiling of schistosome resistance across the 18 chromosomes of the snail RILs A major QTL was detected on chromosome 5 between bin markers BgChr5_39634500:1:1 and BgChr5_39675885:1157240:1117, with a significant LOD score of 6.04. Section title: Gene identification and GO analysis Educational score: 4.324515342712402 Domain: biomedical Document type: Study Language: en A total of 118 protein-coding genes were identified in the BgSRR1, and detailed information about all 118 genes can be found in Table S9 . Among the 118 genes, 73 have homologs with known functions, which are listed in Table 1 (see the discussion section for more information). Table 1 A list of genes in BgSRR1 that encode proteins with homologs No Gene product No Gene product No Gene product 1 cis -aconitate decarboxylase 26 FGGY carbohydrate kinase domain-containing protein 51 trichohyalin-like isoform X1 2 thiosulfate sulfurtransferase 27 ran-binding protein 3 52 splicing factor 3B subunit 5 3 trichohyalin 28 glutathione peroxidase 1 53 TATA box-binding protein-like protein 1 4 WASH complex subunit strumpellin 29 DNA-directed RNA polymerases I, II, and III subunit RPABC1 54 acyl-protein thioesterase 1-like isoform X2 5 exportin-2 30 phosphatidylinositol-glycan-specific phospholipase D 55 NIPA-like protein 2 6 CDK5 and ABL1 enzyme substrate 1-like isoform X1 31 bifunctional polynucleotide phosphatase/kinase 56 transmembrane protein 59 7 apolipophorins-like isoform X1 32 serine-rich adhesin for platelets-like isoform X1 57 D-amino-acid oxidase (2) 8 transmembrane protein 8A-like isoform X1 33 annexin A4 58 kinesin-like protein KIF2A 9 calcium homeostasis endoplasmic reticulum protein 34 protein phosphatase 1 regulatory subunit 7 59 tachykinin-like peptides receptor 99D 10 MAP kinase-interacting serine/threonine-protein kinase 1 35 orexin receptor type 2 (2) 60 BgFReDn19 (4) 11 DNA repair endonuclease XPF-like isoform X1 36 adipocyte plasma membrane-associated protein (4) 61 protein FAM166C A 12 superkiller viralicidic activity 2-like 2 isoform X1 37 E3 ubiquitin-protein ligase HECTD3 62 transcription elongation factor A N-terminal and central domain-containing protein 2-like isoform X1 13 derriere protein 38 ankyrin repeat domain-containing protein 13C (2) 63 mediator of RNA polymerase II transcription subunit 26 14 mRNA export factor 39 ethylmalonyl-CoA decarboxylase-like isoform X2 64 SCL-interrupting locus protein 15 ATP synthase subunit delta mitochondrial 40 transmembrane protein 65 (2) 65 cytidine monophosphate (UMP-CMP) kinase 1 cytosolic 16 diisopropyl-fluorophosphatase 41 zinc-finger protein 451 66 choline transporter-like protein 1 17 acyl-CoA synthetase YngI 42 mediator of DNA damage checkpoint protein 1 67 high-affinity cAMP-specific 3p 5′-cyclic phosphodiesterase 7A 18 DNA-directed RNA polymerase I subunit RPA1 43 defense protein 3 (3) 68 mitochondrial fission regulator 2 19 N-acetyltransferase ESCO1 44 ferric-chelate reductase 1 69 pikachurin 20 ATP-dependent RNA helicase DDX43 45 DNA excision repair protein ERCC-6-like 2 70 histone H2A 21 chromatin modification-related protein EAF7 46 TBC1 domain family member 2B (3) 71 THAP domain-containing protein 6 22 stromal membrane-associated protein 1-like isoform X1 47 leucine-rich repeat-containing protein 72 galectin-4 23 zinc-finger SWIM domain-containing protein 5-like isoform X1 48 protein phosphatase 1 regulatory subunit 7 73 eyes absent 4 24 GTP-binding protein Di-Ras2 49 peroxidasin – – 25 HMG box-containing protein 4 50 regulator of telomere elongation helicase 1 isoform X1 – – Note: The number in parentheses at the end of a gene name is the total number of the genes from the same gene family (with the same gene name) in the BgSRR1. Section title: Gene identification and GO analysis Educational score: 4.313724994659424 Domain: biomedical Document type: Study Language: en Gene Ontology (GO) analysis shows enriched biological processes, molecular functions, cellular components, and pathways . Three significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with double-stranded RNA binding, protein heterodimerization activity, and basal transcription factors, along with 68 enriched GO categories, were revealed. These GO categories comprise 21 biological processes, 15 cellular components, and 32 molecular functions ( Table S10 ). The most highly enriched categories include double-stranded RNA binding for molecular function, nucleotide excision repair for biological processes, and three cellular components (WASH complex, endodeoxyribonuclease complex, and histone deacetylase complex). The functional linkage network analysis revealed the top 10 GO enrichments, including GO:0005634 nucleus, GO:0034654 nucleobase-containing compound biosynthetic process, GO:0019438 aromatic compound biosynthetic process, GO:0018130 heterocycle biosynthetic process, GO:1901362 organic cyclic compound biosynthetic process, GO:0097659 nucleic acid-templated transcription, GO:0006351 transcription DNA-templated, GO:0032774 RNA biosynthetic process, GO:0006139 nucleobase-containing compound metabolic process, and GO:0044271 cellular nitrogen compound biosynthetic process . Figure 7 GO and KEGG pathway analyses (A–D) show biological processes, molecular functions, cellular components, and the top 20 pathways, respectively. (E) The hierarchical clustering tree summarizes the correlation among the top 20 significant pathways. Pathways with many shared genes are clustered together. Larger dots indicate more significant p values. (F) The interactive network plot shows the relationship between the top 10 enriched pathways. Two pathways (nodes) are connected if they share 20% (default) or more genes. Darker nodes represent more significantly enriched gene sets. Larger nodes represent larger gene sets. Thicker edges represent more overlapping genes. Section title: Discussion Educational score: 4.122257232666016 Domain: biomedical Document type: Study Language: en A thorough understanding of the fundamental mechanisms that control the traits of interest is crucial for the development of genetically modified organisms for biomedical research. Extensive genetic analyses have been conducted on anti-parasite traits in disease vectors such as mosquitoes, 56 , 57 , 58 , 59 , 60 leading to active studies on genomic modifications for both basic and applied research. 38 , 39 , 40 However, limited progress has been made with schistosomiasis vector snails, hindering our ability to pursue similar innovative work for schistosomiasis control. Different from the relevant genetic studies conducted on vector snails or other mollusks, the current study is based on our long-term efforts to develop genetic resources (the homozygous iM line and iBS90, F2 segregating population, and RILs) and genomic resources (scaffold- and chromosome-level assembled genomes) for the schistosomiasis model snail B. glabrata , with the main objective of deciphering the mechanisms underlying snail resistance to schistosomes. 45 , 52 Section title: Discussion Educational score: 4.082702159881592 Domain: biomedical Document type: Study Language: en The development of RIL snails represents one of our efforts in this direction, despite the painstaking nature of the work involved. RIL snails were produced using an advanced design. The RILs were obtained through two crosses: a parental outcross and an F2 intercross, followed by 10 generations of selfing . This design differs from the standard RIL design, which involves 6–7 generations of inbreeding starting with F2 offspring resulting from a single outcross (parental cross). 49 , 51 , 61 The increased intercrossing, along with a greater number of generations of inbreeding (selfing), should further enhance mapping resolution and reduce the size of QTL by accumulating additional meiotic crossover events. Section title: Discussion Educational score: 4.095550060272217 Domain: biomedical Document type: Study Language: en Indeed, the resistance QTL size was reduced from approximately 8 Mb using the F2 population to about 3 Mb, as revealed by the current RIL population. Importantly, our current findings from two genetic analyses, GWAS and bin marker-based QTL, unequivocally confirm the 3 Mb resistance locus BgSRR1 and its genomic location on chromosome 5, which are also consistent with our previous F2-ddRADseq mapping 45 (see further discussion in the following). Section title: Discussion Educational score: 4.166312217712402 Domain: biomedical Document type: Study Language: en We carefully determined the resistance phenotype of each RI line, as it is important for genetic mapping. In our genetic studies, resistance or susceptibility was defined based on cercarial shedding rather than snail infection. Cercarial shedding directly contributes to disease transmission and human infection. After exposure to schistosome miracidia, many snails become infected or are penetrated by the miracidia, but not all infected snails shed cercariae. Some parasites experience impeded development but still survive in the snail host for a long time, resulting in no cercariae being released from these hosts. This phenomenon was observed in our recent work, which showed that some resistant snails (without shedding cercariae) possessed a varying number of schistosome reads from DNA extracted from the entire snail body. 45 The laboratory-based finding was confirmed by field observations. A large-scale polymerase chain reaction (PCR)-based surveillance program in coastal Kenya demonstrated that the rates of schistosomes present in snail hosts, as detected by PCR, were significantly higher than those observed through cercarial shedding (28%–54% vs. 0.14%–3.4%). 62 If a snail does not shed cercariae, it plays no role in disease transmission, regardless of whether it is infected. As our goal is to apply our findings to field applications, the focus of our investigations is on the phenotype of cercarial shedding rather than on infections. Section title: Discussion Educational score: 4.250483512878418 Domain: biomedical Document type: Study Language: en We employed WGS as a genotyping assay to sequence the genomes of individual RIL snails instead of using pooled DNA samples from multiple snails with the same phenotype (Pool-seq). For each RIL, deep genome sequencing (∼16X coverage) was conducted on a single RIL snail to represent the corresponding RIL for genetic mapping, as all individuals within the same RIL are nearly genetically identical. 49 , 50 , 51 Although this approach is more costly and labor intensive compared to Pool-seq, it provides data that can be used for accurate analyses or re-analyses. As a result, we were able to identify a large number of SNPs ( n = 7,330,259) and bin markers ( n = 2,190) across the 46 RIL genomes. The genetic mapping conducted with the current linkage map, which has denser markers compared to the F2-based linkage map (0.62 cM vs. 1.73 cM), has revealed a smaller QTL size. Subsequent GWAS and QTL analysis both indicated that the snail B. glabrata has a ∼3 Mb BgSRR1 on chromosome 5. This genomic region shows a peak F ST value across chromosome 5 . Linkage mapping analysis of this region reveals only three recombination points, identifying a large haplotype block spanning approximately 1.16 MB and containing 1,117 SNPs. Section title: Discussion Educational score: 4.2559967041015625 Domain: biomedical Document type: Study Language: en BgSRR1 identified by the current RIL-WGS approach agrees with our previous findings using the F2-ddRADseq analysis (i.e., the resistance locus on chromosome 5). 45 In addition to chromosome 5, SNPs linked to resistance were also detected on chromosomes 4, 12, 15, 16, and 18, despite the limited number of SNPs. These chromosomes, especially chromosomes 12 and 16, deserve attention in future studies. We cannot exclude the possibility that loci on these chromosomes may have an effect or a minor effect on resistance. To compare our current findings with previous reports, we mapped QTLs reported by other laboratories 41 , 42 , 43 , 44 , 46 to the 18 chromosomes based on our chromosome-level assembly of B. glabrata . 52 Surprisingly, the chromosomes containing QTLs identified by other research groups differ from chromosome 5 and from those with a limited number of significant SNPs (i.e., chromosomes 4, 12, 15, 16, and 18) . Please note that the chromosome numbers (i.e., the order from 1 to 18) in Figure 8 from Zhong et al. 52 are not exactly the same as the linkage group numbers in Figure 7 of the paper published by Bu. et al.. 45 Therefore, the genes identified by other groups, including a cluster of genes encoding transmembrane proteins, 41 , 42 , 43 , 44 , 46 are not present in our BgSRR1. The reason for the discrepancy between our findings—both previous and current—and those reported by others is still unknown. Figure 8 Distribution of QTLs reported on the 18 chromosomes based on the chromosome-level assembled genome of iM line B. glabrata The previously reported QTLs, including OPM-0423, 41 GRC, 42 RADres and SOD, 43 PTC2, 44 , 46 and qRS2.1 and qRS5.1, 45 are indicated by solid red bars. The pink star marks the chromosomal location of BgSRR1. The RAPD marker OPZ-11 41 is not indicated due to its repetitive sequence. Section title: Discussion Educational score: 4.1039628982543945 Domain: biomedical Document type: Study Language: en Although the GO analysis of the protein-coding genes in BgSRR1 was conducted, caution is warranted in interpreting the findings because about one-third of the genes in the region lack homologs with known functions and could not be included in the GO analysis. Nonetheless, the analysis offers useful insights into the mechanisms of schistosome resistance in snails. Some previously unrecognized pathways may be involved in the defense responses. For example, the enriched GO categories include many genes and pathways related to metabolism. Immunometabolism has recently emerged as a dynamic field in immunology but has not yet been explored in snail immunology or host-parasite interactions. 63 , 64 , 65 Section title: Discussion Educational score: 4.398678779602051 Domain: biomedical Document type: Study Language: en Focusing on genes with known functions, we are excited to find that BgSRR1 possesses genes known to be involved in cellular immunity in snails. The presence of the mitogen-activated protein kinase (MAPK) gene in BgSRR1 is supported by earlier studies suggesting that MAPK-based signal transduction plays a critical role in hemocyte-mediated encapsulation and H 2 O 2 production, leading to the killing of intramolluscan schistosomes. 66 , 67 , 68 Interestingly, two genes from the peroxidase gene family, glutathione peroxidase and peroxidase , have been identified in BgSRR1. Peroxidases are antioxidative enzymes that scavenge H 2 O 2 and inhibit apoptosis. 69 An earlier study also revealed that a thioredoxin peroxidase or peroxiredoxin gene was highly expressed in resistant B. glabrata snails compared to susceptible ones in response to schistosome infection. 70 Additionally, BgSRR1 contains a gene encoding the enzyme thiosulfate sulfurtransferase, which may also have antioxidative properties. 71 These findings suggest that BgSRR1 is involved in regulating cell-mediated immunity, particularly in relation to redox balance. Section title: Discussion Educational score: 4.283954620361328 Domain: biomedical Document type: Study Language: en BgSRR1, however, does not contain genes previously reported to play a significant role in humoral immunity in B. glabrata , such as biomphalysin , 72 fibrinogen-related proteins ( FREPs ), 73 , 74 , 75 , 76 , 77 macrophage migration inhibitory factor genes, 78 and genes encoding proteins associated with the Toll-like receptor pathway. 79 , 80 Notably, we have identified other humoral immune genes in BgSRR1, including apolipophorin , 81 defense proteins , 82 RNA helicase , 83 and E3 ubiquitin protein ligase genes, 84 although the roles of these genes in schistosome resistance have not been reported in snails. It is worth mentioning that BgSRR1 contains four ficolin genes (only fibrinogen [FBG]) but none of the FREPs . Ficolins, which are important players in innate immunity, have been extensively studied in the mosquito- Plasmodium model, 85 , 86 , 87 but not in the snail-schistosome system. Section title: Discussion Educational score: 4.184762954711914 Domain: biomedical Document type: Study Language: en We have observed genes in BgSRR1 that encode enzymes involved in immune cell metabolism. For example, the enzyme cis -aconitate decarboxylase, encoded by immune response gene 1 , produces itaconate, an intermediate metabolite from the tricarboxylic acid cycle in immune cells. 88 Recent studies have demonstrated that itaconate is an important immunometabolite that regulates host defense and inflammation. 89 , 90 The potential role of immunometabolism in defense is also suggested in the GO analyses discussed earlier. Future investigations into the functions of these genes in BgSRR1, especially those not yet studied in B. glabrata , may reveal unexpected aspects of snail-parasite interactions, provide valuable insights into snail defenses, and help identify key resistant genes. Section title: Discussion Educational score: 4.130929946899414 Domain: biomedical Document type: Study Language: en In conclusion, our approach (RIL-WGS), combined with our RIL genetic resource, powerful genome-wide genotyping, GWAS, and bin marker-assisted QTL analysis, has enabled the identification of the BgSRR1 on chromosome 5 of B. glabrata , an important molluscan vector of human schistosomiasis. The identification of BgSRR1 and the genes conferring schistosome resistance has the potential to advance our understanding of host-parasite interactions and facilitate the development of snail-targeted biocontrol strategies for schistosomiasis, a parasitic disease that infects 251 million people worldwide. 91 Section title: Limitations of the study Educational score: 4.040545463562012 Domain: biomedical Document type: Study Language: en While our current findings are robust and supported by a well-developed genetic resource, reliable phenotype and genotype data, and multiple genetic analyses, it is important to acknowledge their limitations, which are common in genetic studies. Firstly, the use of RILs limits the ability to capture information regarding dominance due to their high homozygosity. 92 In fact, our RIL-WGS approach did not detect the presence of a susceptibility-associated QTL under the dominance effect on chromosome 2. 45 Secondly, it should be recognized that our findings are based on a well-developed laboratory system. Therefore, further evaluation and testing of our findings in other systems, particularly in field settings, are necessary. Section title: Lead contact Educational score: 1.067710518836975 Domain: other Document type: Other Language: en Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Si-Ming Zhang ( zhangsm@unm.edu ). Section title: Materials availability Educational score: 1.0332363843917847 Domain: biomedical Document type: Other Language: en This study did not generate any new unique reagents. Section title: Data and code availability Educational score: 1.2373528480529785 Domain: biomedical Document type: Other Language: en • The Illumina sequence data generated from 46 RIL snails have been deposited at the National Center for Biotechnology Information (NCBI) and are publicly available as of the date of publication. Accession numbers are listed in the key resources table . • This paper does not report original code. • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Section title: Acknowledgments Educational score: 0.9570343494415283 Domain: other Document type: Other Language: en This study was funded by the 10.13039/100000002 National Institutes of Health (NIH) ( https://www.nih.gov ) grant R01 AI170587 to S.-M.Z. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The PR1 strain of Schistosoma mansoni was provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD, USA) through NIH-NIAID Contract HHSN272201700014I . Section title: Author contributions Educational score: 0.9192235469818115 Domain: other Document type: Other Language: en S.-M.Z.: conceptualization, breeding of RIL snails and testing of their phenotypes, DNA extraction and quality evaluation, investigation, writing – original draft, and writing – review and editing; G.Y.: investigation and writing – review and editing; A.L.: writing – review and editing; D.Z.: data analysis, investigation, writing – original draft, and writing – review and editing. All authors read and approved the final manuscript. Section title: Declaration of interests Educational score: 0.8786384463310242 Domain: other Document type: Other Language: en The authors declare no competing interests. Section title: Key resources table Educational score: 3.7172367572784424 Domain: biomedical Document type: Study Language: en REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins CTAB solution Teknova Lot no: C219009G1801 Chloroform: Isoamyl alcohol Sigma-Aldrich Lot no: 1003577830 Proteinase K TermoScientific Lot no: 10198999 RNase A TermoScientific Lot no: 2653498 Isopropyl alcohol Honeywell Lot no: CZ999 200 proof pure ethanol KOPTEC Lot no: A08232309D Deposited data Illumina sequence data National Center for Biotechnology Information (NCBI) ( https://www.ncbi.nlm.nih.gov ) BioProject ID: PRJNA1133633. BioSample accession numbers: SAMN42382410-SAMN42382455. Experimental models: Organisms/strains iM line of Biomphalaria glabrata University of New Mexico, USA Si-Ming Zhang iBS90 of Biomphalaria glabrata University of New Mexico, USA Si-Ming Zhang RILs of Biomphalaria glabrata University of New Mexico, USA Si-Ming Zhang PR1 strain of Schistosoma mansoni Biomedical Research Institute in Maryland, USA Margaret Mentink-Kane Software and algorithms Trimmomatic 3.9 Bolger et al. 93 https://github.com/timflutre/trimmomatic CLC Genomics Workbench 23 QIAGEN Aarhus, Denmark https://digitalinsights.qiagen.com/ bcftools Danecek et al. 94 https://samtools.github.io/bcftools/bcftools.html vcftools Danecek et al. 95 https://vcftools.github.io/index.html PLINK 1.9 Purcell et al. 96 https://www.cog-genomics.org/plink/ JMP 14 SAS Institute Inc., Cary, NC https://www.jmp.com/en_us/home.html Binmarkers-v2 Qin et al. 97 https://github.com/lileiting/Binmarkers-v2 IciMapping Meng et al. 98 http://www.isbreeding.net ShinyGO Ge et al. 99 https://bioinformatics.sdstate.edu/go/ Other A scaffold-level assembled genome of iM line and iBS90 of Biomphalaria glabrata Bu et al. 45 https://doi.org/10.1038/s42003-022-03844-5 A chromosome-level assembled genome sequence of Biomphalaria glabrata Zhong et al. 52 https://doi.org/10.1038/s42003-022-03844-5 Section title: Snails and schistosomes Educational score: 4.001649379730225 Domain: biomedical Document type: Study Language: en The snail Biomphalaria glabrata , a major intermediate host of human schistosomiasis in Neotropical countries, was used for this study. The efforts to generate B. glabrata recombinant inbred lines (RILs) are described in the results section. Breeding, cultivation, and storage of the RIL snails were conducted at the Center for Evolutionary and Theoretical Immunology (CETI), University of New Mexico (UNM), United States. The generation of recombinant inbred (RI) lines (RILs) is also described in the results section. The PR1 strain of Schistosoma mansoni used to infect the snails was collected from the Biomedical Research Institute in Maryland, USA ( https://www.afbr-bri.org ). Section title: Exposure of schistosome miracidia to snails Educational score: 4.065985202789307 Domain: biomedical Document type: Study Language: en To determine the phenotype of the RI lines, 6–8 juvenile snails (0.3–0.6 mm shell diameter) were randomly chosen from each RI line for infection. The snails were individually placed into the wells of a 24-well cell culture plate (one snail per well) and 20 schistosome miracidia were added to each well. The snails were fully submerged in water overnight to ensure complete exposure to the miracidia. Afterward, the exposed snails were transferred to large tanks for continued culturing until cercarial shedding was performed. Section title: Determination of schistosome resistance phenotype Educational score: 4.088736057281494 Domain: biomedical Document type: Study Language: en Examination of the phenotype began at 45 days post-exposure (dpe). The exposed snails were placed individually in the wells of a 24-well plate and exposed to light for 0.5 h (hr). Snails that shed cercariae were considered susceptible to schistosomes, while those that did not shed cercariae were transferred to the aquatic tank and cultured for later examination of shedding. If a snail did not shed cercariae at 60 dpe, it was classified as a resistant snail. This procedure allowed us to determine the phenotype of each RI line. Once the phenotype was determined, the remaining snails from each RI line (those not exposed to schistosomes) were preserved in liquid nitrogen for DNA extraction. Only the RI lines that exhibited the same phenotype in all tested individuals were selected for genetic mapping. Section title: DNA extraction Educational score: 4.176008224487305 Domain: biomedical Document type: Study Language: en A single snail thawed from liquid nitrogen was placed into a 1.5 mL tube and ground in 750 μL of CTAB buffer. 100 After homogenization, 20 μL of proteinase K (20 μg/μL) was added to the homogenate and incubated at 60°C for 1 h (hr). Next, 750 μL of chloroform: isoamyl alcohol (24 : 1) was added and rocked for 0.5 h. Following centrifugation, the supernatant was transferred to a new tube. To degrade and remove RNA, 10 μL of RNase (10 μg/μL) was added to the new tube and incubated at 37°C for 0.5 h. An equal volume (750 μL) of chloroform: isoamyl alcohol was added to the solution and rocked at room temperature for 10 min (min). Genomic DNA was precipitated using isopropyl alcohol, washed with 70% ethanol, and dissolved in nuclease-free water. Section title: Library preparation and WGS Educational score: 4.160333633422852 Domain: biomedical Document type: Study Language: en The genomic DNA was qualified and quantified using agarose gel electrophoresis (1%) and the Qubit 2.0 DNA HS Assay (ThermoFisher), respectively. For library preparation, the KAPA Hyper Prep kit (Roche) was used. In brief, the genomic DNA was sheared into 500 bp fragments using the Covaris LE220-plus. After ligating the adapters, the fragments were amplified by PCR. The quantity and quality of the libraries were assessed using the Qubit 2.0 DNA HS Assay, the Tapestation High Sensitivity D1000 Assay (Agilent Technologies), and the QuantStudio 5 System (Applied Biosystems). Finally, the libraries were sequenced using an Illumina NovaSeq S4. Section title: Trimming, mapping reads, and SNP calling Educational score: 4.1851420402526855 Domain: biomedical Document type: Study Language: en The raw Illumina reads for the two parental lines (iM line and iBS90) were retrieved from GenBank . 45 It is important to note that all Illumina data, including those from the two parental snails and the RIL snails were generated from the same Illumina platform (Admera Health; www.admerahealth.com ) and the same quality control was applied to all samples. All raw reads were trimmed and cleaned using Trimmomatic v0.39 93 with the following parameters: ‘ILLUMINACLIP: TruSeq3-PE-2.fa: 2:30:10 HEADCROP:7 LEADING: 25 TRAILING: 25 SLIDINGWINDOW: 4:25 MINLEN: 36’. The clean reads from each RIL sample were individually aligned to the reference genome of B. glabrata 52 using the Map Reads to Reference tool in the QIAGEN CLC Genomics Workbench 23 (Qiagen Genomics, Denmark) with the default parameters. SNP calling for the iM line and iBS90 was conducted according to the methods described previously. 45 The Identify Known Mutations from Mappings tool of the CLC workbench was used to genotype the RILs individually at SNP loci that were polymorphic in the two parental snails. Subsequently, the SNPs identified from the CLC were exported individually to VCF files and then merged into a single VCF file using bcftools. 94 Finally, the resulting VCF file was filtered using vcftools 95 with the following parameters: ‘--minDP 5; --maxDP 100; --maf 0.1; --max-missing 0.8'. Section title: Genome-wide SNP genotyping and GWAS Educational score: 4.126946926116943 Domain: biomedical Document type: Study Language: en Single SNP genotype and phenotype association analyses were conducted using PLINK software. 96 To identify significant associations, we applied the widely accepted threshold of p < 5 × 10 −8, 101 which is derived from a Bonferroni correction for all independent SNPs in the genome. To measure the divergence between susceptible and resistant populations, we calculated the fixation index (F ST ) using VCF tools. We used a sliding window of 10 kb, with an increment of 5 kb, to perform this calculation. Significant high F ST outliers were identified based on the 95th quantile from the genome-wide distribution and jackknife procedure. Outlier analysis was conducted using the Jackknife Distances in SAS JMP 14. This involved calculating pairwise distances between data points, resampling the data by removing one point at a time, and analyzing the variance of the resulting distances. Points with unusually high variance were flagged as potential outliers and further validated. Section title: Genetic bin marker calling Educational score: 4.171338081359863 Domain: biomedical Document type: Study Language: en To identify genomic intervals in a mapping population with no recombination events, we used the Binmarker-v2.3 tool ( https://github.com/lileiting/Binmarkers-v2 ). 97 This tool employs a sliding window approach of 10 kb to generate genetic bin markers. Missed genotypes were imputed and miscoded genotypes were corrected using strict criteria: a genotype that differed from surrounding genotypes, no missing data in surrounding genotypes, and identical surrounding genotypes. Next, markers with 100% identical markers were merged together. These bin markers were then organized based on the physical position of the chromosome. A change in genotyping within any sample was considered a recombination breakpoint. SNPs between recombination breakpoints were classified as bin markers, indicating that no recombination occurred within that bin. Section title: Construction of the linkage map and QTL analysis Educational score: 4.122276306152344 Domain: biomedical Document type: Study Language: en Bin markers showing significant deviation ( p < 0.001) from the 1:1 segregation ratio were excluded from constructing the linkage map. Heterozygous genotypes were treated as missing data and imputed using the "maxmarginal" method implemented in the R/qtl package. Linkage map construction and QTL analysis were conducted using QTL IciMapping version 4.2.53. 98 Simple interval mapping and inclusive composite interval mapping were employed to detect potential QTLs associated with snail resistance or susceptibility to schistosome parasites. A significant threshold of the logarithm of odds (LOD) (LOD = 4.0) based on 1,000 permutation tests was applied. Section title: Analysis of protein-coding genes in the QTL region Educational score: 4.030220031738281 Domain: biomedical Document type: Study Language: en The coding genes were further verified manually by BLAST searching against NCBI databases. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were performed using the web-based tool ShinyGO 0.80 ( http://bioinformatics.sdstate.edu/go/ ). 99 A flowchart showing bioinformatic and genetic analyses is provided in Figure S1 . Section title: Quantification and statistical analysis Educational score: 4.155148029327393 Domain: biomedical Document type: Study Language: en PLINK was employed for genome-wide association studies (GWASs) with a stringent p -value threshold of 5 × 10 −8 to identify significant associations. Vcftools was used to calculate fixation index (F ST ) statistics, and outlier analysis was performed using Jackknife Distances in SAS JMP 14, with a p -value threshold of <0.05 for significance. A permutation test with 1,000 iterations and a type I error rate of 0.05 was used to establish the significance threshold for QTL LOD scores. Functional enrichment analysis, including GO and KEGG pathways, was conducted with an E-value threshold of < 1e-5. Significant GO term enrichment was assessed using Fisher’s exact test, applying a p -value threshold of <0.05. These analyses identified genetic variants associated with snail resistance and explored their functional implications. | Review | biomedical | en | 0.999995 |
PMC11699760 | Section title: Background Educational score: 3.831063747406006 Domain: biomedical Document type: Study Language: en Xylazine—often referred to as “tranq” or “ anastesia de caballo ” in Spanish—is a α2-adrenergic receptor agonist, used for sedation in veterinary contexts [ 1 – 3 ]. Unlike other α2 agonists, such as clonidine or dexmedetomidine, xylazine has not been approved for human consumption in the United States (US) or other jurisdictions . Xylazine appeared in the illicit drug market of Puerto Rico in the early 2000s where it was used to augment the effects of heroin . It subsequently emerged as an additive to illicit fentanyl in mainland US in the late 2010s and early 2020s . The prevalence of xylazine among fatal overdoses has risen exponentially in subsequent years [ 7 – 9 ], however its causal role in overdose events remains a matter of scientific discussion . Qualitative data suggest that xylazine may increase the complexity and difficulty of life-saving treatment during overdoses . Section title: Background Educational score: 3.8045854568481445 Domain: biomedical Document type: Clinical case Language: en The arrival of xylazine-fentanyl co-use to the North American overdose crisis has been highly notable, and xylazine has been identified as an emerging threat by the government of the US , Chile , by the Organization of American States , and more recently by Mexico . However, the scope of the problem in the Mexican context deserves further study, as robust epidemiological data are not yet available. Preliminary evidence suggest that xylazine may be found in high concentration among fentanyl samples in Tijuana . Additionally, little information is available in the literature about the clinical management of xylazine-involved overdoses, especially in community harm reduction spaces. Here we present a series of clinical cases of xylazine-fentanyl overdose, which exemplify the clinical approach developed, and the lessons learned, by an overdose response team at Prevencasa, a community harm reduction clinic in Tijuana, Baja California, Mexico. The patients whose cases are featured here provided written consent. Section title: Background Educational score: 3.2374563217163086 Domain: clinical Document type: Other Language: en Prevencasa serves a large population of people who inject and smoke illicit opioids, many of whom have been deported from the US. The clinic has more than a decade of experience responding to overdose events in the surrounding neighborhood of Zona Norte (which concentrates a large number of illicit drug sales points and open-air drug use). Although overdose events were initially sporadic, they became increasingly common with the arrival of illicit fentanyl to Tijuana . In response, the clinic trained an overdose response team, with designated clinical members (usually one physician, one nurse, and one or more harm reduction staff members) equipped with naloxone and other supplies. All team members were trained in how to assess for the clinical signs and symptoms of opioid overdose, as well as how to administer naloxone administration. MD and RN team members were additionally trained in how to rapidly assess key vital signs, especially using pulse oximetry, and in the administration of oxygen and other life-saving measures. Section title: Case presentation 1 Educational score: 3.785405158996582 Domain: clinical Document type: Clinical case Language: en A 61-year-old man with opioid and methamphetamine use disorder, well-known to the clinic, who uses a wheelchair due to a complete right lower limb amputation, was found lying face first on the ground, across the street from the harm reduction clinic, bleeding from a head laceration . His respiratory rate was imperceptible, he was cyanotic, and his pupils were pinpoint. A nurse administered a 4.0 mg intranasal dose of naloxone, and the patient quickly resumed visibly respirating, however, he did not regain consciousness. Pulse oximetry showed the patient saturating at 96%, with a pulse of 95 bpm, and auscultation revealed normal breath sounds. The patient remained unarousable for approximately 20 min, during which time the clinical team monitored his oxygenation status, breath sounds, and pulses. He subsequently awoke suddenly in a delirious state and became agitated, endorsing distressing withdrawal symptoms. He pulled himself into his wheelchair and wheeled himself into traffic in a nearby roadway, exposing himself to considerable physical risk. There he once again lost consciousness, falling backwards onto the curb. Several community members placed him back into his wheelchair, and brought him into the clinic, where he accepted post-overdose monitoring. His vitals remained stable for the next two hours. Urine screening revealed positivity for xylazine, fentanyl, and methamphetamine. The patient reported a two-decade history of opioid use disorder, and endorsed currently both smoking and injecting ‘China White’ (a fentanyl powder formulation that increasingly contains xylazine in Tijuana) mixed with methamphetamine every two hours. The patient provided a drug sample to the on-site drug checking program, as well as a urine sample. Both tested positive for xylazine, using two different brands of immunoassay qualitative testing strips, as well as fentanyl. The patient received counseling about the risks of xylazine and fentanyl. Section title: Case presentation 2 Educational score: 3.5972602367401123 Domain: clinical Document type: Clinical case Language: en A 28-year-old man with long-standing methamphetamine use disorder and no other known medical history was found unconscious, in a supine position, on the sidewalk, a few meters from the harm reduction clinic. Community members on scene reported that they observed him smoking a substance from a glass pipe prior to losing consciousness. He was not arousable, and pulse oximetry showed an oxygen saturation of 81% and a heart rate of 92 beats per minute. His blood pressure and blood glucose were within normal limits. He responded to verbal and physical stimuli only by opening his eyes but was not verbally responsive. On several occasions he attempted to sit up but was too sedated to do so. The overdose response team delivered oxygen through nasal cannula via a portable oxygenation concentrator. The patient’s oxygenation saturation immediately responded by rising to 100%. The patient was brought into the clinic for vital sign monitoring and supplemental oxygen. The response team opted against naloxone administration given that his vital signs stabilized with supplemental oxygen. After 40 min the patient awoke calmly, was alert and oriented to self, place, and date, and was informed about the process of his care, as well as the possible exposure to xylazine. He endorsed having used the street opioid mixture “China White” in a recreational fashion, although typically he only consumes stimulants, and it is unusual for him to consume opioids. He denied having used benzodiazepines, alcohol or other psychoactive substances. The patient declined urine or substance testing or any further medical care and left the clinic. He was seen one week later for follow-up care and was found in stable condition and received counseling. Section title: Case presentation 3 Educational score: 3.709930181503296 Domain: clinical Document type: Clinical case Language: en A 36-year-old man with long-standing opioid use disorder was found sitting on a curb, leaning back onto a car, very close to the harm reduction clinic, his eyes were covered by a hat. He was sitting among a group of various community members who were smoking and/or injecting illicit opioids. He did not respond to verbal cues, which prompted a member of the overdose response team to attempt to arouse him with touch, to which he also did not respond. The overdose response team member noted that the patient’s extremities were stiff, cold, and diaphoretic. The rest of the response team was summoned, the patient was laid flat on the ground, and was noted to be cyanotic, with blue lips and nails. Pulse oximetry revealed an oxygen saturation of 20%. A dose of intramuscular naloxone of 0.4 mg was administered, and within the next 2 min, oxygen saturation reached 99%, although the patient remained unresponsive to verbal and physical stimulus. The patient was placed on his side in recovery position, vital signs were assessed for the next 12 min, at which time he began to move his arms and legs in an agitated fashion, with his eyes remaining closed. Shortly thereafter he opened his eyes, was fully oriented, and was able to follow verbal guidance. He accepted post-overdose care in the clinic and offered a sample from the syringe containing the substance he had consumed prior to overdose, still loaded with 0.2 ml of prepared “China White”. The sample tested positive for fentanyl and xylazine. The patient’s urine tested positive for fentanyl, xylazine and methamphetamine. The patient received counseling about the risks of xylazine when combined with fentanyl. Section title: Discussion Educational score: 4.063071250915527 Domain: clinical Document type: Clinical case Language: en These three clinical cases highlight a range of typical scenarios of xylazine-involved overdose seen by the overdose response team at the Prevencasa community harm reduction clinic in Tijuana, Mexico. In the early months of 2024, due to the arrival of xylazine, a very notable shift in the clinical characteristics of overdose events was observed, which required the overdose response team to adapt its practices. Patients who would have previously quickly regained consciousness after a 4.0 mg dose of intranasal naloxone began to exhibit 10–30-minute periods of unconsciousness, despite adequate oxygenation as measured with pulse oximetry. Patients increasingly began to awaken in a confused or delirious state, exhibiting aggression, and often would expose themselves to physically dangerous circumstances (e.g. walking in traffic, laying on roadways) appearing to be unaware of their surroundings, significantly complicating field management. During this time, routine urine toxicology was offered to patients, as was direct testing of substances and drug wrappers, all of which showed high rates of xylazine positivity, using several brands of lateral flow immunoassay testing strips . Section title: Discussion Educational score: 4.117664337158203 Domain: clinical Document type: Clinical case Language: en In response, the clinical team developed a set of strategies to manage xylazine-involved overdoses (Table 1 ). One valuable lesson was the insight to carefully titrate—and at times even reduce or withhold entirely—the dose of naloxone provided to overdose patients. We hypothesized that local sales points decreased the concentration of fentanyl provided to consumers, replacing it with xylazine. Therefore, 4.0 mg intranasal doses of naloxone seemed to induce more problematic withdrawal symptoms in patients than prior to the arrival of xylazine, as seen in case 1. Affected individuals increasingly would awaken expressing concern about physically uncomfortable withdrawal symptoms, which often prompted an immediate search for repeat illicit opioid consumption, and complicated post-overdose clinical monitoring. Instead, the use of smaller intramuscular doses (such as in case 3), titrated to adequate oxygenation—not consciousness—provides more stable patient care and better outcomes. In urgent overdose response situations, there is a pressing temptation to administer additional naloxone to unconscious patients. However, the non-opioid sedation period induced by xylazine requires re-centering the focus on oxygenation and respiratory status, not consciousness. The team has therefore increased its use of field pulse oximetry as the key clinical parameter of interest, re-doubled efforts to focus on field airway management, including proper positioning, and the use of mobile oxygen tanks. In some instances, perhaps when xylazine is the predominant component driving sedation, adequate oxygenation status can be achieved without naloxone, and supplemental oxygen and airway management are the key techniques employed (such as in case 2 above). The assessment of respiratory status has therefore become a key priority for xylazine overdose management. Although community naloxone distribution has become common, and overdoses involving only opioids are often managed in practice by individuals who lack medical training, the prolonged period of sedation with potential airway compromise induced by xylazine may require specialized knowledge to manage. Community-based training in airway management has been suggested as a key response tactic to xylazine among harm reduction practitioners to solve this challenge . Section title: Discussion Educational score: 2.5315558910369873 Domain: clinical Document type: Other Language: en An increased focus on scene safety is also needed. Xylazine induces a level of sedation that many consumers of illicit opioids are unprepared for . During acute overdose events, scene management is paramount, including transporting patients to a safe area. The post-sedation delirium we have observed often requires careful and gentle redirection from the clinical team to ensure patients do not place themselves in harmful scenarios. Section title: Discussion Educational score: 3.7590057849884033 Domain: biomedical Document type: Other Language: en We have also provided xylazine testing strips to patients, and syringe exchange clients, and provided education to the local population about the risks of xylazine. Xylazine testing strips are a relatively new introduction to the market, and are currently approved for forensic purposes, including directly testing substances. However they have been demonstrated, in practice, to work on urine samples . One concern is that the thresholds used in commercially available xylazine strips may be too low, resulting in a risk of false negative results . However, strips can also be given to participants to directly test their own drug supply before consumption. Many individuals seeking illicit opioids do not wish to consume xylazine—especially once they are aware of its associated health risks . Providing participants with xylazine testing strips—coupled with adequate counseling from medical providers—can therefore empower them to shift their demand towards products with fewer health risks. This can also serve as a segue into a helpful conversation between patient and provider about harm reduction in the midst of active substance use, that can often lead to engagement in opioid substitution treatment. Section title: Discussion Educational score: 3.958144426345825 Domain: biomedical Document type: Other Language: en Collectively these strategies have helped the clinical team manage xylazine-fentanyl overdoses in the field and educate the patient population about evolving health risks associated with shifts in the illicit drug supply. However, more research is needed about the clinical management of xylazine-involved overdoses, especially in community clinics relevant broadly to harm reduction contexts. The techniques we propose here should be validated with more formal clinical studies. Beyond the current response to xylazine-fentanyl combinations , many of these techniques may also apply broadly to the evolving polysubstance crisis unfolding in North America , which increasingly also involves nitazenes, novel synthetic benzodiazepines, and other synthetic sedatives mixed with fentanyls. Across these drug categories, naloxone-resistant sedation and field airway management may be core issues. Section title: Discussion Educational score: 4.070295810699463 Domain: biomedical Document type: Other Language: en Table 1 Strategies to manage xylazine-fentanyl overdose in the field Strategy Description Field Pulse Oximetry Portable, battery-powered pulse oximetry can be used in the field to monitor oxygenation status in patients who have not regained consciousness. Titrate Naloxone to Oxygenation Status Naloxone administration, either intramuscular or intranasal, can be titrated to oxygenation status, not level of consciousness, to avoid inducing unnecessary opioid withdrawal symptoms after the xylazine sedation period passes. Portable Oxygen and Airway Management Portable oxygen tanks, and proper positioning of patients (laying on their side, jaw thrust, visualize airway) can assist in improving oxygenation after naloxone administration, assess for vomit and gastric contents, employ portable suction as needed. Scene Safety Patients with prolonged periods of sedation are vulnerable to traffic, assault, and other physical risks. Response team staff members can help secure the scene. Distribute Xylazine Testing Supplies Testing strips for xylazine presence using immunoassay technology are inexpensive (about $1 per strip) and easy to use. They can be distributed to participants to assist them in screening their drugs. In practice many patients show positivity in urine samples, although the strips have not yet been FDA approved for this purpose, and false negatives may occur. Communnity Education Many patients prefer to avoid xylazine once they are aware of it, understand the health risks, and are equipped with testing strips to detect it. Providers can counsel patients about the risks of xylazine and suggest safer options. This can help encourage patients to switch to methadone or buprenorphine treatment. Section title: Discussion Educational score: 2.027021884918213 Domain: biomedical Document type: Other Language: en Fig. 1 Photos From Overdose Response Team – Cases Involving Xylazine. A ) Overdose response, B ) pulse oximetry post naloxone administration, C ) physical danger consequent to delirious state following return-to-consciousness D ) Urine results showing xylazine, fentanyl, methamphetamine positivity, E ) sample of “China White” tested for xylazine F ) xylazine positivity for China White sample | Study | biomedical | en | 0.999997 |
PMC11699763 | Section title: Background Educational score: 2.1108338832855225 Domain: biomedical Document type: Other Language: en There’s an increasing demand for minimally invasive and aesthetically pleasing tooth restorations, this explains the increasing popularity of partial-coverage ceramic restorations, as they offer a conservative approach to restore destructed tooth structure while achieving both cosmetic and functional demands . Section title: Background Educational score: 2.687405824661255 Domain: biomedical Document type: Other Language: en Adhesive dentistry introduced innovative restoration designs, resulting in a greater adoption of partial-coverage ceramic restorations. Overlay restorations have become a prevalent treatment option for posterior teeth with coronal damage. either due to caries or non-caries issues as abfraction [ 3 – 6 ]. Section title: Background Educational score: 3.1714327335357666 Domain: biomedical Document type: Other Language: en Ceramic restorations merge between excellent biocompatibility and optimal optical and material properties, satisfying both patient and clinical demands [ 7 – 9 ], which is indicated for veneers, inlays, onlays, crowns, and FPDs [ 7 – 11 ]. Section title: Background Educational score: 3.1170709133148193 Domain: biomedical Document type: Other Language: en Glass-ceramics, including lithium disilicate and zirconia-reinforced lithium silicate, are aesthetically appealing restorative materials fabricated with computer-aided design (CAD) computer-aided manufacturing (CAM) technologies. They were designed to replace metal-alloy frameworks, providing benefits in optical, physical, and biological properties . Section title: Background Educational score: 3.239819288253784 Domain: biomedical Document type: Other Language: en The durability of ceramic restorations primarily depends on the quality of bonding and the proper application technique. Cementation enhances fracture resistance by filling the irregularities on the etched fitting surface of the restoration, thereby enhancing bonding strength and preventing crack propagation especially the glass ceramics . Section title: Background Educational score: 3.797612428665161 Domain: biomedical Document type: Study Language: en Maintaining fracture resistance is essential for ensuring the effectiveness of ceramic restorations. It is influenced by preparation design, ceramic material properties, restoration thickness, cementation technique, functional load, and internal ceramic defects . Preparation designs, including cavity depth, width of the isthmus, the degree of taper, and the internal line angles, can influence fracture resistance. Additionally, the aging of the ceramic restoration/tooth complex may affect the failure rate . Section title: Background Educational score: 3.204664707183838 Domain: biomedical Document type: Other Language: en The marginal fit of restoration is a critical element that affects the longevity of overlay restorations. Poor adaptation can create gaps between the restoration and tooth, leading to microleakage, plaque buildup, and potential recurrence of decay . Section title: Background Educational score: 3.0838534832000732 Domain: biomedical Document type: Study Language: en It’s worth mentioning that only a limited number of studies have investigated the impact of the design of preparation on the characteristics of overlay restorations, with most of these focusing mainly on the fracture resistance of the restorations [ 1 , 3 , 19 – 22 ]. Most of the previous pertinent research has assessed inlays and/or onlays that provide partial cusp coverage . Section title: Background Educational score: 3.9429378509521484 Domain: biomedical Document type: Study Language: en An information gap exists concerning the impact of preparation configuration on the marginal fit of ceramic overlays. Therefore, this research aimed to examine the marginal gap and fracture resistance of Advanced Zirconia-reinforced Lithium disilicate (ALD) overlays with various preparation designs. The null hypothesis for this study stated that there is no significant difference in marginal fit (before or after thermal aging) or fracture resistance among the studied groups. Section title: Study design and sample size Educational score: 2.637490749359131 Domain: biomedical Document type: Study Language: en The study followed an in-vitro, parallel-controlled design, in which three parallel groups’ marginal fit and fracture resistance were evaluated. It was conducted in the laboratory of the Conservative Dentistry Department at the Faculty of Dentistry, Alexandria University, Egypt. Section title: Study design and sample size Educational score: 3.578390121459961 Domain: biomedical Document type: Study Language: en The sample size was estimated assuming a 5% alpha error and 80% study power. Based on the difference between independent means of previous study , the minimum sample size was calculated to be 7 samples per group, increased to 9 samples to make up for processing errors. Total sample = number per group x number of groups = 9 × 3 = 27 samples. Section title: Study design and sample size Educational score: 2.288026809692383 Domain: biomedical Document type: Study Language: en The sample size was calculated by G*Power 3.1.9.7. Section title: Specimen preparation Educational score: 2.2868714332580566 Domain: biomedical Document type: Other Language: en Three second mandibular molars typodont teeth (Columbia Dentoform Corporation, USA), were prepared using the following designs : Section title: Specimen preparation Educational score: 3.036726236343384 Domain: biomedical Document type: Study Language: en Group (O) a traditional overlay preparation with anatomical occlusal reduction 1.5 mm. Group (OS) an overlay preparation with anatomical occlusal reduction 1.5 mm and 1.0 mm shoulder finish line circumferentially. Group (OG) an overlay with anatomical occlusal reduction 1.5 mm and central groove preparation with a pulpal depth of 1.0 mm and a width of 2.0 mm. Section title: Specimen preparation Educational score: 4.09200382232666 Domain: biomedical Document type: Study Language: en The typodont teeth that had been prepared were subjected to scanning using an extraoral scanner (InEos X5; Dentsply Sirona, USA), to design three dies corresponding to the tooth preparations. Print out of twenty-seven dies made from 3D printing resin (Model Resin, Formlabs, Somerville, MA), which possess a comparable modulus of elasticity to dentin (modulus = 10 GPa). A laboratory printer (FormLab 2, Formlabs) was used . Each resin die was subsequently embedded in a copper metallic mold filled with auto-polymerizing acrylic resin (Acrostone, Egypt), exposing only the crown and 2 mm apical to the cementoenamel junction, simulating the bone level. Section title: Design and fabrication of overlay restorations Educational score: 4.02304220199585 Domain: biomedical Document type: Study Language: en A computer-aided design (CAD) digital software (inLab CAD SW 22.0.0; Dentsply Sirona) was used to design the restorations with a standardized morphology [Fig. 1 ] . Twenty-seven overlays were fabricated using CAM technology (inLab MCXL, Dentsply Sirona) using Advanced Zirconia-reinforced lithium disilicate CAD/CAM blocks (Dentsply Sirona, Tessera ™). Section title: Design and fabrication of overlay restorations Educational score: 2.3766024112701416 Domain: biomedical Document type: Study Language: en Fig. 1 Shows the restoration design created using CAD technology on three distinct preparation designs: ( A ) group O, ( B ) group OS, and ( C ) group OG Section title: Grouping of specimens Educational score: 2.7551050186157227 Domain: biomedical Document type: Study Language: en Twenty-seven specimens were grouped according to the overlay design into three groups, nine for each. Overlay ceramic restorations were glazed and sintered based on the manufacturer’s guidelines. Section title: Cementation of the specimens Educational score: 4.102153778076172 Domain: biomedical Document type: Study Language: en A 9.5% hydrofluoric acid (Porcelain Etch ® , Bisco, USA) was applied on the dry intaglio surface of the restorations for 20 s, rinsed with a copious amount of water for 60 s, and air dried. Silane (Porcelain Primer; Bisco, Schaumburg, IL, USA) was applied on the etched restorations surface and air dried after 60 s. The overlays were then cemented to the prepared dies with a dual-cure resin cement following the manufacturer’s instructions (BisCem; Bisco, Schaumburg, IL, USA). Cement was auto-mixed and applied on both the intaglio surface of the restoration, and the prepared die surface, each restoration loaded with cement was gently seated on its corresponding prepared die, initially with finger pressure followed by application of a constant static load of 200 g , by a specially designed device to ensure seating of the restoration for 60 s before photopolymerization. Then specimens were tack-cured for 2 s and excess cement was eliminated using scalpel, subsequently, a light curing procedure was applied for 20 s on each surface. Finally, the tooth-restoration interface was polished with polishing disks. The specimens were stored in water at room temperature before being tested. Section title: Marginal fit evaluation before thermal aging Educational score: 4.100505828857422 Domain: biomedical Document type: Study Language: en Evaluation of marginal fit was conducted following the methodology outlined by Holmes et al., The fit accuracy of the overlays on their corresponding prepared teeth was measured twice before and after the thermal aging procedure. Six measurements were taken for each: mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual. The measurements were performed at a magnification of X20 and X110 using a stereomicroscope connected to a digital camera [ Fig. 2 ] . The measurements were determined, and a mean value was calculated and performed by a single-blind examiner for each overlay. Section title: Marginal fit evaluation before thermal aging Educational score: 2.688948631286621 Domain: biomedical Document type: Study Language: en Fig. 2 Marginal fit measurements before thermal aging for the three groups: ( A ) Group O, ( B ) Group OG, and ( C ) Group OS. Group OS ( C ) shows the least marginal gap before thermal aging Section title: Thermal aging Educational score: 3.1582539081573486 Domain: biomedical Document type: Study Language: en Thermal aging was performed with a custom-built device for a total of 5000 cycles representing a 6-month clinical service, between 5 and 55 degrees Celsius in water baths with dwell times of 15 Sects. . Section title: Marginal fit evaluation after thermal aging Educational score: 2.8955867290496826 Domain: biomedical Document type: Study Language: en Following thermal aging, the measurements were assessed using a stereomicroscope at the same magnification and predefined measurement points [Fig. 3 ]. The collected data were then analyzed statistically after processing. Section title: Marginal fit evaluation after thermal aging Educational score: 2.9896910190582275 Domain: biomedical Document type: Study Language: en Fig. 3 Marginal fit measurements after thermal aging for the three groups: ( A ) Group O, ( B ) Group OG, and ( C ) Group OS. The marginal gap increased after thermal aging in all three groups following the same pattern as before thermal aging where the maximum marginal discrepancy was noted in ( A ) group O Section title: fracture resistance test Educational score: 4.134007930755615 Domain: biomedical Document type: Study Language: en A universal testing machine (5ST, Tinius oslen, England) was used to evaluate the fracture resistance of the specimens. A stylus with a 6 mm diameter custom-made stainless-steel ball was used to apply the load on the central fossa on the occlusal surface of the overlay along the long axis. A rubber sheet was placed under the sphere indenter to serve as a cushion and distribute forces evenly at the occlusal surface. The crosshead speed was 1 mm/min until fracture [Fig. 4 ]. The software program (version 10.2.4.0; Horizon) automatically recorded the maximum loads for each specimen in newtons (N). The failure mode for each specimen was classified according to the structures involved in the fracture according to Burke’s classification (Table 1 ) . Section title: fracture resistance test Educational score: 3.989980936050415 Domain: biomedical Document type: Study Language: en Fig. 4 Schematic representation of the fracture resistance test setup. A universal testing machine (5ST, Tinius Olsen, England) applied load using a custom-made 6 mm diameter stainless-steel ball on the central fossa of the overlay along the long axis. A rubber sheet was placed beneath the sphere indenter to cushion and evenly distribute forces across the occlusal surface. The load was applied at a crosshead speed of 1 mm/min until fracture occurred Section title: fracture resistance test Educational score: 2.804771661758423 Domain: biomedical Document type: Other Language: en Table 1 illustrates Burke’s classification of failure modes Mode of failure I Slight crack or fracture in restoration II Under 50% of the restoration lost III Fracture of the restoration extending through the midline IV Over 50% of the restoration lost V Serious fracture of the tooth structure and\or restoration Section title: Statistical analysis Educational score: 3.8899738788604736 Domain: biomedical Document type: Study Language: en Normality was checked using Shapiro Wilk test and Q-Q plots. Marginal fit and fracture resistance were not normally distributed thus both were presented mainly using median, minimum, maximum in addition to mean and standard deviation. The Kruskal Wallis test was used to analyze data between groups, followed by Dunn’s post hoc test with Bonferroni correction. Differences in marginal fit before and after thermal aging were analyzed using Wilcoxon Sign Rank test. All tests were two-tailed, with the significance level established at a p-value of ≤ 0.05. Data was analyzed using IBM SPSS, version 23 for Windows, Armonk, NY, USA. Section title: Marginal gap before and after thermal aging Educational score: 4.133249759674072 Domain: biomedical Document type: Study Language: en For marginal gap, the greatest mean marginal gap prior to thermal aging was observed in group O with a median gap value of 76.97 μm followed by group OG with a median gap value of 52.94 μm and the group OS with a median gap value of 35.57 μm. The marginal gap significantly increased after thermal aging in all three groups ( p < 0.05) following the same pattern as before thermal aging where the maximum marginal discrepancy was noted in group O with a median gap 114.84 μm succeeded by group OG with a median gap value 103.01 μm and the minimum marginal gap was noted in group OS with a median gap value 93.50 μm [Fig. 5 ]. Section title: Marginal gap before and after thermal aging Educational score: 2.9520089626312256 Domain: biomedical Document type: Study Language: en Fig. 5 Waterfall Chart showing comparison of marginal fit results of the three studied groups before and after thermal aging. With a significantly increased marginal gap after thermal aging in all three groups following the same pattern as before the thermal aging Section title: Fracture resistance Educational score: 3.7407078742980957 Domain: biomedical Document type: Study Language: en For fracture resistance, the O group had the highest median fracture load value 1809.08 N followed by Os group with median 1632.77 N. The OG group had the lowest median fracture load values of 1379.63 N [Fig. 6 ]. Section title: Fracture resistance Educational score: 3.264051914215088 Domain: biomedical Document type: Study Language: en Fig. 6 Waterfall chart showing a comparison of fracture resistance of the three studied groups. Group O had the highest median fracture load value of 1809.08 N, followed by Group OS, with a median fracture load value of 1632.77 N. The OG Group had the lowest median fracture load value, 1379.63 N Section title: Modes of failure Educational score: 4.034276008605957 Domain: biomedical Document type: Study Language: en Among the fracture modes , there is no statistically significant difference between the three studied groups ( p = 0.027). In the (O) group ( n = 9), 3/9 (33.3%) had mode II of fracture, 2/9 (22.2%) had mode III of fracture, and 4/9 (44.4%) had mode V of fracture. In group (OS) ( n = 9), 3/9 (33.3%) had mode II of fracture, 1/9 (11.1%) had mode III of fracture, and 5/9 (55.6%) had mode V of fracture. In group (OG) ( n = 9), 1/9 (11.1%) had mode II of fracture, 3/9 (33.3%) had mode III of fracture, and 5/9 (55.6%) had mode V of fracture, [ Figs. 7 and 8 , and 9 ] indicates the frequency of various modes of failure within the groups. Section title: Modes of failure Educational score: 2.590276002883911 Domain: biomedical Document type: Study Language: en Fig. 7 Chart showing a comparison of modes of fracture with no statistically significant difference between the three studied groups ( p = 0.027) Section title: Modes of failure Educational score: 2.465958833694458 Domain: biomedical Document type: Study Language: en Fig. 8 Illustrates the fracture mode observed in this study; as ( A ) mode II: less than half of the restoration lost. ( B ) mode III: Fracture of the restoration extending through the midline; half of the restoration lost. ( C ) mode V: Serious fracture of the tooth structure and restoration Section title: Modes of failure Educational score: 2.1163132190704346 Domain: biomedical Document type: Other Language: en Fig. 9 Presents the fracture modes from the occlusal view: ( A ) mode III: Fracture of the restoration extending through the midline and ( B ) mode V: Serious fracture of the tooth structure and restoration Section title: Discussion Educational score: 4.1055498123168945 Domain: biomedical Document type: Study Language: en This research sought to evaluate and compare the marginal fit and fracture resistance of three different overlay designs using advanced zirconia-reinforced lithium disilicate CAD/CAM material. Both null hypotheses were rejected, as the preparation designs used significantly affected fracture resistance and marginal fit of studied groups. This outcome is consistent with prior research findings that demonstrated a substantial influence of preparation design on the marginal fit and the fracture resistance of restorations . In this study, the marginal fit and fracture resistance of the advanced zirconia-reinforced lithium disilicate (ALD) CAD/ CAM material for three different overlay preparation designs on molars were compared. Section title: Discussion Educational score: 4.143202781677246 Domain: biomedical Document type: Study Language: en This study selected advanced zirconia-reinforced lithium disilicate (ALD) over other materials, such as 5Y-Zirconia and novel fully crystallized lithium disilicate, due to its superior combination of mechanical strength and aesthetic properties. While 5Y-Z offers enhanced translucency, its reduced fracture toughness and absence of transformation toughening make it more prone to failure under stress. ALD, by contrast, provides a favorable balance between strength and translucency, making it particularly suitable for posterior restorations. Moreover, novel fully crystallized lithium disilicate materials were not fully stabilized or commercially available when the study was initiated. ALD also simplifies bonding protocols by eliminating the need for sandblasting, which can compromise surface integrity, thereby ensuring both durability and efficiency . Diligent efforts were made to replicate the clinical intraoral environment in this study. A single scanner system was used to standardize the intraoral scanning process, minimizing variations and ensuring more accurate results. Each preparation design was duplicated to produce standardized dies using 3D printing resin (Model Resin, Formlabs, Somerville, MA) possessing an elasticity modulus similar to that of dentin , for evaluation of ceramic overlays’ marginal adaptation and fracture resistance. The stimulation of the clinical environment was also taken into account in various aspects of the research, including tooth preparation, impression creation, procedure for fabrication, and cementation of restorations. Section title: Discussion Educational score: 4.129055976867676 Domain: biomedical Document type: Study Language: en In this research, 3D-printed resin dies (Model Resin, Formlabs) were chosen over natural teeth due to their ability to provide consistent geometry across all specimens, eliminating variations in size and preparation design inherent to natural teeth. This approach also minimizes the challenges and variability associated with collecting, storing, hand-prepping, and handling natural teeth. Resin dies have a tensile strength of 61.0 MPa, which falls within the range of dentin tensile strength (44.4–97.8 MPa), making them a suitable substitute . Studies have demonstrated that crowns cemented to resin die yield fracture strength results comparable to those cemented on natural teeth. For instance, one study concluded that resin dies closely mimic the fracture behavior of crowns on dentin. Furthermore, another study reported that zirconia crowns fractured on resin dies exhibited similar strength values to those fractured on enamel dies . They did not replicate the natural tooth structure for cement adhesion. However, adhesion was not a primary focus of our research. Section title: Discussion Educational score: 3.5601394176483154 Domain: biomedical Document type: Study Language: en Based on Holmes et al., the term “marginal gap” denotes the vertical gap between the cervical edge of restoration and the prepared tooth surface. In simpler terms, the marginal gap pertains to the surface area of the cement, which is subjected to the oral environment and is susceptible to degradation . In this study the marginal gap was evaluated using the direct-view technique using a stereomicroscope. Some earlier research has also utilized this method . Section title: Discussion Educational score: 4.128232002258301 Domain: biomedical Document type: Study Language: en The marginal fit was evaluated in this study before and after thermal aging. The findings indicated that the marginal gap for all groups was below 120 μm before and after thermal aging, falling within the range considered clinically acceptable as determined by the Consensus among most authors suggests . Comparing data on marginal adaptation from various studies poses challenges and potential inaccuracies due to several factors, including variations in preparation design, measurement techniques, the number and location of measurement points, the type of resin cement employed, and the method used to fabricate the restoration. Advanced 4- or 5-axis milling machines, which can enhance the fit between the prepared tooth structure and the restoration’s intaglio surface, also contribute to these variations. However, the precision and reliability of the outcomes improve when more measurement points are included and consistently assessed at the same locations across samples. This approach reduces variability, ensuring that the results accurately reflect the restoration’s adaptation quality. Our study adopted this approach to ensure accurate and reliable results. Therefore, carefully considering these variations is essential when making in vitro comparisons of data . In this research, the restorations were adhered under 200 g applied weight to standardize the pressure applied . However, to ensure consistency in the cementation procedure, all restorations were placed by the same operator. Based on the findings of this research, group OS revealed the minimal marginal gap after cementation with a median gap value of 35.57 μm, succeeded by group OG with a median gap value of 52.94 μm and group O with a median gap value of 76.97 μm. Section title: Discussion Educational score: 4.028284549713135 Domain: biomedical Document type: Study Language: en Therefore, the preparation design of group OS (overlay preparation with 1.5 mm anatomical reduction of the occlusal surface and 1.0 mm circumferential shoulder finish line) has more retentive form and more defined margins, resulting in a reduction of the marginal gap size. These results aligned with the findings of Yang et al., who assessed how tooth preparation design influences the marginal adaptation of composite resin CAD-CAM Onlays by comparing two different preparation designs. They noted that the conventional design, which provided more excellent retention, resulted in greater adaptation. However, this result contradicted the findings of Falahchai et al., and Kim et al., Who determined that preparation designs lacking retention would offer superior adaptation to those with retention features. The variation can be ascribed to the different materials employed for restoration fabrication, variations in preparation designs, cementation techniques, and distinct techniques for gap measurement. Section title: Discussion Educational score: 4.08120059967041 Domain: biomedical Document type: Study Language: en In this research, there was a reduction in marginal fit (increase in micro gap) in all groups after exposure to thermal aging compared to the initial measurements. This observation aligned with the outcomes reported in earlier studies . They reported that the thermal aging affected all groups’ marginal fit and showed decreasing marginal adaptation. This finding was in agreement with the outcomes obtained in the current study. In group O, the statistical significance of the change in marginal gaps was noticeably lower compared to group OG and group OS. This may be due to the nature of 3D printing resin material; the temperature variations cause the resin to undergo expansion and contraction, particularly in thin margins . Section title: Discussion Educational score: 4.219964981079102 Domain: biomedical Document type: Study Language: en In the current study, the fracture resistance was evaluated for all groups after thermal aging. The force required to cause a fracture in the group (O) was 1809.08 N. However, it was 1632.77 N for group (OS). The lower value was recorded in group (OG) 1379.63 N. Studies showed that the occlusal forces generated during chewing and biting usually reach approximately 100 N, with a maximum bite force in habitual occlusion of up to 320 N . These values are considerably lower than the fracture resistance observed in our results, suggesting that partial restorations can comfortably endure occlusal forces without risk of failure. The durability of all-ceramic restorations is impacted by various factors, such as the ceramic material’s microstructure and fatigue, fabrication methods, preparation design, and bonding technique . The results of this study showed that group O (teeth with 1.5 mm anatomical occlusal reduction that received occlusal veneers) and group OS (teeth with 1.5 mm anatomical occlusal reduction and 1.0 mm circumferential shoulder finish line preparation) exhibited the highest fracture resistance of the studied groups under the imposed forces , This observation was consistent with the results obtained from Clausen et al., Who evaluated occlusal overlays and the influencing of ceramic material and preparation design. They reported that all-ceramic full coverage restorations bonded exclusively to enamel exhibited a tendency towards increased fracture resistance compared to those bonded to dentin with a finishing line located in enamel. Falahchai et al. revealed that teeth with less extensive restorations and sound marginal ridges demonstrated a lower incidence of fractures, reporting that increasing the reduction of tooth structure in the central area, such as with a mesio-occluso-distal preparation, leads to compromising the strength of the remaining tooth structure. Alternatively, the intracoronal extension of an overlay may produce a wedging effect. Hence, this clarifies why the fracture resistance outcomes of group (OS) exceeded those of group (OG) (characterized by anatomical occlusal reduction with a central groove), where both the central portion of the tooth and marginal ridges were eliminated. Also, preparation designs that emphasize retention have complex shapes with sharp inner edges, making them more prone to developing predetermined weak points. These geometric changes can lead to localized stress concentrations . This finding aligned with Channarong et al. , Who evaluated the resistance of bonded ceramic overlay restorations to fractures as influenced by different preparation designs. They found that the more natural tooth structure that remains, the more favorable the long-term prognosis for that tooth. Therefore, the conservation of natural tooth structure is crucial for the overall lifespan of the tooth. Also, they demonstrated that overlay restorations utilizing the adhesive system could reinforce defective teeth to a fracture resistance level similar to that of intact teeth. In contrast, the margin type and axial wall length did not affect the final fracture resistance. However, these results contradicted Alberto et al.‘s findings . They reported that the fracture resistance of the full-coverage crown restorations was notably greater than that of the occlusal-cover restorations. Also reported that the effect of the amount of the preparation that was 2.0 mm and 1.5 mm thick needed significantly more fatigue cycles to fail (17 − 15 times fatigue cycles) compared to crowns with a thickness of 1.0 mm. Also, the study has shown that self-adhesive resin cement exhibits weaker mechanical and bonding properties compared to conventional resin-luting cement . However, previous research has demonstrated that a 1 mm thin layer of lithium disilicate ceramic bonded to enamel exhibits a lower risk of fracture compared to bulker restorations (1.5–2 mm as recommended) bonded to dentin, primarily because of reduced mechanical complications . This confirms that other factors like ceramic thickness and type, type of cementation, tooth architecture, and study design can influence fracture resistance in addition to preparation design. Previous research indicated that any loss of tooth structure, whether caused by caries or cavity preparation, reduces the fracture resistance of restorations. The literature suggests that more conservative preparation designs with lower retention features tend to enhance fracture resistance . Building on the previous findings, this explains the results observed in our study. Group O, which featured the most conservative preparation design with no retentive form, demonstrated the highest fracture resistance. In contrast, Group OG, which included a central groove and internal angles, showed increased stress concentration in these areas, leading to reduced fracture resistance. Section title: Discussion Educational score: 3.270718574523926 Domain: biomedical Document type: Study Language: en According to the study’s results, various preparation designs exhibited distinct. frequencies of modes of failure. Nevertheless, these differences were not significant. More than half of the restoration fractures noted in this study involved damage to both the die and the restorative material. This observation is consistent with the results documented in previous studies by Johnson et al., And Alberto et al., . Section title: Discussion Educational score: 2.650472402572632 Domain: biomedical Document type: Study Language: en This study had several limitations. These limitations include that it was conducted in vitro, differing from a clinical setting where factors such as saliva and restricted access for the scanner inside the oral cavity would affect the precision of scanning prepared teeth. The internal adaptability was not evaluated in the study and faced challenges in accurately simulating a clinical setting. Section title: Conclusions Educational score: 1.4544252157211304 Domain: biomedical Document type: Other Language: en According to the results of this research: Section title: Conclusions Educational score: 4.0866217613220215 Domain: biomedical Document type: Study Language: en All tested groups showed marginal fit results within clinically acceptable parameters. All preparation designs utilized in this study for creating overlay restorations from ALD ceramics demonstrated acceptable fracture resistance. Higher fracture resistance was shown with intact marginal ridges and less extensive preparations. Modification of tooth preparation significantly impacted the magnitude of the fracture resistance and the marginal gap observed around ALD overlays. Thermal aging affected the marginal gaps of the three groups following the same pattern as before thermal aging. | Other | biomedical | en | 0.999996 |
PMC11699785 | Section title: Introduction Educational score: 3.9895942211151123 Domain: biomedical Document type: Study Language: en The incidence of anterior cruciate ligament (ACL) tears in the United States has been reported at 6.8 per 100,000 annually, making it the most common knee ligament injury . Failure rates amongst primary ACL reconstruction (ACLR) procedures have been estimated to range from 3.2 to 11.1% . Several factors contribute to postoperative failure, including traumatic reinjury, tunnel malposition, and biological failure . Beyond revisions, secondary injuries such as meniscus tears and contralateral injuries are reasons for overall reoperation after primary ACLR . The presence of concomitant meniscal tears, such as ramp lesions with ACL injuries, has been reported to range from 9 to 40% and is associated with a 7.7% rate of secondary meniscectomy . Because of this, it is important for surgeons to have a guide for predicting risk profiles for secondary injury or revision after ACLR. Section title: Introduction Educational score: 3.9732797145843506 Domain: biomedical Document type: Review Language: en In recent years, the use of artificial intelligence (AI) has become popular in orthopedic research . Specifically, machine learning (ML) models can learn complex patterns and associations between variables and outcomes from large datasets . These relationships can be used to generate predictive models incorporating patient demographics, injury characteristics, and surgical techniques, which can be used in the clinical setting . Machine learning models can be classified into classical machine learning (Random Forest, Gradient Boosted Regression Model (GBM) etc.) and deep learning with neural networks (NN) (Artificial Neural Networks, Multi-Layer Perceptron etc.). Classical machine learning models tend to be faster and require fewer resources, however, they require manual feature selection, whereas NN models are able to automatically learn features from raw data . ML research has been performed in various orthopedic domains, such as hip arthroplasty, hip arthroscopy, and spinal cord injuries [ 10 – 12 ]. Therefore, the application of ML in predicting objective outcomes following ACLR offers great potential to be used to identify and manage patient expectations, tailor rehabilitation regimens to maximize functional recovery, and to identify optimal candidates for specific surgical interventions. Section title: Introduction Educational score: 3.997738838195801 Domain: biomedical Document type: Review Language: en While the use of AI in ACL literature is promising, surgeons must familiarize themselves with the overall results, advantages, and disadvantages of ML models . To date, no review has provided a comprehensive summary of the utilization of ML models in predicting postoperative outcomes after ACLR. Therefore, this systematic review aims to summarize the statistical performance of machine learning in predicting revision, secondary injury, or reoperations in ACLR, and to provide a general overview of findings from these models. It was hypothesized that ML models would be superior in predicting these outcomes compared to standard logistic regression models. Section title: Materials and methods Educational score: 3.9671847820281982 Domain: biomedical Document type: Review Language: en This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Revised Assessment of Multiple Systematic Reviews (R-AMSTAR) guidelines for coordinating and reporting systematic reviews . Section title: Search strategy Educational score: 3.84621262550354 Domain: biomedical Document type: Study Language: en Three online databases (PubMed, Medical Literature Analysis and Retrieval System Online (MEDLINE), Excerpta Medica dataBASE (EMBASE)) were searched from database inception to February 6, 2024, to identify literature on the use of ML to predict revision, secondary injury, or reoperation in ACLR. The search strategy is described in Supplementary Table 1 . Section title: Search strategy Educational score: 4.054714679718018 Domain: biomedical Document type: Study Language: en Inclusion criteria included the following: (1) studies examining machine learning models to predict objective outcome measures (e.g. revision, reoperation, secondary injury) following ACLR, or external validity of established databases using machine learning models to predict the aforementioned outcomes, (2) simulation-based or laboratory studies and (3) studies written in English. Exclusion criteria included (1) systematic reviews or meta-analyses, (2) text-book chapters, (3) conference abstracts, (4) biomechanical studies, (5) levels of evidence V (i.e. case reports), (6) case series with less than five patients, and (7) cadaveric/animal studies. References of included studies and of pertinent review papers were manually searched to ensure all means of study identification were exhausted. If multiple papers reported the same outcomes using identical patient cohorts, only the article with the largest sample size or latest follow-up period was included. If multiple papers presented overlapping but non-identical cohorts, all articles were included as the extent of patient overlap was unable to be determined. Section title: Study screening Educational score: 2.112976312637329 Domain: biomedical Document type: Study Language: en Two authors independently performed title and abstract screening. Disagreements at this stage were resolved amongst reviewers, and a more senior author was consulted for remaining discrepancies. During the full-text stage, independent screening was performed and conflicts were resolved in a similar fashion. Section title: Assessment of agreement Educational score: 4.013981819152832 Domain: biomedical Document type: Study Language: en The inter-reviewer agreement was evaluated using a kappa (κ) statistic for screening. A priori classification was defined according to the following criteria: a κ of 0.91–0.99 was almost perfect agreement; a κ of 0.71–0.90 was considerable agreement; a k of 0.61–0.70 was high agreement; a κ of 0.41–0.60 was moderate agreement; a κ of 0.21–0.40 was fair agreement and a κ or ICC value of 0.20 or less was no agreement . Section title: Quality assessment Educational score: 3.8715672492980957 Domain: biomedical Document type: Study Language: en The Methodological Index for Non-Randomized Studies (MINORS) criteria were used for methodological quality assessment . Based on the MINORS criteria, non-comparative studies could get a maximum score of 16. For non-comparative studies, classification was a priori based on a previous systematic review: 0–4 indicated very low-quality evidence, 5–7 indicated low-quality evidence, 8–12 indicated fair-quality evidence, and scores ≥ 13 indicated high-quality evidence . Section title: Data abstraction and outcomes Educational score: 3.9400413036346436 Domain: biomedical Document type: Study Language: en Two review authors independently extracted and summarized data from included articles using a Google Sheets (Google LLC, Mountain View, CA, USA) spreadsheet. Demographic data such as number of patients, mean age, patient sex, and follow-up times were recorded. Machine learning specifics included the primary outcome of interest (e.g. revision, secondary injury, or reoperation), statistical software used, models assessed, training and test splits, and the handling of missing data. Adherence to the Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis (TRIPOD) guidelines was also assessed . Section title: Data abstraction and outcomes Educational score: 4.111512184143066 Domain: biomedical Document type: Study Language: en Discrimination, or classification accuracy, was assessed using area under the receiver operating curve (AUC) and concordance. AUC values range from 0 to 1, with increasing values representing increased discriminatory capacity . Concordance is another representation of AUC, ranging from 0.5 to 1, with increasing scores indicating a model that more accurately identifies the most true positive results and least false negative results . Calibration was assessed using calibration slope, intercept, and error. The calibration intercept is the tendency of a model to overestimate results, with scores approaching 0 indicating less frequent overprediction or underprediction . Calibration slope identifies if predictions are precise or extreme, with scores closer to 1 indicating better model predictions across the range of possible outcomes . Brier scores combine both discrimination and calibration, with values ranging from 0 to 1, with lower scores indicating higher accuracy . Factors deemed highly predictive for revision, secondary injury or reoperation were also extracted. The level of evidence of each paper was reported according to the authors’ statement or, if unstated, was reported using the Oxford Centre for Evidence-Based Medicine (OCEBM) guidelines . Section title: Outcome reporting Educational score: 2.06999135017395 Domain: biomedical Document type: Study Language: en Results were presented using descriptive statistics. Means, ranges, percentages, and standard deviations (SD) were calculated using Google Sheets software (Google LLC, Mountain View, CA, USA). Section title: Literature search Educational score: 4.083648681640625 Domain: biomedical Document type: Study Language: en The initial search resulted in 780 studies, of which 304 were duplicates. Of the 476 remaining, 20 were selected for full-text screening after abstract and title screening. Nine full-text articles satisfied the eligibility criteria and were included in the final analysis . There was a high level of agreement during title and abstract screening (κ = 0.892, 95%CI 0.799–0.986) and perfect agreement at the full-text stages (κ = 1.00). Section title: Literature search Educational score: 2.8717751502990723 Domain: biomedical Document type: Other Language: en Fig. 1 Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) flow diagram Section title: Study quality Educational score: 2.7634360790252686 Domain: biomedical Document type: Review Language: en All studies included in this review were classified as level IV evidence [ 24 – 32 ]. The mean MINORS score was 11.7 (range: 11–12). On average, the studies included were of fair quality. Section title: Study characteristics Educational score: 4.037515163421631 Domain: biomedical Document type: Study Language: en Five of nine (55.6%) studies reported following the TRIPOD guidelines . Two studies examined the external validity of a previous machine-learning model developed using source data from the Norwegian Knee Ligament Registry (NKLR) database . The other seven studies had their own internal validation [ 24 – 27 , 29 , 31 , 32 ]. Primary model development was performed using the NLKR, DKLR, and Rochester Epidemiology Project databases in three , two , and three studies , respectively. One study each used data from the American College of Surgeons National Surgical Quality Improvement Program database , Shanghai Sixth People’s Hospital , and the STABILITY I trial . Section title: Demographics Educational score: 4.013210296630859 Domain: biomedical Document type: Review Language: en This review included nine studies comprising 125,427 patients with a mean follow-up of 5.82 (range of means: 0.08–12.3) years. Of the eight studies that reported on patient sex, 51,511 female patients (41.5%) were included. The average age of patients at surgery was 26.73 (range of means: 19–32) years. A detailed description of study characteristics and demographics can be seen in Table 1 . Section title: Demographics Educational score: 4.184356212615967 Domain: biomedical Document type: Study Language: en Table 1 Study characteristics Author (Year) Study Design (Level of Evidence) MINORS SCORE (/16) Purpose Source data if external validation study Database/Institution TRIPOD Followed? Number of patients/knees Mean age at surgery Female (%) Follow-up of Outcome (years) Martin Retrospective Cohort (III) 12 Assess sample size effect on accuracy NR NKLR + DKLR Yes 62,955 MEDIAN 26 ( IQR: 20–36) [Missing data 1870] 26,446 7.6 (4.5) Martin Retrospective Cohort (III) 12 External Validation of Cox Lasso NKLR DKLR Yes 10,922 29 (11) 4916 8.4 (4.3) Martin Case-control (III) 12 Determine if machine learning analysis of NKLR can identify the most important risk factors associated with subsequent revision of primary ACL reconstruction NA NKLR NR 24,935 28 (11) 10,916 1, 2, 5 years Johnson Retrospective Cohort (III) 12 Machine learning to predict ACL re-operation NA Rochester Epidemiology Project Yes 1400 27 NR 9 years (min 2 years) Lopez Retrospective Comparative Prognostic (IV) 11 Machine learning (ML) models to predict outcomes following ACLR NR American College of Surgeons National Surgical Quality Improvement Program database NR 21,636 31.8 (10.5) 7638 (35.3%) 30 days Ye Case-control (III) 12 Machine learning to determine objective and subjective clinical outcomes of ACLR and to determine the most important predictors NA Shanghai Sixth People’s Hospital NR 432 26.8 (8.4) 112 (25.9%) 6 years (3.1) Martin Retrospective Cohort (III) 11 To assess the external validity of the NKLR model using STABILITY 1 RCT NKLR NKLR + STABILITY 1 trial NR 591 19.0 (3.2) 304 (51.4) 1, 2 Jurgensmeier Retrospective Cohort (III) 11 Machine learning to determine risk of secondary meniscal injury post primary ACLR NA Rochester Epidemiology Project (REP) Yes 1187 25 (18–34) 502 (42.3%) 12.3 (6.6–17.6) Lu Retrospective Cohort (III) 12 Machine learning to compare risk and timing of secondary meniscal injury between nonoperative, delayed ACLR, and early ACLR patients NA Rochester Epidemiology Project (REP) Yes 1369 28 (18–37) 677 (40.7) min 2 year MINORS: methodological index for non-randomized studies, TRIPOD: transparent reporting of a multivariable prediction model for individual prognosis or diagnosis, NKLR: norwegian knee ligament registry, DKLR: danish knee ligament registry, RCT: randomized controlled trial, NR: not reported, NA: not applicable, ACL: anterior cruciate ligament Section title: Machine learning methods Educational score: 4.146840572357178 Domain: biomedical Document type: Study Language: en Primary outcomes were revision ACLR, secondary meniscus injuries, graft failure, and all-cause reoperation in five [ 26 , 28 – 31 ], two , one , and one study , respectively. The most common model was Random Forest/Random Survival Forest, used in six studies (66.6%). The second most used model was the Cox Lasso model, assessed in four studies [ 28 – 31 ] (44.4%). Two studies used neural networks, MLPClassifier (Multi-Layer Perceptron) and Artificial Neural Network (ANN) (22.2%), respectively. With respect to model evaluation, seven (77.8%) studies used calibration [ 24 , 25 , 27 – 31 ], five (55.6%) used concordance (including C-statistic AUROC) [ 27 – 31 ], four (44.4%) used AUC (including Discrimination - AUROC) [ 24 – 26 , 32 ], and two (22.2%) used Brier Score . Section title: Machine learning methods Educational score: 4.047700881958008 Domain: biomedical Document type: Study Language: en Five (55.6%) studies used a 75/25 training/test split [ 24 , 28 – 31 ], two (22.2%) used 0.632 bootstrapping with 1000 resampled datasets , one (11.1%) used an 80/20 training/test split , and one (11.1%) used a 90/10 training/test split . Seven studies reported on their handling of missing data [ 24 – 27 , 29 – 31 ], of which five (71.4%) studies used multiple imputation . One external validation study included patients with data for features used in all five models from the source study . Another study excluded patients with missing data . A full description of the Machine Learning Methods can be seen in Table 2 . Section title: Machine learning methods Educational score: 4.210958480834961 Domain: biomedical Document type: Study Language: en Table 2 Study methods Author (year) Primary Outcome Statistical Software and ML algorithms Models Model Evaluation Training/Test Split Missing Data Method Martin Revision R (Version 4.1.11 R Core Team) Cox lasso Random survival forest Gradient boosting Super learner Concordance - Harrell C-index Calibration 75/25 Multiple imputation Martin Revision R (Version 3.6.1) Cox Lasso Concordance - Harrell C-index Calibration NR (external validation study - original model 75/25) Patients included if they had data for five predictive models from original model Martin Revision R (Version 3.6.1) Cox Lasso Survival Random Forest Generalized Additive Model (GAM) Gradient Boosted Regression Model (GBM) Calibration Concordance 75/25 Multiple imputation Johnson All-cause re-operation SciPy version 1.6.2 MLPClassifier GaussianNB LogisticRegression KNeighborsClassifier BaggingClassifier RandomForestClassifier AdaBoostClassifier GradientBoostingClassifier XGBClassifier AUC Calibration AUPRC F1 Recall Accuracy Precision 75/25 Multiple imputation Lopez ACLR post-op outcomes (revision included) TensorFlow Python open-source coding platform (Google Brain, Alphabet Inc., Mountain View, CA) Artificial Neural Network ML Logistic Regression AUC Accuracy 80/20 Excluded Ye Graft failure SPSS (Version 25.0; IBM Corp) Logistic Regression Gaussian Naïve Bayes Random Forest XGBoost Isotonic XGBoost Sigmoid XGBoost AUC Accuracy F1 90/10 NR Martin Revision R Cox Lasso Concordance - Harrell’s C-index Calibration 75/25 NA Jurgensmeier Secondary meniscus tear R 4.1.2 using RStudio version 1.4.1717 (RStudio, Boston, MA) SVM Random Forest XGBoost Elastic Net Discrimination - AUROC Calibration Brier score 0.632 bootstrapping with 1000 resampled datasets Multiple imputation Lu Secondary meniscus tear R 4.1.2 using RStudio version 1.2.5001 (RStudio, Boston, MA). Random Survival Forests C-statistic (AUROC) (Concordance) Calibration Brier Score 0.632 bootstrapping with 1000 resampled datasets Multiple imputation ML: machine learning, AUC: area under the curve, AUROC: area under the receiver operating curve, AUPRC: area under the precision-recall graph, ACLR: anterior cruciate ligament reconstruction, NR: not reported, MA: Massachusetts, CA: California, NR: not reported, NA: not applicable Section title: Feature selection Educational score: 4.007091522216797 Domain: biomedical Document type: Study Language: en Various features were assessed when evaluating machine learning models. Age and body mass index (BMI) were examined in eight [ 24 , 25 , 27 – 32 ] (88.9%) and six [ 24 – 27 , 29 , 32 ] (66.7%) studies, respectively. Both sex [ 24 – 27 , 32 ] and time between injury and ACLR [ 28 – 32 ] were considered in five (55.6%) studies. Femur fixation method [ 28 – 31 ] and KOOS QOL [ 28 – 31 ] were assessed in four studies each (44.4%), while graft choice , sports participation , and meniscal injury were examined in three (33.3%) studies each. Other features selected can be seen in Table 3 . Section title: Feature selection Educational score: 4.269613265991211 Domain: biomedical Document type: Study Language: en Table 3 Model Complete Set Author (year) Feature Selection AUC Calibration Intercept Calibration Slope Brier Score Concordance (95 CI) Calibration Error Martin Age at surgery Yrs. injury to surgery KOOS QOL Graft: hamstring Age at injury Femur fix: susp/cort. Graft: QT/BQT KOOS Sport Men. injury: none Activity: pivoting Graft: other Fix. comb: susp/interference Surgery on same knee KOOS All low NR NR NR NR 1 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.65–0.70) SL: 0.67 (0.65–0.69) 2 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 5 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.65–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 1 year: Cox Lasso 7.19, n.s RSF: 5.54, n.s GB: 7.48, n.s SL: 8.67, p = 0.034 2 year: Cox Lasso 8.17, p = 0.043 RSF: 6.42, n.s GB: 4.53, n.s SL: 4.10, n.s 5 year: Cox Lasso: 11.37, p = 0.01 RSF: 9.27, p = 0.026 GB: 11.07, p = 0.011 SL: 11.82, p = 0.008 Martin Patient age at primary surgery KOOS QoL score at primary surgery Graft choice Femur fixation method Years between injury and ACLR NR NR NR NR 1 year: Cox Lasso: 0.678 2 years: Cox Lasso: 0.676 5 years: Cox Lasso : 0.678 1 year: Cox Lasso: 22.24, p < 0.001 2 years: Cox Lasso: 11.82, p = 0.008 5 years: Cox Lasso : 13.98, p = 0.003 Martin Age at surgery Fixation combination Tibia fixation Femur fixation BMI KOOS Sport at surgery KOOS QOL at surgery Years from injury to surgery Age at injury Hospital type Further injury Meniscus injury Injured side NR NR NR NR 1 year Cox Lasso: 0.686 Random Forest: 0.672 GAM 0.687 GBM 0.669 2 year Cox Lasso 0.684 Random Forest: 0.670 GAM 0.685 GBM: 0.666 5 year: Cox Lasso: 0.683 Random Forest: 0.670 GAM: 0.684 GBM: 0.665 1 year Cox Lasso: 4.89, n.s Random Forest: 3.12, n.s GAM 4.79, n.s GBM 4.98, n.s 2 year Cox Lasso 11.35, p = 0.01 Random forest: 11.66, p = 0.009 GAM 11.19, p = 0.011 GBM: 3.76, n.s 5 year: Cox Lasso: 6.19, n.s Random Forest: 3.71, n.s GAM: 6.98, n.s GBM: 0.38, n.s Johnson Age Sex BMI Occupation Sport participation Injury mechanism Occurrence of reoperation after ACLR MLPClassifier: AUC = 0.61 GaussianNB: AUC = 0.58 LogisticRegression: AUC = 0.70 KNeighborsClassifier: AUC = 0.68 BaggingClassifier: AUC = 0.75 RandomForestClassifier: AUC = 0.76 AdaBoostClassifier: AUC = 0.73 GradientBoostingClassifier: AUC = 0.75 XGBClassifier: AUC = 0.77 NR NR NR NR NR Lopez Sex Race BMI (Calculated From The Recorded Height And Weight) American Society Of Anesthesiologists (ASA) Classification History Of Smoking Diabetes Hypertension Requiring Medication Wound Infection Use Of Steroids For A Chronic Condition Bleeding disorders were Abstracted Perioperative Data Such As Anesthesia Type (General, Spinal, IV Sedation, Regional, Other) Surgery Setting (Inpatient Vs Outpatient) Operative Time (Prolonged Operative Time Defined As > 120 min) ANN: Reoperation: 0.842 ACLR-related Readmission: 0.872 Logistic Regression: Reoperation: 0.601 ACLR-related Readmission: 0.606 NR NR NR NR NR Ye Age Sex BMI Time From Injury To Surgery Participation In Competitive Sports Preoperative Lysholm And IKDC Scores Posterior Tibial Slope High-Grade Knee Laxity Graft Diameters Of Anteromedial And Posterolateral Bundles Medial And Lateral Meniscal Resection Follow-Up Period Meniscal Reinjury After ACLR Graft Failure: XGBoost (excellent): AUC = 0.944 (0.001), Accuracy = 0.986 (0.012) Residual Laxity: Random Forest (excellent): AUC = 0.920 (0.014), Accuracy = 0.914 (0.024) NR NR NR NR NR Martin Patient Age At Primary Surgery Knee Injury And Osteoarthritis Outcome Score Quality Of Life Subscale (KOOS-QOL) Score At Primary Surgery Graft Choice Femur Fixation Method Time Between Injury And ACLR NR NR NR NR 1 year: Original Norwegian Algorithm Performance: 0.686 (0.652–0.721) STABILITY data: HT = HT, HT + LET = BPTB: 0.713 (0.634–0.791) HT = HT, HT + LET = Unknown: 0.609 (0.528–0.691) All patients = HT: 0.674 (0.597–07.51) 2 year: Original Norwegian Algorithm Performance: 0.684 (0.650–0.718) STABILITY data: HT = HT, HT + LET = BPTB: 0.713 (0.637–0.789) HT = HT, HT + LET = Unknown = 0.608 (0.530–0.688) All patients = HT: 0.673 (0.598–0.747) 1 year: Original Norwegian Algorithm Performance: 4.9 n.s. STABILITY data: HT = HT, HT + LET = BPTB: 2.6 n.s. HT = HT, HT + LET = Unknown: 10.6 p < 0.01 All patients = LT: 8.7 p < 0.01 2 year: Original Norwegian Algorithm Performance: 11.3 p = 0.01 STABILITY data: HT = HT, HT + LET = BPTB: 11.7 p < 0.01 HT = HT, HT + LET = Unknown: 8.9 p < 0.01 All patients = LT: 10.2 p < 0.01 Jurgensmeier Age Sex Body mass index Sport participation Diagnosis of hypermobility or malalignment Radiographic findings Management SVM: Apparent 0.782 (0.779–0.785), Internal Validation 0.738 (0.736–0.739) Random Forest: Apparent 0.997 (0.994–0.999), Internal Validation 0.790 (0.785–0.795) XGBoost: Apparent 0.814 (0.813–0.816), Internal Validation 0.758 (0.755–0.761) Elastic Net: Apparent 0.673 (0.61–0.736), Internal Validation 0.646 (0.643–0.648) SVM: 0.0161 Random Forest: 0.006 XGBoost: 0.007 Elastic Net: 0.0165 SVM: 1.091 (1.086–1.096) Random Forest: 0.9608 XGBoost: 0.9569 Elastic Net: 0.8926 SVM: 0.14 (0.13–0.15) Random Forest: 0.10 (0.09–0.12) XGBoost: 0.12 (0.11–0.14) Elastic Net: 0.18 (0.17–0.20) NR NR Lu Age Sex Body mass index Activity level Occupation Comorbid diagnosis Radiographic findings Management ACLR: 0.80 (0.76–0.83) Non-op: 0.66 (0.58–0.74) ACLR: 0.0051 (− 0.014–0.024) Non-op: 0.0048 (− 0.059–0.069) ACLR: 0.97 (0.89–1.05) Non-op: 0.97 (0.65–1.30) ACLR: 0.106 (0.029–0.183 Non-op: 0.111 (0.034–0.188) NR NR ACLR: anterior cruciate ligament reconstruction, AUC: area under the curve, CI: confidence interval, KOOS: knee osteoarthritis and outcome score, QOL: quality of life, QT: quadriceps tendon, BQT: quadriceps tendon with a bone -block, GB: gradient boosted regression model, RSF: random survival forest, SVM: support vector machine, HT: hamstrings tendon, BPTB: bone-patellar tendon-bone, LET: lateral extra-articular tenodesis, SL: super learner, GAM: generalized additive model, NR: not reported, n.s: not significant, non-op: non-operative Section title: Area under the curve (AUC) Educational score: 4.066973686218262 Domain: biomedical Document type: Study Language: en Five studies reported an AUC for their chosen models [ 24 – 27 , 32 ]. Overall, AUC for the strongest-performing models in each study ranged from 0.77 to 0.997, indicating that these models ranged from fair to excellent discrimination. The best-performing model was Random Forest (AUC = 0.997) when used to predict secondary meniscus injury . One study found that XGBoost was the best model for predicting graft failure (AUC = 0.944) . When Artificial Neural Network (ANN) was compared with logistic regression, ANN was superior, with an AUC of 0.842 (good discrimination) compared to 0.601 (poor discrimination) for Logistic Regression (LR) . In another study, Random Forest was slightly superior to LR with AUCs of 0.77 and 0.70, respectively . Section title: Concordance Educational score: 4.105146408081055 Domain: biomedical Document type: Study Language: en Four studies reported on concordance [ 28 – 31 ]. Overall, concordance for the best-performing models in each study ranged from 0.67 to 0.71, indicating that these models ranged from poor to fair discrimination. The best-performing model was Cox Lasso (Concordance: 0.71) . One study assessed the STABILITY trial and found that when STABILITY patients with hamstring tendon (HT) autografts in addition to lateral extra-articular tenodesis (LET) were coded as receiving a bone-patellar tendon-bone graft (BPTB) from the NKLR data, this subgroup achieved the highest concordance, with scores of 0.713 (range: 0.634–0.791) and 0.713 (range: 0.64–0.79) at one and two years, respectively . In the same study, the original Norwegian Algorithm reported concordance of 0.686 (range: 0.65–0.72) and 0.684 (range: 0.65–0.72) at one and two years, respectively . Section title: Concordance Educational score: 4.068375587463379 Domain: biomedical Document type: Study Language: en One study reported predicting revision concordance for several models over different time intervals. At the one-year interval, the Cox Lasso model had the lowest concordance, with a score of 0.59 (range: 0.56–0.61) and 0.58 (range: 0.56–0.61) at two and five years. The RSF, GB, and SL models all showed higher concordance scores of 0.67 (ranges, RSF: 0.64–0.69, GB: 0.65–0.70, SL: 0.65–0.69), maintaining their scores at two and five years . Another study reported concordance for the Cox Lasso model of 0.678 at one year, 0.676 at two years, and 0.678 at five years . One study found that the GAM model had the highest concordance across all time points (1-year: 0.687, 2-year: 0.685, 5-year: 0.684) . This was followed by Cox Lasso (1-year: 0.686, 2-year: 0.684, 5-year: 0.683), Random Forest (1-year: 0.672, 2-year: 0.670, 5-year: 0.670), and GBM (1-year: 0.669, 2-year: 0.666, 5-year: 0.665) . Section title: Brier scores Educational score: 4.081099510192871 Domain: biomedical Document type: Study Language: en Two studies reported on Brier Scores . Random Forest was the most accurate model in a study reporting on secondary meniscal injuries, and the studies had Brier scores ranging from 0.10 to 0.18, indicating low deviation of predictions and actual outcomes. One study found that Random Forest was the most accurate, with a Brier score of 0.10 (range: 0.09–0.12) at a mean follow-up of 12.3 (6.6–17.6) years, with key variables being time to return to sport, visual analog scale (VAS) pain score at injury, and time to surgery . Another study predicting secondary meniscal injuries reported a Brier score of 0.106 (range: 0.029–0.183) at a minimum two-year follow-up using the Random Survival Forest model, with key variables being time to return to sport, VAS pain score at injury, and hypermobility . Section title: Calibration intercept and calibration slope Educational score: 4.12282133102417 Domain: biomedical Document type: Study Language: en Two studies looked at calibration intercept and slope . The best-performing models for calibration interval reported scores ranging from 0.0051 to 0.006. The random survival forest model performed best when predicting secondary meniscal injury after ACLR with a score of 0.0051 (− 0.014 to 0.024) at a minimum two-year follow-up . The positive intercept indicates that included ML models tend to underestimate the risk; however, the confidence interval suggests that the systemic underprediction is not statistically significant. The best-performing models for calibration slope reported slopes from 0.96 to 0.97, with Random Survival Forest reporting the highest score (0.97) at a minimum two-year follow-up in a study predicting second meniscal injury . XGBoost was similar with a slope of 0.957 (0.952–0.962) at a mean follow-up of 12.3 (6.6–17.6) years in a study predicting second meniscal injuries . These values suggest that the models used tend to slightly overestimate risk, placing too much importance on predicting features. Overall, both studies revealed that the calibration intercept and slope were most accurate using the predictive features of time to return to sport and VAS pain score. Section title: Calibration error Educational score: 4.117586135864258 Domain: biomedical Document type: Study Language: en Calibration error was measured in four studies [ 28 – 31 ]. One study reported calibration errors for various models at one-, two-, and five-year marks . At one year, the Cox Lasso, Random Survival Forest (RSF), and Gradient Boosting (GB) models all had non-significant calibration errors, whereas the Super Learner (SL) model demonstrated a calibration error of 8.67 ( p = 0.034). At two years, the Cox Lasso model showed a significant calibration error of 8.17 ( p = 0.043). At five years, calibration errors were significant in all models: 11.37 ( p = 0.01) for Cox Lasso, 9.27 ( p = 0.026) for RSF, 11.07 ( p = 0.011) for GB, and 11.82 ( p = 0.008) for SL. One study reported significant calibration errors for the Cox Lasso model, with errors of 22.24 ( p < 0.001) at one year, 11.82 ( p = 0.008) at two years, and 13.98 ( p = 0.003) at five years . One study found significant miscalibration at two years, with calibration errors of 11.35 ( p = 0.01) for Cox Lasso, 11.66 ( p = 0.009) for Random Forest, and 11.19 ( p = 0.011) for Generalized Additive Model (GAM). None of the models showed significant calibration errors at five years. Another study externally validated the original Norwegian Algorithm using the STABILITY trial. They found that the subgroup (HT + LET patients coded as having BPTB grafts) with the highest concordance had a significant calibration error of 11.7 ( p < 0.01) at two years . The original Norwegian Algorithm also showed a significant calibration error of 11.3 ( p = 0.01) at two years. Other subgroups analyzed showed evidence of miscalibration at one and two years, respectively ( p < 0.01). Complete data from the model set can be seen in Table 3 . Section title: Multiple imputation data Educational score: 4.037633419036865 Domain: biomedical Document type: Study Language: en Only one study reported data on multiple imputation analyses . The concordance data from this study was not significantly different ( p < 0.05) from the original set. The specific data can be seen in Table 4 . However, the calibration data revealed an increased statistically significant calibration error in the multiple imputation cohort. At one year, two of the four models showed miscalibration ( p > 0.05), and at two and five years, all models showed significant miscalibration ( p < 0.05). The calibration error at one year ranged from 4.17 to 8.35. At two years, it ranged from 8.34 to 8.98; at five years, it ranged from 8.30 to 14.05. Section title: Multiple imputation data Educational score: 4.21002721786499 Domain: biomedical Document type: Study Language: en Table 4 Multiple Imputation Data Multiple Imputation Data Set Author Concordance (95 CI) Calibration Martin 1 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.66 (0.64–0.69) GB: 0.68 (0.65–0.70) SL: 0.67 (0.65–0.70) 2 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.67 (0.65–0.70) GB: 0.67 (0.65–0.70) SL: 0.67 (0.65–0.70) 5 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.65–0.70) GB: 0.67 (0.65–0.69) SL: 0.67 (0.65–0.70) 1 year: Cox Lasso 8.35, p = 0.039 RSF:4.17, p = 0.244 GB: 7.57, p = 0.056 SL: 7.99, p = 0.046 2 year: Cox Lasso 8.81, p = 0.032 RSF: 8.96, p = 0.030 GB: 8.98, p = 0.030 SL: 8.34, p = 0.039 5 year: Cox Lasso: 8.30, p = 0.040 RSF: 8.95, p = 0.030 GB: 11.53, p = 0.009 SL: 14.05, p = 0.003 Original Data Set Martin 1 year: Cox Lasso 0.59 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.65–0.70) SL: 0.67 (0.65–0.69) 2 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.64–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 5 year: Cox Lasso 0.58 (0.56–0.61) RSF: 0.67 (0.65–0.69) GB: 0.67 (0.64–0.69) SL: 0.67 (0.64–0.69) 1 year: Cox Lasso 7.19, n.s RSF: 5.54, n.s GB: 7.48, n.s SL: 8.67, p = 0.034 2 year: Cox Lasso 8.17, p = 0.043 RSF: 6.42, n.s GB: 4.53, n.s SL: 4.10, n.s 5 year: Cox Lasso: 11.37, p = 0.01 RSF: 9.27, p = 0.026 GB: 11.07, p = 0.011 SL: 11.82, p = 0.008 KOOS: knee osteoarthritis and outcome score, CI: confidence interval, GB: gradient boosted regression model, RSF: random survival forest, SL: super learner, GAM: generalized additive model, n.s: not significant Section title: Factors predicting outcomes Educational score: 3.372377634048462 Domain: biomedical Document type: Study Language: en Various features were considered most important predictive features (top three) by the assessed models. Years from injury to surgery were considered most important by four models (Random Forest, SL, GBM, GAM). Graft choice was considered most important by three models (Cox Lasso, GBM, GAM). Three models considered age at surgery most important (Random Forest, SL, GBM). Femur fixation was considered most important by three models (Cox Lasso, GBM, and GAM). A comprehensive list of the importance of the other features can be seen in Table 5 . Section title: Factors predicting outcomes Educational score: 4.175801753997803 Domain: biomedical Document type: Study Language: en Table 5 Model performance Author (year) Factors predicting outcomes (in order of importance) Martin Random forest : age at surgery age at injury years from injury to surgery KOOS QOL Cox model (lasso) : femur fix - susp/cort graft qt/BQT fix comb: interfer/susp Graft other Femur fix interf. Grand boosted regression : age at surgery years from injury to surgery graft age at injury KOOS QOL Super learner : age at surgery years from injury to surgery KOOS QOL Graft hamstring age at surgery Martin NA - External validation study Martin Cox-Lasso : graft choice femoral fixation KOOS QoL at time of surgery Years from injury to surgery Age at the time of surgery Random Forest : age at time of injury tibial fixation device fixation device combination GAM : Graft Years from injury to surgery Femur fixation other KOOS QOL at surgery GBM : Age at surgery Years from injury to surgery Femur fixation KOOS QOL at surgery Lopez Surgery Setting Operative Time BMI Age Race Johnson Systemic Inflammatory Disease Distal Tear Location Concomitant MCL Repair VAS Proximal Tear Location Ye Medial Meniscal Resection Participation In Competitive Sports Posterior Tibial Slope Graft Diameter Of PLB Male Gender Martin NA - External Validation Study Jurgensmeier Time to RTS VAS Pain Score at injury Time to surgery Age at injury Tear location Lu Time To RTS VAS At Injury Consultation Hypermobility Involvement In Noncontact Sports African American Race RTS: return to sport, VAS: visual analogue scale, PLB: posterolateral bundle, MCL: medial collateral ligament, BMI: body mass index, KOOS: knee osteoarthritis and outcome score, QOL: quality of life, GBM: gradient boosted regression model, GAM: generalized additive model, NA: not applicable Section title: Discussion Educational score: 4.0755743980407715 Domain: biomedical Document type: Review Language: en The primary finding of this systematic review was that existing machine learning models to predict secondary injury or surgery after ACLR are variable in terms of discriminatory performance. Overall, Random Forest models were the most effective at predicting outcomes when using AUC, Brier, Calibration slopes and intercepts. Cox-Lasso was the most effective model when using concordance. Of the four studies reporting on AUC, values were relatively high, ranging from 0.77 to 0.997. However, of the four studies reporting on concordance, the mean values of all studies were closer to 0.5 than 1.0. Furthermore, there was variability when evaluating calibration. While the two studies reporting on Brier scores, calibration slope, and intercept reported minimal evidence of miscalibration in highest performing models, the four studies reporting on calibration error found significant evidence of miscalibration at either two and five-year follow-ups amongst 10 of 14 models assessed. Factors deemed important for secondary ACLR or injury (e.g. secondary meniscus injury, graft failure) were also variable from model to model and study to study. Section title: Discussion Educational score: 4.071986198425293 Domain: biomedical Document type: Review Language: en Machine learning has become incredibly popular in developing models to predict postoperative outcomes, and there is immense potential benefit in using these analyses to generate prediction models and calculators. However, this review demonstrates that there is still room for improvement in model performance. One recent study of 104 patients reported AUC values for several factors predictive of revision, ranging from 0.756 to 0.813 . These values fall in the range of the predictive models that reported on AUC included in this review. Some models in this review had AUCs over 0.95 for predicting secondary meniscus injuries and revision, suggesting strong discriminatory power . However, it is notable that other studies providing C-statistics reported relatively low discrimination, with mean values all being under 0.75. The findings in this review align with a recent systematic review on ML models in various orthopaedic sub-specialties . They found that in spine surgery, hip arthroscopy, total joint arthroplasty, and shoulder arthroplasty, the C-statistics ranged from 0.65 to 0.92, 0.51–0.94, 0.63–0.89, and 0.70–0.95, respectively. While some models from this review had low C-statistics (e.g., closer to 0.5), others had values closer to 1 . They also noted the lack of external validation and inconsistent adherence to predictive modeling guidelines. Therefore, it is possible that existing models investigating revision and secondary injury risk may be missing key important factors. Section title: Discussion Educational score: 4.109770774841309 Domain: biomedical Document type: Study Language: en External validation studies are essential to assess the generalizability of machine learning models. One included study attempted to externally validate the revision prediction model from the original NKLR dataset using the Danish Knee Ligament Registry (DKLR) . Concordance was similar between populations (DKLR: 0.68; NKLR: 0.68–0.69); however, there was significant evidence of miscalibration at one, two, and five years when evaluating the DKLR group. Furthermore, compared to the NKLR dataset, calibration error at one and five years was greater (4.89 versus 22.24 and 6.19 and 13.98 respectively) . The other external validation study assessing the STABILITY trial using the NKLR model reported a concordance of 0.71; however, it found significant evidence of miscalibration at two years. While two of six studies demonstrated strong calibration, these models have not been externally validated like that of the NKLR database. Having models demonstrate strong calibration and concordance at the two-year mark is incredibly important as one in 17 (6%) of ACLR patients will suffer a second ACL injury within two years of the index operation. Furthermore, rates of secondary ACL injury (e.g. ipsilateral or contralateral) at five, ten, and fifteen years have been reported to be 12%, 27%, and 31% . Continuous evaluation of established and novel machine learning algorithms is incredibly important for prediction calculators to translate effectively into clinical practice. Section title: Discussion Educational score: 4.124019622802734 Domain: biomedical Document type: Review Language: en Factors that were not included in the current review that may be important when considering secondary injury risk include concomitant lateral extra-articular tenodesis (LET) procedures, meniscus status, medial collateral ligament (MCL) injuries, and elevated posterior tibial slope (PTS) (or effects of bone morphology). The STABILITY I study, a large multicenter randomized controlled trial (RCT) comparing ACLR with and without LET, found that at 24 months postoperative, the LET group had a rupture rate of 4.1% compared to 11.2% in the non-LET group ( p = 0.004) . A secondary analysis from this trial demonstrated that younger age, greater posterior tibial slope, high-grade knee laxity, and earlier return to sport all contributed to increased odds of rupture. Larger hamstring autograft diameter was protective in reducing the odds of knee laxity in the form of asymmetric pivot shift . Several of these factors were not a part of the risk calculators developed from machine learning algorithms. Specifically, some studies suggest that an increased PTS may place strain on the ACL, increasing the risk of failure . Certain groups have proposed a threshold of 12 degrees, and have advocated for the use of slope-reducing osteotomies to reduce the PTS, especially in revision settings . Section title: Discussion Educational score: 4.247040748596191 Domain: biomedical Document type: Review Language: en Machine learning analyses offer immense potential in terms of predictive capacity, however it is clear that there is much room for improvement, especially in the field of predicting revision or secondary knee injury after ACLR. With the risk of revision still being an issue, this review advocates for including factors such as the inclusion of LET procedures, graft diameter, meniscus status, and elevated posterior tibial slope in developing these models. Furthermore, future studies are encouraged to continue to attempt to externally validate existing and novel models to assess generalizability. Demonstrating strong concordance or AUC and little evidence of miscalibration both in the short-term and long-term is essential in order to implement risk-calculators in the clinical setting. There are a few limitations to this review. First, there were only two inclusions that served as external validation studies, which limit the generalizability of the reported findings. Second, only 55% of studies reported adhering to the TRIPOD guidelines for diagnostic studies, indicating high variability in the quality of individual datasets and reporting of results. This limitation is also noted in a recent systematic review on ML models in orthopaedic trauma, which reported a TRIPOD statement adherence of 62% , highlighting the need for better adherence to reporting guidelines. Third, there were limited amounts of comparisons with traditional multivariate logistic regression analyses, preventing the ability to make conclusive statements about the superiority or inferiority of machine learning models when the two methods are compared. Only two studies in this review included NN models, which is another source of weakness. NN modelling would allow for the inclusion of image data and, thus, the creation of multimodal models that incorporate images and clinical variables. In this review, the NNs did not perform better than classical models, which may be because NNs require more resources to create and larger datasets to avoid overfitting. Ultimately, current ACLR prediction models mainly incorporate classical ML, as opposed to multimodel prediction models. Multicenter collaboration based on high-quality prospective databases and registries, with agreement between investigators on feature inclusion, is needed for high-quality ML prediction algorithms. Only nine studies were included in this review, all of which were level IV evidence, preventing the ability to perform a meta-analysis and pool machine learning performance statistics. The average quality of the included studies was fair, which limits the reliability of the findings and highlights the need for further high-quality research in this domain. Finally, it is important to note that these predictive models are preliminary and have not been assessed in a prospective cohort of patients. Future adequately powered longitudinal studies testing these models are needed to ascertain their external validity. Section title: Conclusion Educational score: 4.016915321350098 Domain: biomedical Document type: Study Language: en Machine learning models designed to predict the risk of revision or secondary knee injury demonstrate variable discriminatory performance when evaluated with AUC or concordance metrics. Furthermore, there is variable calibration, with several models demonstrating evidence of miscalibration at two or five-year marks. A key limitation of this study is the lack of external validation of existing models, which restricts their generalizability. Future efforts should focus on validating current models in addition to developing and integrating multimodal neural networks to improve predictive accuracy and reliability. Further comparisons with traditional multivariate logistic regression analysis are also needed to validate the benefit of more advanced models. Section title: Electronic supplementary material Educational score: 0.9769887328147888 Domain: other Document type: Other Language: en Below is the link to the electronic supplementary material. Section title: Electronic supplementary material Educational score: 1.2997462749481201 Domain: biomedical Document type: Other Language: en Supplementary Material 1 | Study | biomedical | en | 0.999996 |
PMC11699795 | Section title: Introduction Educational score: 2.8048970699310303 Domain: biomedical Document type: Other Language: en It is well known that edentulous ridges progressively diminish in size throughout the patient’s life, especially after complete loss of all natural dentition; consequently, all denture wearers are required to have regular follow-up visits to maintain optimal denture retention and adaptation which reflects on denture efficient serviceability. Denture correction by relining or even rebasing may be considered the most common option of treatment . Section title: Introduction Educational score: 3.8910491466522217 Domain: biomedical Document type: Other Language: en It was reported that, after relining procedures for ill-fitting dentures, patients expressed a greater level of comfort with their new dentures . On the other hand, as a result of multiple laboratory procedures, material dimension changes, polymerization shrinkage, and residual monomer that may cause pain and mucosal reactions, many edentulous patients are not completely pleased with their dentures and maintain complaining from discomfort, pain, and inadequate retention while using the denture . Patients with compromised medical history that contra-indicate the use of dental implants require to have more valid and reliable treatment options for constructing more satisfactory complete dentures, as a result, providing patients with alternative treatment options and different manufacturing techniques is one of the main goals of merging modern innovations into dental clinics . Section title: Introduction Educational score: 3.6069352626800537 Domain: biomedical Document type: Other Language: en Digital dentistry plays an extremely important role in removable dentures fabrication recently, it always requires a very accurate impression in order to produce retentive and well adapted denture. To digitally fabricate a prosthesis, a digital impression is obtained either from intraoral scanning of the residual ridge or extra-oral scanning of the master cast and send to the dental laboratory to create a digital model on which the denture is designed and manufactured either by additive or subtractive method . Section title: Introduction Educational score: 3.8696677684783936 Domain: biomedical Document type: Study Language: en Computer-aided design (CAD) computer-aided manufacturing (CAM) systems prove to reduce complicated laboratory steps, time and effort consumed by the laboratory technicians, the edentulous patients and the dentists . Patient’s data is recorded and stored for future use if duplicate dentures are needed . Digital dentures showed satisfying clinical outcomes with a smoother, more regular surface than conventionally processed ones [ 9 – 14 ]. The digitally fabricated complete denture from resin discs that were produced under elevated temperature and pressure to overcome the problem of dimension changes of heat-polymerized acrylic resin . Section title: Introduction Educational score: 2.0488903522491455 Domain: biomedical Document type: Other Language: en There were many objective and subjective factors that influence patient satisfaction as age, gender, denture past experience, mastication and chewing ability, appearance, denture retention and adaptation, patient’s pain threshold and the psychological and behavioral manners of the patient . Section title: Introduction Educational score: 3.737359046936035 Domain: biomedical Document type: Other Language: en Complete denture retention and adaptation are greatly affected by many factors as method of fabrication, denture extension, shape and compressibility of the supporting tissues, the thickness of the denture base, the material from which denture constructed. The digital superimposition of scanned denture fitting surface with either the scanned oral tissues or a cast is a recently introduced approach to evaluate and compare the adaptation of the constructed dentures . Section title: Introduction Educational score: 2.968287706375122 Domain: biomedical Document type: Other Language: en Hence, the method of denture fabrication directly affects its retention and clinical performance, which consequently affects the patient’s satisfaction. A question may arise regarding the effectiveness of incorporating digital technology with the conventional relining technique which may overcome the drawbacks of denture flasking that deform the denture during processing, and to enhance denture retention and adaptation to reach patient satisfaction. Section title: Introduction Educational score: 2.7366883754730225 Domain: biomedical Document type: Study Language: en The null hypothesis of this study that, there were no significant differences between digitally and conventionally constructed dentures regarding denture retention and adaptation and patient satisfaction. Section title: Materials and methods Educational score: 1.6413511037826538 Domain: clinical Document type: Clinical case Language: en Twelve patients, both males and females, were selected from the outpatient clinic of the dental faculty, the British University in Egypt (BUE) with complete dentures constructed in the last two years. The patient’s main chief complain was loose ill-fitted maxillary complete denture. Section title: Inclusion criteria Educational score: 2.8050811290740967 Domain: biomedical Document type: Other Language: en Patients having completely edentulous maxillary and mandibular arches accompanied by normal skeletal relationship (class I) with well-developed maxillary edentulous ridge, and without severe undercuts to minimize the effect of undercuts on retention values. The patients were suffering from a loose or “falling” denture but with proper esthetics, centric relation, and the correct vertical dimension of occlusion (VDO). Section title: Exclusion criteria Educational score: 3.372701406478882 Domain: biomedical Document type: Other Language: en Patients with significant skeletal problems (class II or III) or who went through severe osseous surgery or any hereditary or acquired osseous abnormality find it challenging to place the denture properly on the supporting ridge. If the patient suffers from xerostomia, excessive flabby ridges, any pathology of the oral mucosa, or any inflammatory changes, candidiasis, hyperplasia, neurological disorders, and malignancies If the denture creates a major speech problem, poor esthetics, or an unsatisfactory jaw relationship. Section title: Exclusion criteria Educational score: 1.1520813703536987 Domain: biomedical Document type: Other Language: en The current study has been accepted by the Committee of Ethics, Faculty of Dentistry, British University in Egypt with approval number (23–067). All the participants have been informed about the treatment plan, the follow-up recalls needed and signed written consents. Section title: Steps for relining the maxillary denture Educational score: 3.0281171798706055 Domain: biomedical Document type: Other Language: en Tissue preparation: Before making the relining impression, patients were advised to remove their previous dentures from their mouth for at least three to four days and massage their soft tissues two to three times per day to simulate blood flow and improve the recovery of any small ulcers or inflammations with the application of proper medicaments. Section title: Steps for relining the maxillary denture Educational score: 3.6357669830322266 Domain: biomedical Document type: Other Language: en Denture preparation: Occlusion must be balanced before the relining process to guarantee even occlusal contact and that the denture will not move or tilt when the patient is asked to close his mouth during impression material setting. To prevent contact with the reflected tissues, denture borders were lowered by around 2 mm, except the denture posterior border (post dam). The denture tissue surface was relieved by about 1–2 mm to create even space for impression material. Section title: Steps for relining the maxillary denture Educational score: 3.3679487705230713 Domain: biomedical Document type: Other Language: en Border molding with putty consistency rubber base impression material (Speedex addition silicon, Coltene, Switzerland) was done, then the denture fitting surface was loaded with light body rubber base impression material, which should flow evenly to cover the entire fitting surface and the borders of the denture with a thin continuous layer. The denture was then inserted inside the patient's mouth, and border molding was completed manually and functionally. Section title: Steps for relining the maxillary denture Educational score: 2.6508753299713135 Domain: clinical Document type: Other Language: en To stabilize the upper denture throughout the impression material setting process, patients were advised to close their mouth, and that therefore a "closed mouth reline protocol" was followed. After the material setting, the denture was taken out of the patient’s mouth, and the impression was examined for any imperfections and should show an accurate reproduction of the denture-bearing area. Fig. 1 Patient closed in centric while relining impression setting Section title: Digital technique of denture construction Educational score: 4.050464630126953 Domain: biomedical Document type: Study Language: en Before boxing and pouring the relining impression, the maxillary denture was scanned from all surfaces, fitting (impression surface), polished, and occlusal surfaces using a desktop scanner (3Shape E2, Copenhagen, Denmark.) the scanned images were exported to the computer software as standard tessellation language file (STL) . STL file was imported into a dental CAD/CAM milling machine software program (Exocad DentalCAD; exocad, GmbH, Germany) to fabricate a new maxillary complete denture after finalizing the denture design digitally from PMMA discs of 25 mm length (Yamahatchi dental MGF, Japan) . After milling, finishing, denture characterization were completed using three shades of composite. (Visio,lign, Bredent, GmbH, Germany) . Fig. 2 Scanned maxillary denture with relining impression Fig. 3 CAD/CAM milled denture Fig. 4 A Finished milled denture, B Finished relined denture Section title: Conventional technique of denture construction Educational score: 2.233557939529419 Domain: biomedical Document type: Other Language: en After boxing the impression, an extra hard stone material (Gypsum type VI, BMS, Italy) was used for impression pouring making sure that any extra material on the base's teeth or facial surfaces was eliminated. The denture and the cast shouldn't be separated. Section title: Conventional technique of denture construction Educational score: 3.2346739768981934 Domain: biomedical Document type: Other Language: en Denture processing was completed by flasking the denture into a metal flask, immersed it into boiling water for wax elimination, packing the heat cure acrylic resin (Acrostone, Egypt), curing the resin by long cycle in a hot water bath at 72 ◦C for 6.5 h, then deflasking, finished and polished was done . Section title: Sample size calculation Educational score: 3.9712774753570557 Domain: biomedical Document type: Study Language: en A pilot study was conducted measuring retention and adaptation of 5 samples of the relined dentures group and the CAD/CAM milled dentures group. The Cohen’s d effect size was measured from the pilot study. Based on the Cohen’s d effect size with assuming a type I error of 0.05 and a study power of 0.9, the sample size required to detect a significant difference between the two groups was increased to include 12 samples. Section title: Patient’s randomization Educational score: 2.798414707183838 Domain: biomedical Document type: Study Language: en This study was designed to be a crossover study were the twelve patients had received one denture from both groups, a block randomization was done by the author to determine the order of dentures insertion (which denture group to deliver first). Section title: Denture delivery Educational score: 2.7535455226898193 Domain: biomedical Document type: Other Language: en Dentures were delivered after examination of fitting and polished surfaces for any nodules or sharpness, occlusal adjustments were done if needed by selective grinding. Post-insertion instructions were the same as given for any removable prosthesis. After the end of the two weeks follow up period of the first group of dentures, a resting period of one week was given to all patients before delivering and evaluating the other group of dentures. Section title: Denture delivery Educational score: 2.425354242324829 Domain: biomedical Document type: Study Language: en Randomization and resting period were needed to avoid the effect of the order of the prosthesis on the measurements of denture retention and evert the influence of muscle adaptation on the obtained results. Section title: Denture delivery Educational score: 2.0929481983184814 Domain: biomedical Document type: Study Language: en Data were collected at the appointment of denture insertion and two weeks later and used to evaluate patient satisfaction, denture retention, and adaptation. Section title: Denture delivery Educational score: 1.494344711303711 Domain: biomedical Document type: Other Language: fr I. Patients’ satisfaction evaluation through a questionnaire Section title: Denture delivery Educational score: 2.533585786819458 Domain: biomedical Document type: Study Language: en The questionnaire used was developed especially for this study and designed as a version of the Likert scale with ranges from 1 (completely unsatisfied) to 5 (completely satisfied), representing the denture satisfaction scale at denture insertion time and two weeks later regarding denture retention, mastication, speech, and esthetics . Section title: Denture delivery Educational score: 1.525739312171936 Domain: biomedical Document type: Other Language: fr Example of the questionnaire for patient satisfaction measurement. Section title: Denture delivery Educational score: 1.2486777305603027 Domain: other Document type: Other Language: en On a scale from 1 to 5, how do you feel about your denture retention? Section title: Denture delivery Educational score: 1.3698421716690063 Domain: biomedical Document type: Other Language: en At insertion time 1 2 3 4 5 Completely unsatisfied Satisfied Completely satisfied Two weeks later 1 2 3 4 5 Completely unsatisfied Satisfied Completely satisfied Section title: Denture delivery Educational score: 1.709282398223877 Domain: biomedical Document type: Study Language: en The questions were repeated regarding mastication, speech, and esthetics. Section title: Denture delivery Educational score: 2.080857276916504 Domain: biomedical Document type: Study Language: en Patient-based outcomes data were collected. A total satisfaction score was calculated for each item; higher scores mean greater satisfaction. II. Denture retention evaluation Section title: Denture delivery Educational score: 3.0971145629882812 Domain: biomedical Document type: Other Language: en A (19-gauge) orthodontic wire in the form of a hook was secured on the center of the palate at a point of intersection of the lines connecting the hamular notches, the right and left canines with the midline by self-cured acrylic resin material (Acrostone, Egypt), then the denture placed on the supporting tissues intraorally . Fig. 5 Denture with the hook seated intraoral Section title: Testing procedure Educational score: 3.1403770446777344 Domain: biomedical Document type: Study Language: en The patient was in an upright position. A digital force gauge (Force Gauge HF- 50N) was linked to the hook and a downward pulled force was applied slowly in a vertical direction opposite to that of the denture path of insertion until the denture was displaced, the force value recorded at the time denture displacement was registered. After repeating this procedure three times, the average was determined . Fig. 6 Retention measurement Section title: Testing procedure Educational score: 1.8654030561447144 Domain: biomedical Document type: Other Language: ro I. Denture adaptation evaluation Section title: Testing procedure Educational score: 2.755113124847412 Domain: biomedical Document type: Study Language: en Maxillary arches of all patients were scanned using an intraoral scanner (3 shape TRIOS 4 wired, Copenhagen, Denmark), fitting surfaces of both denture groups were also scanned using desktop scanner ( 3Shape E2, Copenhagen, Denmark). Section title: Testing procedure Educational score: 4.122657775878906 Domain: biomedical Document type: Study Language: en After scanning of the denture fitting surface, the STL file was imported to a three-D measurement program (Geomagic Control X, 3D Systems, United States) software to flip the fitting surface of the dentures to resemble the intra-oral tissue surface to build and superimpose STL files using first initial alignment and then best-fit alignment . For every measurement point, the root mean square (RMS) (mm) was computed considering both positive and negative-values, The adaptation deviations of the repaired dentures were calculated as done by the previous study . Fig. 7 A Color map of group A adaptation. B Color map of group B adaptation. Pressure from the denture fitting surface to the oral tissues is indicated by yellow to red colors. The denture fitting surface and oral tissues are separated by a blue color. The green color denotes that the oral tissues and the denture fitting surface are in contact Section title: Testing procedure Educational score: 3.734208822250366 Domain: biomedical Document type: Study Language: en Statistical analysis for retention values and data was tested for normality using the Shapiro–Wilk test. Between-group comparisons were conducted using an independent t test, while within-group comparisons were conducted using paired t test. The significance level was set at p = 0.05. statistical analysis was completed by SPSS software (version 25). While statistical analysis for adaptation, data descriptive statistics consisted of mean, standard deviation, median, minimum, and maximum values. Data was tested for normality using the Shapiro–Wilk test and an independent t test was used for between-group comparison. The significance level was set at 0.05. Statistical analysis was completed by SPSS software (version 25). Section title: Patient satisfaction results Educational score: 2.2685863971710205 Domain: biomedical Document type: Study Language: en All patients of both groups A &B were satisfied with their dentures regarding retention, chewing ability and speech. All the patients of group B (relined dentures) and half the patients (50%) of group A (digitally constructed dentures) were satisfied regarding denture appearance and esthetics. Section title: Retention results Educational score: 4.148055076599121 Domain: biomedical Document type: Study Language: en Retention values were significantly higher after two weeks than at the insertion time ( p < 0.001) within both groups. Group B showed significantly higher retention than group A at the insertion time ( p = 0.007) and 2 weeks later ( p < 0.001). As showed in Table 1 Fig. 8 . Table 1 Descriptive statistics, the results of independent t test in the rows for between-group comparison and the results of paired t test in the columns for within group comparison at different time intervals Group A Group B p- value Insertion Time Mean (SD) 5.0 (0.2) 5.2 (0.2) 0.007* Median (Range) 5.1 (4.7—5.3) 5.3 (5.0—5.4) After 2 weeks Mean (SD) 5.5 (0.1) 6.1 (0.2) < 0.001* Median (Range) 5.4 (5.3—5.7) 6.1 (5.8—6.4) p- value < 0.001* < 0.001* Fig. 8 Bar chart representing the retention values of both groups at denture insertion time and two weeks after Section title: Denture adaptation results Educational score: 4.1248321533203125 Domain: biomedical Document type: Study Language: en Group A showed significantly higher RMS (less adaptation) than group B ( p < 0.001) with a mean difference 0.47 and 95%CI: (0.46,0.48). as showed in Table 2 Fig. 9 . Table 2 Comparison between the results of independent t test of RMS values of the two groups Descriptives Milled Group Relined Group p- value Mean 1.22 0.74 < 0.001* SD 0.01 0.01 Median 1.22 0.75 Min 1.20 0.72 Max 1.24 0.76 Fig. 9 Bar chart representing the mean RMS in both groups Section title: Discussion Educational score: 4.046900749206543 Domain: biomedical Document type: Study Language: en The success of any prosthesis in terms of speech, function, appearance, and denture retention is thought to be greatly influenced by patient happiness. The primary factor considered when assessing the clinical performance of complete dentures is their ability to remain in the mouth during function. Wearers of CDs always have one basic complain: insufficient retention . The current in-vivo study was conducted to assess the degree of patients’ satisfaction and compare the adaptation and retention gained from relining loose maxillary denture conventionally using flasking procedure versus a newly CAD/CAM milled maxillary denture obtained from scanning the relining impression to fabricate complete denture digitally. Section title: Discussion Educational score: 2.8371150493621826 Domain: biomedical Document type: Study Language: en Many parameters, including physical factors (adhesion, cohesion, surface tension and peripheral seal) and mechanical factors (mechanical locking into undercuts and ridge anatomy), have a significant impact on the retention of complete maxillary dentures . Therefore, patients with xerostomia, viscous saliva and/or severe bony undercuts were excluded from this study. Section title: Discussion Educational score: 2.58516788482666 Domain: biomedical Document type: Study Language: en In this study, closed mouth impression technique of relining was carried out. The main point of creating a suction-effective denture is completely sealing the entire border of oral mucosa to prevent air invasion so negative pressure will be established . Section title: Discussion Educational score: 4.05134391784668 Domain: biomedical Document type: Study Language: en The current study findings stated that all patients were satisfied with both of their dentures regarding retention, speech and chewing ability (occlusion) this is mainly because the digital dentures were milled from the scanned old denture with the same teeth setting and relationship, so the occlusion and speech weren’t changed. On the contrary, Kang YJ et al. did a cross-over single-blinded randomized in-vivo study to compare between the digitally fabricated complete dentures versus conventionally fabricated complete dentures. It was found that masticatory efficiency and speech with the digitally fabricated complete dentures were less favorable than the conventionally fabricated complete dentures, however, comparable results were obtained for denture adaptation and patient satisfaction between the two denture groups. Section title: Discussion Educational score: 3.748569965362549 Domain: biomedical Document type: Study Language: en Saponaro et al. had studied and compared patient satisfaction of digitally constructed dentures and it was shown that, 3 (6.25%) of the 48 participants reported having bad esthetic from their CDs, while 1 of the 48 individuals (2.08%) suffered from altered phonetics. On the second visit, 24.44% of the digital dentures were not placed because of poor retention, an improper centric relationship, poor aesthetics, and altered phonetics . Section title: Discussion Educational score: 2.371018409729004 Domain: biomedical Document type: Study Language: en At the present study, retention values were recorded at insertion time and two weeks after, no additional follow up periods were needed as it was claimed that no significant changes in retention values between conventional dentures and digital dentures were recorded for longer follow up periods than 2 weeks . Section title: Discussion Educational score: 4.12194299697876 Domain: biomedical Document type: Study Language: en Group B (relined dentures) showed a statistically- significant higher retention values at all follow up periods and also more denture adaptation compared to group A (milled dentures), this may be explained by the fact that relining impression was taken under patient’s biting force, so it is muco-compressive impression recorded the areas of the peripheral soft tissues under compression. The same outcome was revealed from an in-vivo study comparing digital and conventional impression technique in edentulous maxillae and it was revealed that conventional impression-taking is more precise than digital impressions in recording the peripheral soft tissues responsible for creating strong peripheral seal areas to ensure retentive removable prostheses . Section title: Discussion Educational score: 3.9489991664886475 Domain: biomedical Document type: Study Language: en Over-extended borders of digitally constructed maxillary dentures was found to be another reason that could explain their reduced retentive values compared to conventionally constructed dentures as shown in a study done to compare the digitally and the conventionally constructed complete denture, it was claimed that over-extended borders of the digital dentures were obvious at the time of denture insertion caused denture displacement and reducing the retention of digital denture, however, after two weeks and three months follow up periods, no significant differences were found between both denture groups . However, in this study overextended denture borders was minimal and not considered the main cause of reduced retention of digitally constructed dentures. Section title: Discussion Educational score: 2.469508171081543 Domain: biomedical Document type: Other Language: en Many authors stated that the larger the scanned distance along the edentulous arch the more the horizontal error occurred . This may be due to a progressive error acumination while producing the final image from collecting and translating single separate images. The wider areas of the edentulous arch are captured the more numbers of images would be superimposed, so the final data will be distorted as a result of the inaccuracies accumulation generated. On the other hand, laboratory (desktop) scanners don’t show this similar pattern of deviation . Section title: Discussion Educational score: 4.0951337814331055 Domain: biomedical Document type: Study Language: en The scanner employed in this study was able to accurately scan the borders of the existing dentures, occlusal surface, and the relining impression surface (fitting surface) of the old denture with precise capturing of complete denture contours and soft tissue details . however, the fitting surface of the milled denture revealed a highly smooth texture that hinders improved appropriate retention, border seal, and physical adaptability to the underlying tissues. Another study evaluated denture retention of digitally constructed dentures (milled and printed). Retention values of both denture groups after 2 weeks follow up period were significantly improved from baseline, the reason for this might be related to the settling of the denture base, functional adaptation, and neuromuscular coordination of the patients . Section title: Discussion Educational score: 3.84334135055542 Domain: biomedical Document type: Study Language: en It should be known that there is no intraoral scanner could produce a true precise functional impression with proper borders extension and shape. Nonetheless, without functional molding of the vestibular mucosa and slight compression on the highly-resilient posterior palatal seal areas, an acceptable border and palatal seal wouldn’t be achieved, which consequently, affect denture retention negatively . With the same concept, a study was done to compare the accuracy of scanning edentulous maxillary arch using different types of intraoral scanners versus scanning the conventional impression and the referring casts (laboratory scanners) and the results showed that laboratory scanning revealed more accurate results . Section title: Discussion Educational score: 3.165724992752075 Domain: biomedical Document type: Study Language: en Other adverse outcomes reported with digital complete dentures regarding differences in VDO measurements, poor esthetics, bad teeth setting, and impaired speech . In the present study, patients had their existing loose dentures constructed from 2 years or less with proper VDO and correct centric relation to avoid teeth wear, low VDO and to ensure proper occlusion for the digitally constructed denture. Section title: Discussion Educational score: 3.0668628215789795 Domain: biomedical Document type: Study Language: en Moreover, many other studies confirmed that milling burs size, shape, and sharpness played a significant role in the shape of the final milled prothesis regarding its surface roughness and surface adaptation which limits the accurate reproduction of denture fitting surface details that will negatively affect the intimate adaptation with oral mucosa . Section title: Discussion Educational score: 4.000837802886963 Domain: biomedical Document type: Study Language: en However, in pilot cohort research done by Bidra et al., it was found that while patients expressed great satisfaction with phonetics and esthetics, just 50% of them were dissatisfied with the retention, stability, or adaptability of their digitally produced dentures . In contrast, Kattadiyil et al. reported no discernible change in phonetics or esthetics and discovered a markedly higher retention rate for milled maxillary complete dentures when compared with conventional ones made for the same patient. Patients who had digital dentures scored better overall, and for denture base contour, fit, stability, retention, and extension. This was explained by the fact that, in contrast to traditional ones, there was no polymerization shrinkage throughout the fabrication process, which resulted in greater fit and retention [ 42 – 44 ]. Section title: Discussion Educational score: 2.620292901992798 Domain: biomedical Document type: Study Language: en A prospective cross-over study on clinical effectiveness and patient satisfaction with digital dentures against conventional dentures was conducted by other studies. They found that while the method of manufacture had no effect on patient satisfaction, there was a tendency for digital dentures to be more clinically successful than traditional dentures , which was not proved in this study. Section title: Conclusion Educational score: 1.9051505327224731 Domain: biomedical Document type: Study Language: en Within the limitation of this study, it could be concluded that: Section title: Conclusion Educational score: 2.99871563911438 Domain: biomedical Document type: Study Language: en Ill-fitted maxillary complete dentures corrected by conventional relining procedure showed better esthetics, retention, and adaptation than digitally constructed maxillary dentures which showed acceptable esthetics, retention, and adaptation with better time and data saving. Section title: Clinical impact Educational score: 3.141256332397461 Domain: biomedical Document type: Other Language: en The idea of incorporating relining impression of loose old denture with scanning, designing and fabricating complete dentures digitally is reliable and could result in successful denture. although this digital manufacturing method could not improve denture retention and adaptation compared to the conventional relining procedure, but it has the advantages of avoiding material discrepancies during flasking and curing, in addition to avoiding complicated laboratory steps and long processing time. Section title: Limitations and future prospectives Educational score: 3.7626683712005615 Domain: biomedical Document type: Study Language: en This study has been tried to replace conventional laboratory steps of denture relining with more digital, easy, and time saving ones, but some limitations were observed; considering that intraoral scanners are reliable devices for imaging oral tissues with great accuracy, however, recording the tissues with slight pressure is not achieved till now. Future studies are recommended to investigate the possibility of substituting the conventional clinical steps of denture construction with digital ones to convert prolonged clinical appointments to be less time consuming and more reliable with analysis related to the functional effectiveness of the resultant dentures should be considered. Section title: Supplementary Information Educational score: 1.4485911130905151 Domain: biomedical Document type: Other Language: en Supplementary Material 1. | Other | biomedical | en | 0.999995 |
PMC11699797 | Section title: Introduction Educational score: 3.9260575771331787 Domain: biomedical Document type: Other Language: en Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an acquired multi-system disease characterized by persistent fatigue and exertional intolerance with a disproportionate worsening after physical or cognitive exertion referred to as post-exertional malaise (PEM). Furthermore, it is accompanied by a variety of other symptoms that are related to immunological, cognitive, and autonomic dysfunction. Section title: Introduction Educational score: 3.6286137104034424 Domain: biomedical Document type: Study Language: en The worldwide prevalence was estimated at 0.89% in a pre-pandemic meta-analysis, though estimates vary according to the case definitions used. 1 While there is evidence for a genetic predisposition to ME/CFS, 2 it is most often triggered by viral infections. 3 Since the COVID-19 pandemic, SARS-CoV-2 has become the leading viral trigger for ME/CFS. 4 Given the high incidence of COVID-19, the prevalence of ME/CFS is expected to strongly increase. Section title: Introduction Educational score: 3.831017255783081 Domain: biomedical Document type: Review Language: en The diagnosis of ME/CFS is based on established diagnostic criteria, with the most frequently used being the Canadian Consensus Criteria (CCC) and the Institute of Medicine (IOM) criteria. These require the presence of key symptoms including lingering fatigue, PEM, unrefreshing sleep, cognitive impairment, and/or orthostatic intolerance. 5 ME/CFS diagnosis relies so far on these clinical criteria, although most patients present with objective clinical findings including diminished handgrip strength or autonomic dysfunction. 4 Section title: Introduction Educational score: 4.111706733703613 Domain: biomedical Document type: Study Language: en The pathophysiological mechanism of ME/CFS is not yet known, but numerous studies suggest dysregulation of the immune system. Elevated antibodies were found in ME/CFS patients in several studies, most frequently against adrenergic and muscarinic receptors. 6 , 7 Elevated β1 and β2 adrenergic receptor autoantibodies (β1/β2 AR-AB) and M3/M4 acetylcholine receptor autoantibodies (M3/M4 AChR-AB) are likely to modulate the autonomic nervous system function and vasoregulation. 7 , 8 Section title: Introduction Educational score: 4.114206790924072 Domain: biomedical Document type: Study Language: en Levels of these autoantibodies were found to be associated with symptom severity and structural alterations in the central nervous system in both post-COVID syndrome (PCS) and ME/CFS. 9 , 10 , 11 , 12 , 13 The β2 AR-AB was the best marker for distinguishing PCS from recovered patients, and levels of β2 AR-AB were associated with both fatigue and vasomotor symptoms in PCS-ME/CFS patients. 10 , 14 Apart from elevated autoantibodies a chronic activation of inflammatory pathways and an alteration in memory B-cells were shown. 15 , 16 Section title: Introduction Educational score: 3.9599905014038086 Domain: biomedical Document type: Other Language: en The goal of immunoadsorption (IA) is to improve the clinical condition of patients with autoantibody-mediated diseases by selectively removing immunoglobulins from circulation via extracorporeal adsorption from their plasma. 17 , 18 Unlike plasmapheresis, IA employs a high-affinity column that specifically binds and eliminates immunoglobulins including autoantibodies and immune complexes, leaving other plasma components behind. 18 Section title: Introduction Educational score: 4.110810279846191 Domain: biomedical Document type: Study Language: en Given the previous evidence for a potential role of autoantibodies in PCS and ME/CFS and the effectiveness of IA in postinfectious ME/CFS, we here aimed to evaluate the effectiveness of IA treatment specifically in post-COVID ME/CFS patients. 19 , 20 Effectiveness was measured by assessing patient-reported outcomes and hand grip strength. We hypothesized that IA would lead to clinical improvement in ME/CFS patients four weeks after treatment, with an additional beneficial effect from repeat IA in responders with relapse. Section title: Study design Educational score: 3.7308554649353027 Domain: biomedical Document type: Study Language: en This prospective cohort study, conducted at the outpatient department for immunodeficiencies at the Institute of Medical Immunology, Charité - Universitätsmedizin Berlin, recruited patients between October 2022 and October 2023. Patients received IA treatment and follow-up visits for 12 months. Responders to the first cycle of IA were offered a second cycle. An interim report of the first 10 patients was published in October 2023. 21 This study was being conducted within the National Clinical Studies Group (NKSG), a clinical trial and translational research platform focused on developing therapies for PCS and ME/CFS, funded by the German Ministry of Education and Research (BMBF). 22 Section title: Patients Educational score: 3.9955174922943115 Domain: biomedical Document type: Study Language: en Patients were diagnosed and recruited at the outpatient department for immunodeficiencies at the Institute of Medical Immunology at the Charité - Universitätsmedizin Berlin. The diagnosis of ME/CFS was based on the modified Canadian Consensus Criteria (CCC) and a minimum of 14 h of PEM. 23 , 24 Inclusion criteria encompassed elevated β2 AR-AB at the time of screening and SARS-CoV-2 infection at the time of disease onset. All patients had to provide proof of SARS-CoV-2 infection by positive PCR, antigen test, or serology (SARS-CoV-2 nucleocapsid protein antibodies). Section title: Patients Educational score: 2.577512264251709 Domain: biomedical Document type: Study Language: en Patients were excluded from this study if they had relevant comorbidities, 25 pre-existing fatigue, evidence of organ dysfunction, or acute or chronic infections such as HIV or hepatitis. Additionally, patients who were unable to leave their homes due to the severity of their illness were also excluded. Section title: Patients Educational score: 1.1914311647415161 Domain: biomedical Document type: Other Language: en All patients signed informed consent before study inclusion. The Ethics Committee of the Charité - Universitätsmedizin Berlin approved this study in accordance with the 1964 Declaration of Helsinki and its later amendments . Section title: Procedures Educational score: 3.7673532962799072 Domain: biomedical Document type: Study Language: en Five sessions of IA were administered at the Department of Nephrology at Charité - Universitätsmedizin Berlin. IA treatment was conducted in an outpatient setting over a ten-day period, with sessions spaced no more than two days apart. The TheraSorb® – Ig omni 5 adsorber (Miltenyi Biotech B.V. & Co. KG, Bergisch Gladbach, Germany) was used for removal of human lambda and kappa chains containing immunoglobulins IgG (subclasses IgG1-IgG4), IgA, IgM, IgE, and immune complexes as well as free lambda and kappa light chains from the plasma. Section title: Procedures Educational score: 4.12596321105957 Domain: biomedical Document type: Study Language: en To ascertain the efficacy of IA, total serum immunoglobulin levels were assessed via immunoturbidimetry before, during, and after treatment. Antibodies against β1, β2, M3, and M4 receptors were determined using ELISA technology by CellTrend GmbH, Luckenwalde, Germany, both before and after treatment. Intra- and inter-assay coefficients of variation for the ELISAs provided by CellTrend were: b1 AR-AB 9.6%/12.0%, b2 AR-AB 4.2%/3.8%, M3 AchR-AB 5.9%/10.1%, and M4 AchR-AB 7.3%/12.5%. Pre- and post-treatment samples were analyzed in the same assay run. The upper normal levels of autoantibodies were determined based on validation studies of a healthy control group and defined as being larger than the 90th percentile of a healthy control group (>14U/l for β2 AR-AB). Section title: Procedures Educational score: 4.22354793548584 Domain: biomedical Document type: Study Language: en For patient-reported outcomes, questionnaires were filled out before, during, and after the treatment in monthly intervals and validated by physicians. Patients’ health-related quality of life was assessed using the 36-Item Short-Form Survey (SF-36), with scores ranging from 0 to 100, with 100 indicating no limitations. 26 Response to IA treatment was defined as a minimum increase in the SF-36 physical functioning domain (SF-36 PF) of 10 points from baseline to four weeks post IA, indicating a clinically relevant improvement. 27 Fatigue was evaluated using the Fatigue Severity Scale (FSS), ranging from 9 to 63, with a total score of 36 or more suggesting relevant fatigue. 28 Additionally, disease-related disability was scored according to the Bell score, rating the restriction in daily functioning on a scale from 0 to 100, with 100 indicating no restriction. 29 PEM was evaluated using the DePaul Symptom Questionnaire (PEM-DSQ), ranging from 0 to 20 for both severity and frequency, PEM duration was assessed ranging from 0 to 6, with higher values indicating higher PEM severity. 24 Further cardinal symptoms of both PCS and ME/CFS, including fatigue, muscle pain, immunological symptoms, and cognitive impairment, were scored on a numeric rating scale (NRS) from 1 to 10, with 10 indicating maximum symptom severity (not formally validated). Autonomic dysfunction was evaluated according to the Composite Autonomic Symptom Score (COMPASS31), ranging from 0 to 100, with 100 indicating maximum autonomic dysfunction. 30 Section title: Procedures Educational score: 4.134043216705322 Domain: biomedical Document type: Study Language: en Handgrip strength (HGS) of the dominant hand was measured using a digital hand dynamometer (EH101, Deyard, Shenzhen, China) in two separate sessions. Rest time between sessions was 60 min, in which no strenuous physical activity took place. Before starting the measurement, patients were shown two separate demonstrations of how the dynamometer should be used. Patients sat in an upright position facing a standard table during measurements of HGS. The forearm of the dominant hand was placed on the table in full supination holding the dynamometer. Under supervision and verbal motivation, the handle was pulled 10 times with maximum force for three seconds, followed by a five-second relaxation phase. The dynamometer displayed the highest value reached within these three seconds (measurement in kg), this single value was then recorded. The attempt with the highest reading out of ten repetitions was recorded as the maximum strength (Fmax), and the average strength (Fmean) of each session was calculated. 31 Section title: Procedures Educational score: 4.124344825744629 Domain: biomedical Document type: Study Language: en Further, the Reactive hyperemia index (RHI), which is a measure for endothelial function, was assessed using a peripheral arterial tonometry device . The technology measures the pulsatile volume changes in the vascular beds of the finger using optical sensors. The subjects were in supine position for a minimum of 15 min before measurements, in a quiet, temperature-controlled room. Occlusion of the brachial artery was performed on the nondominant upper arm using a standard blood pressure cuff. The occlusion pressure was at least 60 mmHg above the systolic blood pressure. Upon release of the cuff, the resulting surge in blood flow causes vessel dilation. Each recording consisted of five minutes of baseline measurement, five minutes of occlusion measurement, and five minutes post-occlusion measurement. The post-occlusion dilation relative to pre-occlusion levels is calculated as the RHI. Endothelial dysfunction was defined as an RHI ≤ 1.67 based on previous cohort studies. 32 Section title: Procedures Educational score: 1.50717294216156 Domain: biomedical Document type: Study Language: en Study data were collected and managed using the REDCap electronic data capture tools hosted at the Charité - Universitätsmedizin Berlin. 33 , 34 Section title: Statistical analysis Educational score: 4.117046356201172 Domain: biomedical Document type: Study Language: en Statistical analyses were conducted using R version 4.3.0 and RStudio version 2023.03.1. A linear mixed-effects model (LMM) was employed to assess changes in multiple outcome variables across different time points. The analysis was performed using the lmer function from the lme4 package (version 1.1-35.5), and ggplot2 (version 3.5.0) was utilized for data visualization. For each outcome variable, the LMM included time as a fixed effect and patient number as a random effect to account for within-patient correlation. The mixed model was fitted using restricted maximum likelihood (REML), and statistical significance was evaluated using t-tests with p -values approximated through Satterthwaite's method for degrees of freedom, implemented via the lmerTest package (version 3.1-3). Missing data were accounted for by using all available observations in the model, allowing for the estimation of fixed and random effects without listwise deletion, assuming data are missing at random. Section title: Statistical analysis Educational score: 3.528712511062622 Domain: biomedical Document type: Study Language: en For comparisons between groups the non-parametric Whitney-U test was used. Correlation analysis was performed using the nonparametric Spearman coefficient. A two-tailed p -value of <0.05 was considered to provide evidence of a statistically significant result. Section title: Role of the funding source Educational score: 0.9788644909858704 Domain: other Document type: Other Language: en The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. Section title: Results Educational score: 3.869976282119751 Domain: biomedical Document type: Study Language: en The β2 AR-AB levels of 402 patients, who met the CCC for post-infectious ME/CFS, were measured between June 2022 and March 2023. Out of these 402 patients, 189 (47%) had elevated β2 AR-AB levels. From this group, patients who planned IA treatment and fulfilled inclusion criteria were offered to participate in this trial and 23 were recruited for the study between September 2022 and October 2023. Three patients withdrew their consent to participate prior to the treatment due to concerns about the burden of study participation. 20 patients completed a six-month follow-up resulting in an 87% retention rate, suggesting that the protocol was generally acceptable to those who proceeded with the treatment. Section title: Results Educational score: 3.5990958213806152 Domain: clinical Document type: Study Language: en The five IA treatment sessions were completed for all patients within a 10-day period in an outpatient setting. Sessions lasted between 4.5 and 9 h and were followed by a minimum of one day and a maximum of two days of rest. In five out of 20 patients, a Shaldon's catheter was required for vascular access; for the other patients peripheral venous puncture was sufficient. One patient experienced a thrombosis of the internal jugular vein as a side effect of the catheter. Otherwise, no severe side effects of IA treatment were reported. Section title: Results Educational score: 1.7956820726394653 Domain: clinical Document type: Other Language: en The treatment was generally reported as physically exhausting and frequently triggered PEM. To mitigate this, we aimed to minimize external stressors, ensured proper oral hydration, and offered lorazepam for up to three days as supportive therapy. Section title: Results Educational score: 4.0628533363342285 Domain: biomedical Document type: Study Language: en The median disease duration was 22 months (IQR: 15–31) at the time of inclusion. Seven patients were male and 13 were female with a median age of 40 (IQR: 36–51). All patients had a severe degree of disability with a median Bell score of 30 (range 20–40), corresponding to reduction of the functional state to 50% and being usually housebound. Individual patient characteristics are displayed in Table 1 . Table 1 Cohort characteristics and response to treatment as assessed by the SF36 PF. Patient Gender (M/F) Age (years) Time since COVID-19 (months) SF36 PF pre IA SF36 PF post IA (M1) ΔSF36 PF Responder to IA (yes/no) 1 F 36 39 15 70 55 Yes 2 M 41 25 25 70 45 Yes 3 F 56 15 25 65 40 Yes 4 F 37 21 45 85 40 Yes 5 F 52 23 45 80 35 Yes 6 M 59 31 35 65 30 Yes 7 F 51 38 15 35 20 Yes 8 F 31 19 40 60 20 Yes 9 F 59 32 20 35 15 Yes 10 F 50 31 15 30 15 Yes 11 F 36 10 15 25 10 Yes 12 M 36 9 40 50 10 Yes 13 M 46 32 20 30 10 Yes 14 M 57 14 50 60 10 Yes 15 M 33 23 25 30 5 No 16 F 36 15 25 30 5 No 17 F 44 14 5 5 0 No 18 M 33 39 60 60 0 No 19 F 37 15 30 25 −5 No 20 F 39 19 50 45 −5 No F: female; IA: immunoadsorption; M: male; M1: month 1 post immunoadsorption; SF36 PF: Short Form-36 physical functioning. Section title: Results Educational score: 4.063675880432129 Domain: biomedical Document type: Study Language: en There was a positive correlation between the immune score, which depicts the severity of the lymph node pain, throat pain, and flu-like symptoms, and the levels of antibodies against β1 AR ( r = 0.53, p = 0.017), β2 AR ( r = 0.46, p = 0.040), M3 AChR ( r = 0.60, p = 0.006), and M4 AChR ( r = 0.60, p = 0.006) at baseline. Otherwise, no significant correlation was found between the autoantibody levels and the clinical presentation. Section title: Results Educational score: 4.166182041168213 Domain: biomedical Document type: Study Language: en There was a mean increase in the SF-36 PF of 17.75 points ( CI : 13.41–26.16, p < 0.001) four weeks post IA. The corresponding Cohen's d was calculated as 1.19, indicating a large effect size. There were, however, four patients with an increase of 10 points, which is considered as a clinically meaningful but small effect. 27 As seen in Fig. 1 , though the SF-36 PF scores tended to decrease between three and six months post IA, at month six post IA a significant mean improvement of 12.81 points ( CI : 4.99–20.61, p = 0.002) remained. Fourteen out of 20 patients (70%) responded to the treatment as defined by an increase of at least 10 points in the SF-36 PF four weeks post IA suggesting a clinically meaningful improvement. Seven patients were non-responders according to this definition; however, two of these showed a delayed response at month 2. Fig. 1 Course of the mean 36-Item Short-Form Survey physical functioning domain (SF36 PF) over the study period. The duration of IA therapy is indicated by the green bar. A higher score indicates less restriction in physical functioning. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. From top to bottom, the panels display the trajectories of the SF36 PF for: a) the whole cohort ( n = 20), b) responders to the treatment defined by a ≥ ten-point increase in the SF36 PF at month 1 post IA ( n = 14), and c) non-responders to the treatment defined by a ≤ ten-point increase in the SF36 PF at month 1 post IA ( n = 7). Section title: Results Educational score: 4.103903770446777 Domain: biomedical Document type: Study Language: en There was no significant difference in age, symptom severity, disease duration, or level of β2 AR-AB between responders and non-responders. However, among female patients, responders had a significantly higher maximum HGS at baseline ( Mdn = 23.5 kg, IQR: 17.7–25.5 kg) compared to non-responders ( Mdn = 9.8 kg, IQR: 8.53–11.05 kg) ( z = −2.62, p = 0.006, r = 0.73). Section title: Results Educational score: 4.167166233062744 Domain: biomedical Document type: Study Language: en Serum IgG, IgA, and IgM levels were collected from all patients at baseline, before each treatment, and after treatment. Compared to baseline, IgG, IgA, and IgM were significantly decreased during IA treatment ( p < 0.001) with a mean IgG reduction of 8.66 g/l ( CI : 8.06–9.26 g/l) (79%, CI : 73–84%), mean IgA reduction of 1.3 g/l ( CI : 1.2–1.51 g/l) (68%, CI : 63–78%), and mean IgM reduction of 1.1 g/l ( CI : 0.84–1.34 d/l) (76%, CI : 58–93%) at day five of the treatment. Fig. 2 a–c shows the course of immunoglobulin levels over time. β2 AR-AB decreased in parallel with the immunoglobulins with a mean reduction of 26.57 U/l ( CI : 20.11–33.02 U/l) (77%, CI : 58–95%). Fig. 2 Course of immunoglobulin and autoantibody levels over the study period ( n = 20), the duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. From left to right, the panels display the trajectories of: a) immunoglobulin G (IgG), b) immunoglobulin A (IgA), c) immunoglobulin M (IgM), d) β1 adrenergic receptor autoantibodies (β1 AR-AB), e) β2 adrenergic receptor autoantibodies (β2 AR-AB), f) M3 acetylcholine receptor autoantibodies (M3 AchR-AB), and g) M4 acetylcholine receptor autoantibodies (M4 AchR-AB). Section title: Results Educational score: 4.189100742340088 Domain: biomedical Document type: Study Language: en Patients reported improvements in several key clinical symptoms at four weeks post IA. There were significant improvements in fatigue, as measured by the SF-36 energy/fatigue domain, and in pain, as measured by the SF-36 pain domain, following IA treatment. These improvements remained significant through month six. The maximal change was observed two months after IA, with a mean increase of 19 points ( CI : 11.61–26.39, p < 0.001) and 22.63 points ( CI : 13.28–31.96, p < 0.001) from baseline, respectively, as shown in Fig. 3 . Fig. 3 Clinical symptom progression over the study period ( n = 20). The duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. From left to right, the panels display the trajectories of: a) 36-Item Short-Form Survey energy/fatigue domain (SF36 energy/fatigue), where a higher score indicates less fatigue, b) 36-Item Short-Form Survey pain domain (SF36 pain), where a higher score indicates less pain, c) post-exertional malaise (PEM) as assessed by the DePaul Symptom Questionnaire (DSQ-PEM), higher scores indicate more severe PEM, and d) Composite Autonomic Symptom Score (COMPASS31), where a higher score indicates more autonomic symptoms. Section title: Results Educational score: 4.144842624664307 Domain: biomedical Document type: Study Language: en Furthermore, patients reported a lasting improvement in autonomic symptoms, as shown in Fig. 3 d. Improvements were most noticeable in the orthostatic, secretomotor, and gastrointestinal domains of the COMPASS31, which are shown in Figure S4 in the Supplement , as well as in the total score with a mean decrease of 12.23 points ( CI : 19.06–5.4, p = 0.001) at month one. Section title: Results Educational score: 3.405431032180786 Domain: biomedical Document type: Study Language: en All patients experienced severe PEM lasting at least 14 h, as specified by the inclusion criteria. Both frequency and severity of post-exertional symptoms significantly decreased after IA according to the DSQ-PEM. The total score is shown in Fig. 3 c, remaining significantly decreased through month six. Section title: Results Educational score: 4.115742206573486 Domain: biomedical Document type: Study Language: en Additionally, there was a significant mean decrease of 4.9 points ( CI : 1.34–8.46, p = 0.009) in fatigue as assessed by the FSS at month one after IA as shown in Figure S3 in the Supplement . According to the symptom scores (NRS 1–10), there were also improvements in muscle pain, immunological as well as cognitive symptoms, that remained significant through month 6 with their maximum decrease between months two and three after IA as shown in Figure S1 in the Supplement . Section title: Results Educational score: 4.07893180847168 Domain: biomedical Document type: Study Language: en The improvement in the degree of disability according to the Bell score post IA reached statistical significance only at months two, four, five, and six, with a maximum mean increase of 5.18 points ( CI : 1.24–9.12, p = 0.013) at month two post IA as shown in Figure S2 in the Supplement . Section title: Results Educational score: 4.1325554847717285 Domain: biomedical Document type: Study Language: en To further quantify physical improvement, we measured repeat HGS as an objective marker of muscle fatigue. There were improvements, especially in repeat HGS before and after treatment in female patients as shown in Fig. 4 . The mean HGS during the second measurement was significantly increased six months post IA by a mean of 20% ( CI : 2–39%, p = 0.042) indicating a better recovery of strength within the 60 min of rest time between HGS measurements. In female responders ( n = 9), both mean and maximum HGS were significantly increased as early as four weeks post-IA (not shown). Fig. 4 Course of mean handgrip strength (Fmean) in % in female patients ( n = 13) measured a) initially and then b) repeated after one hour over the study period. The duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML), with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Section title: Results Educational score: 3.956465244293213 Domain: biomedical Document type: Study Language: en As a measure of endothelial dysfunction, the reactive hyperemia index (RHI) was assessed using an EndoPAT device. Six patients, three responders and three non-responders, had a reduced RHI (<1.68). There was no significant change in the RHI at six months post IA. Section title: Results Educational score: 4.1536173820495605 Domain: biomedical Document type: Study Language: en Responders to the first IA treatment were offered a second IA treatment within six to 12 months after completing the initial cycle, when their physical function or symptoms worsened again. The course of physical functioning according to the SF-36 PF in the seven patients receiving a repeat IA is shown in Fig. 5 . Symptoms improved again four weeks after the second IA treatment but then remained at a similar level as after the first IA treatment. Fig. 5 Course of 36-Item Short-Form Survey physical functioning domain (SF-36 PF) over the study period in patients who received a second cycle of IA therapy ( n = 7). The duration of IA therapy is indicated by the green bar. Error bars represent 95% confidence intervals. Data were analyzed using a linear mixed-effects model fitted by restricted maximum likelihood (REML) for each cycle, with t-tests computed using Satterthwaite's method for degrees of freedom with significance levels indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Section title: Discussion Educational score: 4.086522579193115 Domain: biomedical Document type: Study Language: en There is increasing evidence that autoantibodies, including those targeting β-adrenergic and muscarinic acetylcholine receptors, may contribute to the pathophysiology of PCS and ME/CFS. We here provide evidence from an observational study that depletion of autoantibodies by IA can lead to improvement in overall physical functioning, as well as the severity of several key symptoms, including PEM, fatigue, pain, immunological, cognitive, and autonomic symptoms in a subset of post-COVID ME/CFS patients. Section title: Discussion Educational score: 3.917349100112915 Domain: biomedical Document type: Study Language: en The clinical improvements observed after autoantibody removal via IA support the hypothesized involvement of these autoantibodies in the pathophysiology of ME/CFS and PCS in the responders. Meanwhile, a smaller group of patients did not show a response to the treatment. This highlights the potential diverse mechanisms underlying this condition, and indicates that autoantibodies may play a role only in a subgroup of patients. Section title: Discussion Educational score: 4.048442363739014 Domain: biomedical Document type: Study Language: en IA has demonstrated clinical improvements in post-infectious ME/CFS patients in two small pre-pandemic trials conducted by our group. 19 , 20 Although in most patients symptoms worsened after a few months, IA can induce longer lasting improvement for more than 12 months in a subset as observed in our previous study in postinfectious ME/CFS. 20 In this study, clinical improvements generally peaked between months two and three, then gradually declined. However, even after six months, statistically significant improvements compared to baseline were still evident. Section title: Discussion Educational score: 4.125699996948242 Domain: biomedical Document type: Study Language: en It is important to acknowledge that the observed improvement in SF36 physical functioning does not apply to all patients as there were several non-responders to the treatment and four patients had only a small improvement. Among these, some patients showed improvement only after a delay of two or three months, while others did not demonstrate any noticeable improvement. Thus, it is important to study the efficacy of autoantibody-depleting therapies in larger controlled trials. Further identifying factors predicting individual responses would support the clinical findings. To address this question, we are currently conducting several additional analyses to identify potential biomarkers for treatment response. These analyses include a comprehensive B cell subtyping using CyTOF analysis, the measurement of further autoantibodies, and markers of immune activation, hypoperfusion, microclots, and SARS-CoV-2 persistence. Although the main effect of IA is the depletion of autoantibodies, there is some evidence that memory B cells can be affected by IA. 19 A potentially important finding is that responders had a higher baseline maximum HGS, suggesting they have less severe muscular or mitochondrial impairment. 35 We could not identify any other significant differences in the clinical phenotypes of responders compared to non-responders. However, ME/CFS patients are a heterogeneous group, and there may be comorbid conditions, such as structural neuroanatomic abnormalities, that may have been overlooked in our patient cohort. Section title: Discussion Educational score: 4.338312149047852 Domain: biomedical Document type: Study Language: en Growing evidence suggests that SARS-CoV-2 can trigger autoimmune processes, contributing to long-term effects of COVID-19. Dobrowolska et al. summarized recent findings indicating that PCS can develop autoantibodies against a range of antigens, including those specific to the immune and cardiovascular systems, the thyroid, and rheumatoid-specific targets, G-protein coupled receptors, and more. However, the clinical significance of most of these autoantibodies remains unclear. 36 Several studies found elevated autoantibodies against β-adrenergic and muscarinic receptors in PCS patients and demonstrated an association between the levels of these autoantibodies with fatigue, neurological symptoms and pain. 9 , 10 , 11 , 12 , 13 , 37 , 38 Importantly, two recent studies by Chen et al. as well as Santos Guedes de Sa et al. were able to induce similar symptoms in mice by transferring IgG from PCS patients. 39 , 40 Remarkably, Chen et al. showed that patterns of transferred symptoms varied depending on the plasma proteome signature of the patients. IgG from those with neuronal or immune involvement induced pain, while IgG from those with muscular involvement impaired motion in the mice. 39 In the study by Santos Guedes de Sa et al., patients showed a broad pattern of autoantibodies reactive with neuronal and endothelial tissues. IgG from individual patients induced distinct symptoms such as pain, hypersensitivity or loss of coordination in mice. 40 These findings provide further evidence for the diversity of autoantibodies in PCS, possibly driven by prolonged inflammation, infection of various cell types and tissues, and reactivation of Epstein–Barr virus. Our IA study shows that removing autoantibodies generally led to disease improvement in a subset of patients; however, it remains unclear whether the benefits are primarily due to the removal of β2 AR-AB or other specific autoantibodies. Furthermore, decreases of β2 AR-AB and immunoglobulins were seen in all patients, independent of response. Section title: Discussion Educational score: 4.272243499755859 Domain: biomedical Document type: Study Language: en The clinical improvement observed after autoantibody removal via IA in a majority of patients supports the hypothesized role of these autoantibodies in the pathophysiology of PCS and ME/CFS in the responders. Although IA has a rapid and high clinical efficacy in a subset of patients, the clinical benefits of IA are in some patients temporary, and the procedure is strenuous. During our trial, one case of internal jugular vein thrombosis occurred as a side effect of the catheter. Although the patient experienced no long-term effects following temporary anticoagulation, the risk of such complications should be considered. Furthermore, it is a highly specialized procedure, available only in select medical centers with the necessary equipment and expertise. B-cell and/or plasma cell depletion thus emerges as a promising treatment option, addressing the urgent need for more effective sustained therapeutic strategies. B cell depletion with the CD20 monoclonal antibody rituximab proved effective in a subset of ME/CFS patients, and its efficacy was found to be associated with the depletion of β2 AR-AB. 41 However, the results from the phase II rituximab trials could not be confirmed in a multicenter trial, which had several limitations. 42 We are currently initiating a trial with inebilizumab in ME/CFS and PCS patients, a monoclonal antibody directed against CD19 on B cells and plasma blasts showing high clinical efficacy in neuromyelitis optica. 43 In this study, only responders to IA will be included in order to select patients with strong evidence of an autoantibody-mediated disease. Further novel treatment approaches are currently being tested in clinical trials for PCS and ME/CFS patients. These include targeting plasma cells by a monoclonal antibody to CD38, enhancing degradation of autoantibodies by Fc receptor inhibition, and neutralizing antibodies against G-protein coupled receptors with the aptamer BC007 . Section title: Discussion Educational score: 4.020514488220215 Domain: biomedical Document type: Study Language: en Limitations of this study include the small sample size and non-controlled study design. The study was designed to detect larger effect sizes, smaller effects may not be captured with this sample size. Further studies are therefore required; we and two other sites in Germany are currently conducting randomized controlled trials of IA in PCS and ME/CFS with larger sample sizes. 44 Further, the outcomes are primarily patient-reported. The DSQ-PEM is designed for diagnostic purposes, and not validated for tracking changes in PEM in a clinical trial. While PCS encompasses a very heterogeneous patient population with presumably different pathomechanisms, our focus was solely on adult PCS patients meeting the ME/CFS criteria. Therefore, our findings may not be applicable to all PCS patients. Particularly our findings regarding the feasibility and safety of this procedure cannot directly be applied to the most severe, home bound patient group, since they were excluded from the study. Section title: Discussion Educational score: 4.079062461853027 Domain: biomedical Document type: Study Language: en In conclusion, our study suggests that IA treatment may result in rapid clinical improvement in a subset of patients. Further trials with both more patients and clinical outcomes reported are needed to confirm our findings. Our study serves as proof of concept for the initiation of clinical trials using drugs specifically designed to target autoantibodies. Targeting B cells may offer a promising approach to long-term symptom relief by addressing the underlying mechanisms driving autoantibody generation. Section title: Contributors Educational score: 0.8831198215484619 Domain: other Document type: Other Language: en ES conducted the main part of the study together with CK, KW, CS, and LK. RR helped with patient recruitment. CH prepared the figures of the report with input from LK and managed electronic data collection together with HF. CS did the conceptualization and provided resources and supervision. AK and MT managed IA therapy. FS managed laboratory analysis. LK wrote the first draft of the report and performed statistical analysis. Section title: Contributors Educational score: 0.9533646702766418 Domain: other Document type: Other Language: en All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. CH and LK have accessed and verified the data. Section title: Data sharing statement Educational score: 0.9123384356498718 Domain: other Document type: Other Language: en The data presented in this study will be available upon request from the corresponding author. Due to the sensitive nature of the data and the ongoing data collection and analysis, the data are not publicly available. Section title: Declaration of interests Educational score: 0.970641016960144 Domain: other Document type: Other Language: en Charité holds, together with CellTrend, a patent for the diagnostic use of autoantibodies against β2 AR-AB. CS has a consulting agreement with CellTrend and Berlin Cures. MT has received grants from the DFG, BMBF, Weidenhammer Zöbele Stiftung, Baxter, and Cytosorbents. MT has a consulting agreement with AstraZeneca and has received honoraria for lectures from Aey-Congress, AstraZeneca, Boehringer Ingelheim, Bayer, Baxter, Cytosorbents, DGK, DHL, Fresenius, Medical Tribune, MedPoint, Novartis, Pfizer, Sanofi, and Vifor. MT has also received support for attending meetings from AstraZeneca and Vifor. Additionally, MT serves on data safety monitoring or advisory boards for AstraZeneca, Boehringer Ingelheim, and Takeda. PG has received honoraria for lectures and travel support from Miltenyi Biotec GmbH. The other authors declare no conflict of interest. | Other | biomedical | en | 0.999995 |
PMC11699804 | Section title: Introduction Educational score: 3.8725690841674805 Domain: biomedical Document type: Clinical case Language: en The differential diagnosis for pathogens in osteomyelitis varies depending on anatomic location and host risk factors. Vertebral osteomyelitis classically stems through hematogenous spread from a distant primary source . The etiology for vertebral osteomyelitis is typically gram-positive pathogens, regardless of age, gender, or level of spinal involvement. In males younger than 60, Staphylococcus , Streptococcus , and Enterococcus combine to cause 86 % of cases , with Staph. aureus accounting for most of these cases , . Less than 2 % of cases are due to anaerobes . We present a rarely encountered culprit of osteomyelitis. Section title: Case Educational score: 2.9954493045806885 Domain: clinical Document type: Clinical case Language: en A 55-year-old-male with a chronic left uretero-pelvic junction (UPJ) obstruction presented to the ED in 2023 with complaints of 2–3 weeks of low back pain. He reported it was sudden onset, and there was no mechanism of injury. It had been gradually worsening over that timeframe. He described it as midline, sharp, 9/10 stabbing pain with radiation into bilateral hips. It worsened with movement and lying flat in bed. He experienced minimal relief with ibuprofen and started seeing a chiropractor without notable improvement. He missed a week of work related to these complaints. Section title: Case Educational score: 1.5667797327041626 Domain: clinical Document type: Clinical case Language: en He denied any systemic complaints, including fevers/chills, night sweats, other joint pain, bowel/bladder incontinence, or lower extremity weakness. He reported some blood-tinged urine recently but denied dysuria. He had a urinary tract infection about a year prior and has never required IV antibiotics. Section title: Case Educational score: 3.1620328426361084 Domain: clinical Document type: Clinical case Language: en He was known to have a UPJ obstruction since his 20 s, when it was incidentally identified during trauma work-up after he was involved in a car accident. He did not have any intra-abdominal or thoracic injuries related to this accident. Since that time, he had this UPJ obstruction managed with chronic stent exchanges. He did undergo two endoscopic balloon dilation procedures for this, one in the late 1990 's and one in the early 2000's, neither of which were successful. Recently he was evaluated by urology, and it was recommended that the patient undergoes left nephrectomy due to nonfunctional left kidney after a kidney scan was done. Surgery had not yet been performed. He has had several urinary tract infections throughout his adult life, for which he was treated with oral antibiotics. He has not required any antibiotics within the last year. Section title: Case Educational score: 2.625424385070801 Domain: clinical Document type: Clinical case Language: en Upon presentation, the patient appeared well. His temperature was 98.8°F (37.1°C). He was normotensive with heart rates in the 80 s and maintaining oxygen saturations > 90 % on room air. Palpation of the cervical, thoracic, and lumbar spine showed no major tenderness on palpation. On exam the patient had difficulty moving around and sitting due to the low back pain. His exam was otherwise normal. Section title: Case Educational score: 3.759202480316162 Domain: clinical Document type: Clinical case Language: en His white blood cell count was 11.2 k/µL (reference range 4.5–11.0) with 60 % neutrophils (normal 41–77 %). CRP was 3.88 mg/dL (reference range <1.00), and ESR was 43 mm/hour (reference range 0–20). Urinalysis was nitrite negative and had 3 + leukocytes (reference negative). There were 2–10 white blood cells/hpf seen on microscopy (reference 0–2), in the setting of 0–2 squamous epithelial cells/hpf (reference 0–5). Section title: Case Educational score: 3.8242568969726562 Domain: clinical Document type: Clinical case Language: en Urine cultures and blood cultures were obtained. Given overall clinical stability, he was monitored off antimicrobials. CT abdomen/pelvis was obtained and revealed the development of cortical irregularity and osteolysis involving the L4–5 endplates and posterior superior aspect of the L5 vertebral body. There was also paravertebral edema about the L4 and L5 levels with new intermediate density material posterior to the L5 vertebral body, which resulted in moderate central canal narrowing. It was also noted that the left nephro-ureteral stent was mispositioned with the superior cope loop in the mid ureter, with some mild wall thickening of the proximal left ureter. MRI of the Lumbar Spine revealed loss of normal disc height with an abnormal fluid signal and diffuse contrast enhancement of the majority of the L4 and L5 vertebral bodies. There was enhancement in the epidural space, likely contiguous inflammation. Findings were compatible with spondylodiscitis at L4-L5 with contiguous phlegmon into the epidural space. Fig. 1 MRI Lumbar Spine. Fig. 1 Section title: Case Educational score: 3.829925298690796 Domain: clinical Document type: Clinical case Language: en Urine culture ultimately resulted with < 100k CFU/mL urogenital/skin microbiota. On day 2 of admission, the patient underwent a L4/5 disc biopsy in interventional radiology. Pathology revealed focal necrosis and acute inflammation (discitis) with adjacent granulation tissue. AFB stains were negative for acid-fast bacilli, and GMS stains were negative for fungal organisms. Biopsy cultures remained negative on day 5 of admission. Two sets of peripheral blood cultures were also negative at 5 days. Transthoracic echocardiogram was obtained and was negative for vegetations. Section title: Case Educational score: 3.3271939754486084 Domain: clinical Document type: Clinical case Language: en On day 5, he underwent a repeat L4/5-disc biopsy. Results of repeat L4/5 disc biopsy were pending at the time of discharge on hospital day 5. He was started on empiric IV Daptomycin and IV Cefepime. The biopsy culture resulted with Gardnerella vaginalis on both routine and anaerobic cultures 12 days after initial admission. At that time, IV Daptomycin and IV Cefepime were stopped. Alternatively, oral Metronidazole 500 mg three times daily was started. He completed 10 weeks of Metronidazole therapy, with significant improvement in his pain. He ultimately underwent left nephrectomy 3 months following this and had no re-admissions since. Section title: Discussion Educational score: 3.7456376552581787 Domain: biomedical Document type: Clinical case Language: en To our best knowledge, we present the first case of G. vaginalis vertebral osteomyelitis in a male. G. vaginalis is a rare cause of bone and joint infections. On average, anaerobes account for < 2 % of cases of vertebral osteomyelitis , and < 1 % of osteomyelitis in general . Furthermore, G. vaginalis is even less common, particularly in males . G. vaginalis ’ main association is with bacterial vaginosis . Several case reports to-date of G. vaginalis bone and joint infections have occurred in concurrence with G. vaginalis colonization of the genitourinary tract , . Section title: Discussion Educational score: 4.1894450187683105 Domain: biomedical Document type: Study Language: en Table 1 outlines all identified cases of G. vaginalis bone and joint infections in literature. To date, there have only been 3 cases of vertebral osteomyelitis reports in females , , , 1 case of native-joint septic arthritis in a female , 3 cases of prosthetic joint infections in females , , , 1 case of prosthetic joint infection in a male , and 1 case of skull osteomyelitis in a male infant whose gestation was complicated by bacterial vaginosis . All cases of joint infections have been in the hip, and all adult cases of vertebral osteomyelitis in adults have been in the lumbar spine, suggesting a possible anatomic association with the GU tract. A variety of mechanisms have been proposed for genitourinary colonization/infection leading to lumbar spondylodiscitis, including hematogenous spread through the posterior venous plexus or Baston’s plexus , . Table 1 Case reports of Gardnerella Vaginalis bone/joint infections in literature. Table 1 Reference Reference # Year * Age Gender Lesion GV colonization Polymicrobial Surgical Management Antibiotic Therapy ** Outcome Nightingale et al. 7 1986 Infant M Parietal OM Yes (in mother) No Debridement Ampicillin Cure Hodge et al. 9 1995 50 F Vertebral OM Unknown No No Ampicillin Cure Graham et al. 10 2009 38 F Vertebral OM No No No Clindamycin Cure Sivadon-Tardy et al. 11 2009 48 F Hip Septic Arthritis No Yes Debridement Amoxicillin Cure Hoarau et al. 6 2012 71 F Hip PJI Unknown Yes 1-stage revision TMP-SMX Cure Thomas et al. 8 2019 68 F Hip PJI Yes No 1-stage revision Amoxicillin Cure Thomas et al. 8 2019 32 F Hip PJI Yes No 1-stage revision Clindamycin Cure Kim et al. 3 2021 61 F Vertebral OM No Yes No Metronidazole Cure Saricaoglu et al. 5 2022 45 M Hip PJI No Yes Debridement/retention Clindamycin Cure Belote et al. present case 2024 55 M Vertebral OM Unknown No No Metronidazole Cure M: Male F: Female OM: Osteomyelitis GV: Gardnerella vaginalis PJI: Prosthetic Joint Infection * Year of publication ** Antibiotic used for main treatment course Section title: Discussion Educational score: 4.014730453491211 Domain: biomedical Document type: Clinical case Language: en G. vaginalis is a small, facultative anaerobic gram-variable rod , . It is difficult to identify, difficult to culture, and less virulent , , than more common bacteria associated with native vertebral osteomyelitis. G. vaginalis can form biofilms , , including on contraceptive intravaginal ring, but there is no data on extra-vaginal foreign bodies, or in males . It can colonize the urethra in up to 4.5–11.4 % of males , and has been shown to colonize extravaginal mucosa in women . We suspect that our patient had developed colonization of his left ureteral stent, given the noted wall thickening of the adjacent ureter on CT scan. It also may have been the “urogenital flora” grown on urine culture. Unfortunately, this specimen was not available for further analysis as his biopsy culture resulted 12 days after his original urine culture, and routine urine samples at our institution are discarded in that timeframe. We suspect that colonization of his ureteral stent with G. vaginalis is likely what predisposed him to develop native vertebral osteomyelitis. He improved on culture directed therapy, suggesting that the identified G. vaginalis was truly pathogenic. Section title: Discussion Educational score: 4.02351713180542 Domain: clinical Document type: Clinical case Language: en Our case also highlights the diagnostic importance of withholding antibiotics for osteomyelitis in the setting of clinical stability, as per Infectious Disease Society of America (IDSA) Guidelines . Biopsy results should guide therapy. Image-guided biopsy for lumbar spondylodiscitis has variable sensitivity, with reports as low as 40 % . In the setting of a negative initial biopsy, IDSA guidelines recommend a 2nd biopsy to be sent for culture , as was performed in this case. Were repeat biopsy not pursued in this case, he likely would have developed empiric treatment failure and developed worsening symptoms, and potentially hospital re-admission. Holding antibiotics after initial biopsy in this case proved highly beneficial. Section title: CRediT authorship contribution statement Educational score: 0.9146555066108704 Domain: other Document type: Other Language: en Alex Belote: Investigation, Writing – original draft. Kassem Hammoud: Conceptualization, Data curation, Investigation, Writing – review & editing. Section title: Declaration of Competing Interest Educational score: 1.0032367706298828 Domain: other Document type: Other Language: en The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: co-author serves as an editor for the journal ID Cases. KH If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | Clinical case | biomedical | en | 0.999996 |
PMC11699842 | Section title: Introduction Educational score: 4.316468238830566 Domain: biomedical Document type: Review Language: en Organophosphate (OP) compounds are formed through esterification between phosphoric acid and alcohol. Their uses range from pesticides and herbicides to nerve agents in chemical warfare . An example is diazinon, which was industrialised in 1952 by a Swiss chemical company and became the most widely used pesticide in the United States of America. It was cancelled for residential use in 2000 by the US Environmental Protection Agency to reduce the risk of pesticides at home, especially to children . Such acts have reduced the prevalence of OP poisoning in more developed countries, but it remains prevalent in less developed countries often presenting with non-accidental poisonings . An increased interest in OP poisoning in the United Kingdom (UK) stems from recent poisonings such as the Skripel family in March 2018 in Salisbury . In June 2022, the North Atlantic Treaty Organisation (NATO) identified that the deliberate use of chemical, biological, radiological and nuclear materials had the potential to “sow panic and strain national response capabilities” . OP causes irreversible phosphorylation of the acetylcholinesterase enzymes leading to a spectrum of pathology, depending on the time and dose of the agent. These range from cholinergic crisis and intermediate syndrome to OP-induced delayed neuropathy and chronic OP-induced neuropsychiatric disorder . The method of intoxication is by absorption through the skin, gut or bronchi following inhalation as in this case. The suggested management of OP poisoning involves atropine, pralidoxime (a cholinesterase reactivator), benzodiazepines and supportive care . The evidence and advice regarding these treatments can vary and management of such patients is heterogeneous from country to country. Specifically, OP poisoning raises challenges in muscle relaxant dosing with limited literature to support a specific agent or dose. Section title: Case presentation Educational score: 1.4756382703781128 Domain: biomedical Document type: Other Language: en Patient background Section title: Case presentation Educational score: 1.8935778141021729 Domain: clinical Document type: Clinical case Language: en A 40-year-old female weighing 70 kilograms presented to the Accident and Emergency (A&E) department. Her past medical history included ischaemic heart disease for which she took aspirin and atorvastatin, hepatitis B, vitamin D deficiency and lower back pain. She slept in a bedroom treated with diazinon for a bed bug infestation next to her husband; however, unlike her husband, covered her face with the bed sheet. Section title: Case presentation Educational score: 1.0157606601715088 Domain: clinical Document type: Other Language: en Day 0 of admission Section title: Case presentation Educational score: 1.2735061645507812 Domain: clinical Document type: Clinical case Language: en 05:00: The patient woke feeling unwell with vomiting and headache before returning to sleep. Section title: Case presentation Educational score: 0.9203888177871704 Domain: other Document type: Other Language: en 07:30: Family, unable to wake her, called the London Ambulance Service (LAS). The family were all asymptomatic. Section title: Case presentation Educational score: 3.1617910861968994 Domain: clinical Document type: Clinical case Language: en 08:16: LAS arrived. Examination at the scene found the patient to be tachypnoeic at 40 breaths per minute, tachycardic at 123 beats per minute and with a Glasgow Coma Scale score of 11 (E3, V3, M5). Further findings included a blood sugar of 10.1 millimoles per litre (mmol/L), diaphoresis, pallor, hypothermia (36.1 degrees Celsius), shivering and miosis. Fasciculations were noted but she had no focal neurology. LAS administered 400 µg naloxone intramuscularly with no response and transported via blue light to A&E. Section title: Case presentation Educational score: 2.751126527786255 Domain: clinical Document type: Clinical case Language: en 09:20: Assessment in the resuscitation area by A&E Consultant had the same findings and revealed OP poisoning. Additional pertinent signs included fasciculations in the upper chest and increased secretion load. An electrocardiogram revealed sinus tachycardia and venous blood gas showed lactate of 3.8mmol/L with normal pH and bicarbonate levels. Section title: Case presentation Educational score: 2.882030725479126 Domain: clinical Document type: Other Language: en 10:00: The toxicology consultant from the National Poisons Information Service (NPIS) suggested the following: measure serum and red cell cholinesterase levels; hold atropine unless high secretion load and also pralidoxime unless repeated atropine use; consider early intubation due to neuromuscular junction (NMJ) involvement; implement decontamination and donning of surgical mask, visor, gloves and apron. Section title: Case presentation Educational score: 3.480851173400879 Domain: clinical Document type: Clinical case Language: en 10:20: The patient continued to exhibit widespread fasciculations, confusion, and tachycardia with a heart rate of 120 bpm. Oxygen saturations were 92% on room air, and her blood pressure was 99/65 millimetres of mercury (mmHg). Oxygen was administered via a Mapleson C circuit before induction using fentanyl (1 microgram per kilogram) and 2 mg/kg of ketamine and rocuronium. A grade 1 view on laryngoscopy showed fully relaxed vocal cords. Section title: Case presentation Educational score: 2.737776517868042 Domain: clinical Document type: Clinical case Language: en 14:00-18:00: The patient remained in A&E for a prolonged period of time due to bed availability. Frequent suctioning of thick, yellow secretions was required. She remained tachycardic, especially during fasciculations, and developed hypertension (170/100mmHg). For sedation, she required propofol 340mg/hr, and boluses of fentanyl and midazolam before starting a morphine infusion. Section title: Case presentation Educational score: 2.1239397525787354 Domain: clinical Document type: Clinical case Language: en 18:30: Admitted to ICU on 30% inspired oxygen (FiO2). Her blood pressure settled to 100mmHg systolic. Her pupils were 2 millimetres and reactive. She experienced recurrent two-minute episodes of fasciculations. Figure 1 shows ICU observations. Section title: Case presentation Educational score: 1.0365513563156128 Domain: clinical Document type: Other Language: en Day 1 of admission Section title: Case presentation Educational score: 1.698306679725647 Domain: clinical Document type: Clinical case Language: en 02:00: The fasciculations decreased in frequency, haemodynamics stabilised and regular respiratory efforts started. Section title: Case presentation Educational score: 0.9757885336875916 Domain: clinical Document type: Clinical case Language: en 10:00 Following the ICU ward round, she was extubated and discharged the following day. Section title: Case presentation Educational score: 1.5584300756454468 Domain: biomedical Document type: Study Language: en Blood results from the admission are seen in Table 1 . Section title: Case presentation Educational score: 1.2003302574157715 Domain: clinical Document type: Other Language: en Follow-up after one month Section title: Case presentation Educational score: 1.530993938446045 Domain: clinical Document type: Clinical case Language: en The patient was fatigued for three days following discharge but has now made a full recovery. She is back at work. Serum anti-cholinesterase levels were very low in keeping with severe OP poisoning. Section title: Discussion Educational score: 3.6682655811309814 Domain: biomedical Document type: Study Language: en Other cases report non-accidental ingestion of OP with 30% of suicide cases globally due to OP intoxication and most patients received atropine and pralidoxime. A UK-based report treated with atropine and intubated achieved a similar recovery to this patient, without pralidoxime . One paper suggested OP poisoning mortality rates ranged from 3% to 25% worldwide . The UK NPIS report 2023 found, from 579 cases of pesticide poisoning, 81.3% were unintentional acute cases and only 11% of cases were graded severe, which this patient would be categorised into . Section title: Discussion Educational score: 1.9941976070404053 Domain: biomedical Document type: Other Language: ca Muscle relaxant Section title: Discussion Educational score: 4.0345659255981445 Domain: clinical Document type: Clinical case Language: en Ketamine 2 mg/kg due to the patient’s haemodynamic instability and fentanyl 1 mcg/kg were administered. The challenging discussion was the type and dose of muscle relaxant. Suxamethonium is a more potent agonist with greater affinity for the cholinergic receptors due to its structure of two acetylcholine (ACh) molecules joined. Its effects will be exacerbated in the presence of a cholinesterase inhibitor . It is contraindicated due to the risk of prolonged respiratory paralysis and extra junctional receptor side effects following muscle injury suggested by a rising creatine kinase, such as hyperkalemia. Rocuronium at 2 mg/kg achieved suitable vocal cord relaxation within 45 seconds of administration. An increased dose was necessary to overcome increased ACh at the NMJ. In the unlikely event that extubation was required, sugammadex could be considered as a reversal agent. It would be interesting to assess the level and type of muscular blockade prior to and post administration of muscle relaxant using a train-of-four nerve stimulator. This could be tested in a future similar situation if the patient is in a condition where pain from stimulation will not be felt, such as post-induction and pre-muscle relaxant, if starved and a modified rapid sequence induction is justified. Section title: Discussion Educational score: 1.296372413635254 Domain: biomedical Document type: Other Language: pt Antidotes Section title: Discussion Educational score: 4.107892036437988 Domain: clinical Document type: Clinical case Language: en The toxicology advice was to administer atropine only if there was high secretion load or severe bradycardia, due to the risk of worsening agitation, and initially, pralidoxime was recommended as per ToxBase guidelines; however, later re-advised to hold due to the risk of hypotension and limited evidence. The mainstay of treatment for this patient was intubation and ventilation followed by supportive level 3 care in the ICU. It is interesting that despite an anti-cholinesterase activity of <1000 units/litre, no antidote was required in this patient. The authors question whether the type of OP may affect the nicotinic and muscarinic receptors differently and therefore the traditional treatment should be tailored to the receptor-type effects, as in this case. Muscarinic effects include SLUDGE symptoms (salivation, lacrimation, urination, defecation, gastric upset and emesis) and bradycardia whilst nicotinic effects include skeletal muscle contraction, weakness, cramps, tachycardia and seizures . The intermediate syndrome has a varying level of severity and often is undiagnosed unless significant respiratory insufficiency. The fatigue symptoms reported in the days following discharge by this patient may have been the effects of mild intermediate syndrome, and further suggestion of nicotinic involvement . Section title: Conclusions Educational score: 4.095584869384766 Domain: clinical Document type: Clinical case Language: en This case study demonstrated the importance of proactive supportive management for patients with OP poisoning and underscored the need for early intubation when NMJ involvement is suspected. Unlike other published case reports, no "traditional" antidote was given in this case of severe OP poisoning with an anticholinesterase level <1000 units/litre. The patient's condition improved within two days after which she was discharged home. There were no staff symptoms from contamination following appropriate personal protective equipment (PPE) and decontamination. Intubation was easy and successful with an increased dose of muscle relaxant. Further investigation of the type and density of neuromuscular blockade in the future may aid in further tailoring of muscle relaxant dosing as opposed to a significantly increased dose. The symptoms and signs exhibited by this patient may suggest the varying effects of OP on the ACh receptors, specifically nicotinic rather than solely muscarinic symptoms. | Review | biomedical | en | 0.999995 |
PMC11699872 | Section title: Introduction Educational score: 4.049757480621338 Domain: biomedical Document type: Clinical case Language: en Uterine mesothelial cysts are extremely rare. Intra-abdominal mesothelial cysts were first described in 1979 , and approximately 150 cases have been reported in the literature to date. To the best of our knowledge, only five of these cases are associated with the uterus, and only one published in 2019, as in our case, developed within the myometrium . The factors triggering the growth of mesothelial cysts remain unknown, although developmental anomalies are often suspected. A history of abdominal surgery, pelvic inflammation, endometriosis, or mesothelial cysts of the round ligament are considered potential associations . Herein, we present a case of a uterine mesothelial cyst that was surgically treated under the preoperative diagnosis of a degenerated leiomyoma. Section title: Case presentation Educational score: 3.7350828647613525 Domain: clinical Document type: Clinical case Language: en A 41-year-old female patient presented to the gynecology clinic with complaints of abdominal pain. Her medical history revealed two pregnancies, one ending in cesarean delivery years ago and the other in curettage, with no additional comorbidities or surgical history. Transvaginal ultrasonography demonstrated an enlarged uterus with a hypoechoic intramural cystic mass measuring 7.2 × 3.3 × 3.6 cm in the posterior uterine corpus. The endometrium appeared thin and regular, and the bilateral adnexal regions were unremarkable. Computed tomography further identified the lesion as a hypodense mass in the uterine corpus measuring 7.2 × 3.3 × 3.6 cm . Section title: Case presentation Educational score: 2.95304799079895 Domain: clinical Document type: Clinical case Language: en Under the preliminary diagnosis of a degenerated leiomyoma, the patient underwent total laparoscopic hysterectomy and bilateral salpingectomy. Macroscopic examination of the uterus revealed a 6 cm cystic lesion containing serous fluid within the posterior uterine wall . Section title: Case presentation Educational score: 4.1537885665893555 Domain: biomedical Document type: Study Language: en Upon incision, no solid or papillary structures were observed on the cyst wall, although septation was noted in one area. Microscopic evaluation revealed a cystic lesion lined with a single layer of benign cuboidal mesothelial cells located within the myometrium, unrelated to the endometrium or serosa. Immunohistochemical studies showed positive staining of the cyst epithelium with calretinin, D2-40, WT-1, ER, and PR . Section title: Case presentation Educational score: 2.3422048091888428 Domain: biomedical Document type: Clinical case Language: en Extensive sampling was performed to exclude degenerated leiomyoma, but no areas indicative of leiomyoma were identified. The case was diagnosed as a uterine mesothelial cyst based on the histomorphological and immunohistochemical findings. No pathology was observed during the patient's follow-up. Section title: Discussion Educational score: 3.9995360374450684 Domain: biomedical Document type: Study Language: en Since 1985, no cases of mesothelial cysts in the uterine myometrium have been reported in the literature, except for one case in 2019, which, similar to our case, involved a mesothelial cyst developing within the myometrium . Of the 150 cases of mesothelial cysts reported in the literature, only five were uterine-related. One was described as a multicystic lesion attached to the serosa by a pedicle between the left uterine fundus and ovary , another presented as multiple small cysts on the serosal surface of the uterine corpus , one resembled our case within the myometrium , and the remaining two were reported as lesions adhering to the myometrium on the posterior uterine wall . Section title: Discussion Educational score: 4.0631561279296875 Domain: biomedical Document type: Study Language: en Based on the current literature, our case represents the second instance of a mesothelial cyst developing within the myometrium. Due to their prevalence in women of reproductive age, some studies suggest a potential relationship between sex hormones and mesothelial cysts. However, since mesothelial cysts of the round ligament do not demonstrate immunohistochemical expression of ER or PR, some authors propose that these lesions are independent of sex hormones . In the five uterine mesothelial cyst cases, the patients were aged 27-47 years, but the menopause status of women in their 40s is unknown, and while ER and PR expressions were negative in one of the five articles, immunohistochemical studies related to ER and PR were not reported in the other four articles . Section title: Discussion Educational score: 4.565388202667236 Domain: biomedical Document type: Review Language: en The developmental process of these cysts remains controversial due to the limited number of reported cases and the need for further research. Mesothelial cysts share similar morphological and histopathological features with benign cystic mesothelioma . Both can manifest as inclusion cysts within the pelvic cavity. While mesothelial cysts are typically solitary, unilocular, and benign, with epithelia composed of well-differentiated mesothelial cells, benign cystic mesothelioma is often multilocular and considered a reactive lesion or a neoplasm with low malignant potential and a tendency for recurrence . Uterine mesothelial cysts lack specific clinical features. They may be asymptomatic or present with a palpable abdominal mass or pelvic pain in some patients. Radiological diagnosis is challenging due to their similarities with leiomyomas exhibiting cystic degeneration, and they may be misdiagnosed preoperatively as degenerated leiomyomas, as in this case . The characteristic microscopic appearance of a single layer of cuboidal mesothelial cells lining the cyst wall, along with positive staining for specific mesothelial markers such as calretinin, WT-1, mesothelin, and HBME, aids in the diagnosis of pelvic mesothelial cysts . Estrogen and progesterone expression remain variable, and no definitive conclusions have been reached . Although most authors describe mesothelial cysts as benign lesions with low recurrence and malignant potential, cases with recurrence rates of up to 27% in benign cystic mesotheliomas and malignant transformation have been documented in the literature . Treatment options include cyst excision; however, the thin cyst walls often complicate complete excision and result in high recurrence rates. To minimize recurrence, total or partial hysterectomy is considered the optimal approach, especially for older patients or those who do not plan to have children . Section title: Conclusions Educational score: 3.8413732051849365 Domain: biomedical Document type: Review Language: en In conclusion, mesothelial cysts are extremely rare lesions in the uterus, and definitive diagnosis is typically established through pathological examination. Further case reports and advanced studies will help us better understand the development process of this lesion. Awareness of this uncommon lesion and its inclusion in the differential diagnosis of pelvic pain in women of reproductive age can facilitate patient management. Although complete excision of the cyst wall may be challenging, the prognosis is favorable, and hysterectomy, when indicated, significantly reduces the risk of recurrence. | Clinical case | biomedical | en | 0.999996 |
PMC11699873 | Section title: Introduction Educational score: 3.7107508182525635 Domain: biomedical Document type: Other Language: en The internal medicine clinic provides primary care to a diverse population, serving residents of neighboring nations near the border. Effective record-keeping is critical in this dynamic community to ensure continuity of care and facilitate decision-making during emergencies. Despite the importance of advanced care planning (ACP) and advanced medical directives in supporting informed decision-making, a meta-analysis from 2011 to 2016 found that only 36.7% of patients, and 38.2% of those with chronic illnesses, had documented directives . The number of patients with such documentation at this clinic is unknown, and there is no standardized method to educate patients on ACP. This gap highlights the need for early physician-patient discussions and structured resources to improve patient care and preparedness for future healthcare decisions. Section title: Materials and methods Educational score: 3.7868573665618896 Domain: biomedical Document type: Study Language: en This study was conducted at the internal medicine clinic and aimed to address ACP and advanced medical directives with patients to improve overall patient care. The objective was to initiate discussions about ACP and provide advance medical directive instructions and resources to all willing patients. This aimed to enhance awareness, education, and the prevalence of advanced medical directives among clinic patients. The study included all patients aged 18 years and older with appointments at the clinic from February 1 to April 1, 2024. Patients under 17 years, those who refused to participate, those who did not complete the post-intervention survey before discharge, and those who answered “No” to Question 6 of the pre-appointment survey were excluded from the study. This single-group, cross-sectional study was conducted without patient randomization, with participants assigned to the study group based on inclusion and exclusion criteria. Section title: Materials and methods Educational score: 3.138840436935425 Domain: biomedical Document type: Study Language: en Resident physicians conducted the study within the internal medicine residency program, and institutional board review (IRB) exemption was granted on January 31, 2024 , after initial submission on December 6, 2023, and necessary modifications. Data collection took place from February 1 to April 1, 2024. Data collected included the patient's age, comorbidities, advance medical directive completion status, and responses to a post-intervention survey when applicable. A numerical code was assigned to each patient, recorded separately in a Coded Identifier List to link pre-appointment and post-intervention surveys, without containing identifying information. The Excel (Microsoft Corp., Redmond, Washington, DC) file containing the data was saved on a secured, password-protected computer in the principal investigator's locked office. All personnel received training on proper data handling to minimize privacy risks, and data were stored on an encrypted device. Additionally, paper documents were destroyed after being digitized. Section title: Materials and methods Educational score: 2.1249568462371826 Domain: biomedical Document type: Study Language: en Data confidentiality was maintained by recording information in a Microsoft Excel document, with columns representing variables and each row representing individual participants. No identifiable information, such as names or biographical data, was recorded. Each patient received a random numerical code to link surveys, ensuring anonymity. The Excel document was stored on a secure device accessible only to IRB-approved personnel. Paper surveys were kept in the principal investigator’s (PI) locked office, and all documents identifying subjects were destroyed upon study completion per IRB procedures. Section title: Materials and methods Educational score: 3.677323818206787 Domain: biomedical Document type: Study Language: en During the study, all eligible patients who agreed to participate received an anonymous pre-intervention survey in English or Spanish at intake to capture baseline information on ACP. This survey assessed whether the patient had an advance medical directive and whether they wanted to receive education on ACP (Appendix A). Patients had three to four minutes to complete this survey to establish baseline knowledge of ACP and determine if they had completed an advance medical directive. Patients could opt out of the study at any time. For those interested in additional information on ACP, internal medicine residents provided standardized education from the National Institute on Aging website. This educational material explained ACP, the nature and importance of advanced medical directives, and the process for completing them. Patients had five to 10 minutes to review the information during the visit. Section title: Materials and methods Educational score: 2.678758144378662 Domain: clinical Document type: Study Language: en Patients who expressed interest in learning about ACP received printed copies of an out-of-hospital do-not-resuscitate (OOH-DNR) order and a Medical Power of Attorney form, along with bilingual instructions for form completion (Appendices B, C). Following the educational intervention, patients completed a post-intervention survey in English or Spanish, providing feedback on the education and their opinions on end-of-life planning (Appendix D). This survey, completed within three to four minutes, allowed patients to express their views on the education provided, maintaining anonymity and excluding any identifying characteristics. Section title: Results Educational score: 2.467177152633667 Domain: biomedical Document type: Study Language: en During the three months, 52 patients completed the ACP pre-intervention survey. The average age of the responding patients was found to be 59 years. The survey revealed that the three most prevalent diseases were hypertension (n=28, 53.8%), dyslipidemia (n=28, 48.1%), and diabetes (n=22, 42.3%). These results, along with the other surveyed comorbidities, are represented in Figure 1 . Section title: Results Educational score: 2.9654219150543213 Domain: biomedical Document type: Study Language: en The fourth question of the ACP pre-intervention survey asked if patients had advanced medical directives prior to the visit. Of the 52 responding patients, three reported having advanced directives (1.2%), 10 were unsure if they had advanced directives (19.2%), and 39 reported not having advanced directives (75%) . A t-test was performed and found no statistically significant difference between patients' age or number of comorbidities and having an advance medical directive (p > 0.05). Section title: Results Educational score: 2.5282576084136963 Domain: biomedical Document type: Study Language: en The ACP pre-intervention survey also captured patients' attitudes toward learning more about advanced medical directives during their appointments. Twenty-two patients expressed interest in learning more about advanced medical directives, while 30 declined . The performed t-test suggested no statistically significant difference between patients' number of comorbidities or age and their interest in learning more about advanced medical directives (p > 0.05). Section title: Results Educational score: 3.9067037105560303 Domain: biomedical Document type: Study Language: en Of the 52 patients who completed the ACP pre-intervention survey, 23 completed the ACP post-intervention survey. The initial question in the survey asked if patients had ever had discussions about advanced medical directives with a physician prior to the visit. Ten patients (43.5%) reported having such discussions in the past, while 13 (56.5%) stated they had never had advance medical directive discussions before . The performed t-test suggested a statistical significance between the patient's age and the likelihood of having had an advanced medical directives discussion prior to this study (p = 0.013). There was also a significant correlation between the number of comorbidities and having had past discussions (p = 0.025). Section title: Results Educational score: 2.8874166011810303 Domain: biomedical Document type: Study Language: en Questions 2 through 5 were Likert scale questions. Question 2, “This advance medical directive conversation was helpful,” had 15 patients (65.2%) respond “strongly agree,” seven (30.4%) respond “agree,” and one (4.3%) respond “neither agree nor disagree.” Question 3, “I am uncomfortable with end-of-life discussions," had six patients (26.1%) respond “strongly agree,” eight (34.8%) respond “agree,” two (8.7%) respond “neither agree nor disagree,” five (21.7%) respond “disagree,” and two (8.7%) respond “strongly disagree.” Question 4, “I see the importance of having advanced medical directives,” had 17 patients (73.9%) respond “strongly agree” and six (26.1%) respond “agree.” Question 5, “I now plan on completing my advanced medical directives in the future,” had 13 patients (56.5%) respond “strongly agree,” nine (39.1%) respond “agree,” and one (4.3%) respond “neither agree nor disagree.” These results are represented in Figure 5 . As for the reasons for not pursuing advanced directives, three patients (13.0%) responded that they did not know how to start, and one patient (4.3%) stated they did not find it necessary. The remaining patients did not respond. Section title: Discussion Educational score: 4.017829895019531 Domain: biomedical Document type: Study Language: en Publishing these findings is extremely significant for several reasons. Firstly, by standardizing ACP discussions, this study demonstrates a clear pathway to improving patient care through better preparation for end-of-life decisions . Ensuring that patients' wishes are known and respected can reduce stress for both patients and their families during critical times, particularly when patients are unable to communicate their preferences . This aligns with prior research by Silveira et al., which highlighted that patients with advanced directives are more likely to receive care consistent with their values and wishes, reducing the burden on surrogate decision-makers . Section title: Discussion Educational score: 4.023279190063477 Domain: biomedical Document type: Study Language: en This intervention also provided valuable education to patients, many of whom may not have been previously aware of or understood advanced directives . Education is a critical component in ACP, as demonstrated by Bravo et al., who found that structured educational interventions significantly increased the completion rates of advanced directives among community-dwelling older adults . The inclusion of culturally and linguistically appropriate materials, as implemented in this study, is crucial for ensuring that diverse patient populations can fully engage in ACP discussions . Sudore et al. emphasized the need for culturally sensitive ACP discussions, particularly in diverse populations similar to those served in this study . Section title: Discussion Educational score: 3.6490111351013184 Domain: biomedical Document type: Study Language: en Moreover, the project’s methodology and findings can serve as a model for other clinics and healthcare institutions . A systematic approach to ACP discussions promotes the integration of standardized practices into routine care, leading to widespread improvements in how advanced directives are handled across different healthcare settings . Studies by Morrison et al. and Detering et al. have further supported that culturally tailored ACP interventions lead to higher engagement and completion rates among minority groups . Section title: Discussion Educational score: 3.927417278289795 Domain: biomedical Document type: Study Language: en This is particularly important in primary care settings, where long-term patient-provider relationships can facilitate ongoing ACP discussions, as suggested by Tung et al. . The standardized approach used in this study is in line with best practices outlined by Lund et al., who found that systematic implementation of ACP in clinical settings can lead to higher rates of advance directive completion and greater patient satisfaction . Similar findings were reported by Jimenez et al., who highlighted the global lessons learned from systematic reviews of ACP . Rietjens et al. also emphasized the importance of continuity of care in successful ACP implementation . Section title: Discussion Educational score: 4.074590682983398 Domain: biomedical Document type: Study Language: en The results of this study further highlight its significance to current literature on ACP . During the three months, 52 patients completed the pre-intervention survey, with an average age of 59 years . The survey revealed that hypertension, dyslipidemia, and diabetes were the most prevalent diseases among participants, conditions often associated with higher healthcare utilization and potential complications that necessitate clear advance care plans . Notably, only 1.2% of patients reported having advanced directives prior to the study, underscoring a significant gap in ACP documentation in this population . This finding is consistent with national data reported by Yadav et al., which showed that approximately one-third of U.S. adults have completed some form of advance directive . Furthermore, a study by Rao et al. indicated that chronic conditions like those prevalent in this population often correlate with a higher desire for ACP, yet the completion rates remain low due to systemic barriers . Section title: Discussion Educational score: 4.058476448059082 Domain: biomedical Document type: Study Language: en The post-intervention survey results showed a substantial increase in the number of patients who had discussed advanced directives, with 43.5% of participants engaging in these conversations . A significant number of patients found the discussions helpful and recognized the importance of having advanced directives, which is supported by evidence from Jimenez et al., who demonstrated that ACP discussions improve patient and family satisfaction with end-of-life care . These results align with findings by Wright et al., who demonstrated that early ACP discussions significantly reduced anxiety and depression among patients with serious illnesses . Similarly, a randomized controlled trial by Houben et al. showed that ACP discussions are associated with better alignment of care with patients' wishes and fewer unwanted hospitalizations near the end of life . Section title: Discussion Educational score: 4.018272399902344 Domain: biomedical Document type: Study Language: en Furthermore, the study addresses significant gaps in the literature regarding ACP in specific demographics, particularly older adults with multiple comorbidities . Research by Tung et al. suggests that older adults with multiple comorbidities are more likely to engage in ACP discussions, a finding that was corroborated by this study's data, which highlighted the association between comorbidities and prior ACP discussions . Additionally, studies such as those by Song et al. have shown that tailored interventions, like the ones implemented in this project, can significantly increase the completion rates of advanced directives among patients, particularly in populations with chronic illnesses . Zhang et al. also noted that patients with multiple chronic conditions benefit most from ACP, as it helps align their complex care needs with their values . Section title: Discussion Educational score: 3.9111030101776123 Domain: biomedical Document type: Study Language: en The implications of these findings are broad, extending beyond individual patient care to inform policy and training programs for healthcare providers . Integrating ACP discussions early in the patient care process, particularly in primary care settings, is critical for ensuring that patients' preferences are documented and respected throughout their healthcare journey . Kavalieratos et al. found that early integration of palliative care, including ACP, is associated with improved patient and caregiver outcomes, including reduced hospitalizations and enhanced quality of life . This is particularly pertinent in a clinic setting where continuity of care and clear communication are crucial for effective patient management. The importance of healthcare provider training in ACP was emphasized by Lum et al., who found that well-trained providers are more likely to initiate and complete ACP discussions . Section title: Discussion Educational score: 3.8074021339416504 Domain: biomedical Document type: Study Language: en The importance of addressing ACP during public health crises, such as the COVID-19 pandemic, has also been highlighted in recent literature. It was noted that the pandemic has brought ACP to the forefront, emphasizing the need for healthcare providers to engage in these discussions proactively . This aligns with the findings from this study, which underscore the necessity of standardized ACP practices to prepare for unforeseen circumstances and ensure that patient's wishes are respected even during crises . A study by Curtis et al. during the pandemic also highlighted the critical role of ACP in reducing the burden on healthcare systems by ensuring that patient care aligns with their preferences, particularly when resources are limited . Section title: Discussion Educational score: 3.8759002685546875 Domain: biomedical Document type: Review Language: en In addition to primary care and public health settings, ACP has been shown to have significant implications in specialty care. For instance, Mack et al. demonstrated that end-of-life discussions in oncology settings are associated with better patient outcomes and reduced distress . Similarly, Mullick et al. highlighted the importance of integrating ACP into routine practice across various specialties to ensure that patients' wishes are consistently respected . Studies in cardiology and nephrology settings, such as those by Schell et al., have further validated the positive impact of ACP on patient satisfaction and care quality . Section title: Discussion Educational score: 3.990432024002075 Domain: biomedical Document type: Study Language: en This project on improving ACP discussions demonstrates a significant advancement in patient care practices . The standardized approach not only educates and empowers patients but also provides a replicable model for other healthcare institutions . The findings, supported by recent literature, underscore the importance of targeted ACP interventions and their positive impact on patient outcomes and healthcare practices . Future research should explore the long-term effects of such interventions on healthcare utilization and patient satisfaction, particularly in diverse and underserved populations. Moreover, the ongoing development of ACP tools and resources, as discussed by Bristowe et al., will be essential in supporting both patients and healthcare providers in the future . Section title: Discussion Educational score: 1.559439778327942 Domain: biomedical Document type: Other Language: it Limitations Section title: Discussion Educational score: 3.5928432941436768 Domain: biomedical Document type: Study Language: en This study has several limitations that should be acknowledged. First, the sample size was relatively small, with only 52 patients completing the pre-intervention survey and 23 patients completing the post-intervention survey. This small sample size may limit the generalizability of the findings to the broader population served by the internal medicine clinic. Larger studies with more diverse patient populations are needed to confirm these results and determine whether the observed improvements in ACP discussions can be replicated in different settings. Section title: Discussion Educational score: 3.5322325229644775 Domain: biomedical Document type: Study Language: en Second, the study was conducted over a short duration of only three months. This limited timeframe may not have been sufficient to capture the long-term effects of the intervention on patient behavior and outcomes. For instance, while the post-intervention survey indicated an increased likelihood of patients completing advanced directives, it remains unclear whether these intentions translated into actual behavior change over time. A longer follow-up period would be necessary to assess the sustainability of the intervention's impact. Section title: Discussion Educational score: 3.9979002475738525 Domain: biomedical Document type: Study Language: en Finally, the study relied on self-reported data from patients, which may be subject to response bias. Patients may have provided socially desirable answers, particularly regarding their comfort with end-of-life discussions and their intentions to complete advanced directives. Additionally, the study did not include a control group, making it difficult to determine whether the observed changes were solely attributable to the intervention or if other external factors may have influenced the results. Future studies should consider including a control group and using objective measures, such as the actual completion rates of advanced directives, to strengthen the validity of the findings. Section title: Conclusions Educational score: 2.6337666511535645 Domain: biomedical Document type: Other Language: en This project on improving ACP discussions demonstrates a significant advancement in patient care practices. The standardized approach not only educates and empowers patients but also provides a replicable model for other healthcare institutions. The findings, supported by recent literature, underscore the importance of targeted ACP interventions and their positive impact on patient outcomes and healthcare practices. | Other | biomedical | en | 0.999996 |
PMC11699881 | Section title: Introduction Educational score: 4.256564140319824 Domain: clinical Document type: Clinical case Language: en Carotid cavernous fistulas are a serious condition that involves spontaneous or trauma-induced disruption in the carotid arteries within the cavernous sinus. This disruption causes blood to leak into the sinus, which impedes the function of cranial nerves III, IV, V1, V2, and VI . Early signs of CCF often include proptosis, glaucoma, severe headaches, partial blindness, and conjunctival hyperemia, especially in patients with preexisting conditions such as atherosclerosis, hypertension, and diabetes. If left untreated, patients can develop periorbital disfigurement and permanent vision loss . Despite effective methods of diagnosis using angiography and blood vessel repair, this condition is often misdiagnosed due to it making up only 1.5% of endovascular-treated ruptured aneurysms and its similarity in presentation to cerebrospinal fluid (CSF) leaks . Therefore, CCFs have been under-investigated, and additional research is required to delineate the cause, unique features, and proper treatment of this phenomenon. Often misdiagnosed due to its ambiguous presentation, we present a successful clinical workup and treatment of a 78-year-old female patient who developed a CCF in the setting of spinal fusion. We suggest a potential relationship between prolonged prone positioning during surgical intervention and the occurrence of CCF. Section title: Case presentation Educational score: 3.814563512802124 Domain: clinical Document type: Clinical case Language: en A 78-year-old female patient presented for bilateral lateral radicular leg discomfort and back pain refractory to conservative management including physical therapy and anti-inflammatory medications. Magnetic resonance imaging (MRI) of the lumbar spine revealed significant degenerative spondylosis of L4-S1 levels including severe lateral recess stenosis at L4-L5 and bilateral pars defects at L5-S1 . She was neurologically intact with no remarkable findings on physical exam. Operative management was pursued due to her symptoms and imaging findings. She underwent a successful L4-S1 posterior instrumented fusion with partial laminectomies at L4 and L5 along with an L4-L5 bilateral facetectomy. There was no significant intraoperative fluid leak. Estimated blood loss was minimal, and the procedure lasted approximately 228 minutes with the patient prone on a Jackson spine table. Section title: Case presentation Educational score: 2.1879994869232178 Domain: clinical Document type: Clinical case Language: en The patient’s postoperative course was uneventful until postoperative day 3 when the patient developed severe throbbing headaches and neck and shoulder pain/stiffness that resolved with laying flat. The patient’s symptoms resolved the following day, so she was discharged. Two weeks following her surgery, she presented for suture removal and had no complaints of headaches or neck and shoulder pain. Section title: Case presentation Educational score: 3.326328992843628 Domain: clinical Document type: Clinical case Language: en Six weeks following the surgery, the patient presented for a follow-up with complaints of bifrontal headaches that were worse in the morning for the past two weeks. She also reported neck discomfort and sharp discomfort whenever she turned her head to one side or extended her neck. The headaches were not positional nor particularly severe. There was no evidence of a spinal fluid leak from the incision nor palpable fluid collection appreciated on the physical exam. An x-ray of the cervical spine demonstrated degenerative changes from C4 through C7 with complete loss in disc space height at each level from C4 inferiorly . Section title: Case presentation Educational score: 3.6421091556549072 Domain: clinical Document type: Clinical case Language: en The patient presented to the Emergency Department on two separate occasions in the following two weeks; first, she had a pounding headache in bilateral temples with swelling below her left eye and an episode of emesis the evening prior. She was also noted to have conjunctivitis in both eyes and was given antibiotic eye drops. She presented the second time with complaints of a constant headache in the middle of the night, nausea, and new onset blurry vision in her left eye. The patient was also hypertensive with 170 systolic blood pressure. A brain CT scan showed no evidence of an acute intracranial process. Basic lab workup was unremarkable on both visits. The patient was discharged the second time with prescriptions for Percocet and Zofran and was recommended to follow up with her neurologist. Eight weeks following her fusion, the patient was struggling with worsening headaches and nausea. A lumbar spine MRI showed a fluid collection adjacent to the dura with a possible dural tear . Her worsening symptoms prompted a return to the operating room for wound exploration and repair of a suspected CSF leak. There was no apparent CSF leak. Valsalva was performed with no obvious egress of spinal fluid. Given that there may have been an occult non-identifiable leak the dura was covered with two layers of Surgicel (Ethicon, Johnson & Johnson, USA) followed by Adherus (HyperBranch Medical Technology, Durham, NC, USA) tissue sealant. A lumbar drain was placed through the next most rostral intact interspinous space brought out through a separate stab incision. A 10-round Jackson-Pratt drain was tunneled into the sub-fascial space and brought out through an additional stab incision. Section title: Case presentation Educational score: 3.599426031112671 Domain: clinical Document type: Clinical case Language: en Following the surgery, she had two episodes of emesis with continued headaches and nausea. However, she experienced significant improvement in her headaches and nausea over the subsequent days. She was discharged one week later following drain removal and complete resolution of her symptoms. Two weeks following discharge, she presented to the emergency department again for continued headaches, left conjunctival hemorrhage, mild proptosis, diplopia, and a cranial nerve (CN) III palsy. Given the unclear etiology for her headaches and new exam findings, there was a concern for Tolosa-Hunt syndrome or a carotid-cavernous fistula which prompted a brain MRI. Imaging demonstrated prominent superior ophthalmic veins, with the right being greater than the left . Section title: Case presentation Educational score: 3.7498724460601807 Domain: clinical Document type: Clinical case Language: en A left carotid-cavernous fistula was confirmed using diagnostic angiography. The patient underwent transvenous embolization for carotid-cavernous fistula in the intercavernous and left cavernous sinus. The fistula was nearly completely occluded with a significant reduction of flow into the fistula and gradual occlusion noted during angiography from the arterial supply . She had a successful coil embolization of a Barrow Type C carotid-cavernous fistula. Following the procedure, the patient had a significant improvement in her symptoms and was discharged three days later. Her postoperative course was significant for an improvement in her headaches and a lingering CN III and VI palsy predominantly of the left eye. Section title: Discussion Educational score: 4.060612201690674 Domain: biomedical Document type: Other Language: en Carotid-cavernous fistulas (CCF) are pathological shunts between the cavernous sinus and the carotid artery or its meningeal branches. The Barrow classification system anatomically describes four types of CCFs. Type A CCFs are direct, high-flow communications between the cavernous sinus and the internal carotid artery (ICA) as it traverses the sinus. Low-flow fistulas indirectly connect the carotid artery to the cavernous sinus via the ICA meningeal branch (Type B), external carotid artery meningeal branch (Type C), or both branches (Type D) . Section title: Discussion Educational score: 4.121010780334473 Domain: biomedical Document type: Review Language: en Several etiologies regarding the pathogenesis of CCF have been described. Type A CCFs are often associated with trauma, ruptured ICA aneurysms, connective tissue diseases, or iatrogenic injury . Type A CCF has been reported after various procedures, particularly those involving direct carotid manipulation or involvement of adjacent structures; procedures reported in association with Type A CCF include carotid endarterectomy, mechanical thrombectomy, transsphenoidal exploration, craniotomy and ocular surgery . Traumatic CCFs may occur as a result of bony fragment damage to the vessel wall secondary to basilar skull fracture or shear forces directly damaging the vessel wall . Indirect CCFs most commonly affect elderly women and are classically associated with ICA dissection, hypertension, and connective tissue diseases, including atherosclerosis, fibromuscular dysplasia, and Ehlers-Danlos syndrome . Section title: Discussion Educational score: 4.223630428314209 Domain: biomedical Document type: Clinical case Language: en The presenting signs and symptoms of CCF are variable, depending on the characteristics of the CCF. Direct CCF often presents with conjunctival injection, blurred vision, headache, pulsating proptosis, and strabismus, most often affecting the sixth cranial nerve (the only cranial nerve traversing the interior of the cavernous sinus) . Low-flow CCFs often follow an insidious course, and conjunctival injection is typically the predominant physical complaint . Notably, low-flow CCFs are especially subject to misdiagnosis; our case represents a delayed diagnosis due to the patient’s recent spinal surgery and symptomatology mimicking a dural tear. The diagnosis of CCF is dependent upon imaging. Computed tomography (CT) and magnetic resonance (MR) imaging with or without angiography are often the first modalities utilized in assessing possible CCF and may be useful in diagnosing CCF; however, digital subtraction angiography (DSA) is currently considered the “gold standard” imaging modality for CCF because it demonstrates the cavernous sinus filling and drainage patterns . Strong clinical suspicion or CT/MRI evidence of possible CCF should prompt DSA . Section title: Discussion Educational score: 4.146782875061035 Domain: biomedical Document type: Clinical case Language: en The exact treatment strategy is dependent upon the characteristics of the CCF. Direct CCFs are most often treated through endovascular approaches, with embolic materials, such as coils, acrylic glue, or ethylene vinyl alcohol copolymer, injected into the cavernous sinus to close fistula patency . Endoluminal stenting may be utilized to prevent subsequent embolism and promote endothelial proliferation . Twenty to sixty percent of indirect CCFs spontaneously resolve; thus, conservative management is usually attempted before intervention . External manual carotid compression with the contralateral hand several times per day has been shown to promote fistula closure . If surgical intervention is indicated, the same endovascular techniques used to repair direct CCFs are the first-line treatment modalities . However, because indirect CCFs involve smaller, tortuous carotid branches, endovascular therapy is not as effective compared to direct CCFs . Overall, endovascular treatment has a greater than 80% cure rate for CCF . If first-line treatments fail, open surgical ICA ligation can be attempted . Our patient was successfully treated with CCF coil embolization. Section title: Discussion Educational score: 4.030633926391602 Domain: biomedical Document type: Clinical case Language: en CCFs are easily misdiagnosed, as CCF presentations can be highly variable and may be similar to several different diseases . Specific clinical histories may further confound the diagnosis of CCF. For example, Jain et al. describe a CCF that was initially misdiagnosed as hemorrhagic choroidal detachment following ocular surgery . Similarly, our patient's history of recent spinal surgery made a CSF leak a more plausible diagnosis than CCF. CSF leaks can present very similarly to CCF, with headaches, nausea, neck or back tightness, and cranial nerve palsies, being commonly shared symptoms . The diagnosis of CCF in our case was delayed because the patient’s recent surgical history made a dural tear more plausible. However, surgical exploration and drainage did not improve the patient’s symptoms, suggesting a dural tear was not present. After ruling out a dural tear, subsequent workup led to the diagnosis and treatment of CCF. Section title: Discussion Educational score: 3.656050443649292 Domain: biomedical Document type: Clinical case Language: en To our knowledge, there is no association between CCF and spinal surgery or prolonged periods of prone positioning. There have been no reported cases of CCF following surgery not involving the carotid artery or related structures; all reported iatrogenic causes of CCF involve direct carotid involvement or surgery to adjacent structures . The patient has no known vascular diseases and suffered no recent trauma. Indirect CCFs (such as the Type C CCF seen in this patient) disproportionately affect elderly women . We hypothesize that perhaps a prolonged period spent in the prone position during her spinal surgery is responsible for the CCF in a patient whose demographics predisposed her to CCF. Section title: Conclusions Educational score: 3.655388116836548 Domain: clinical Document type: Clinical case Language: en Our case represents the first reported CCF occurring in conjunction with spine surgery. Although unsubstantiated, we believe there may be an association between a prolonged period in the prone position and the development of CCF. Furthermore, we would like to document our clinical workup; in which the clinical history and symptomatology suggested a dural tear, although subsequent workup led to the diagnosis of CCF. This case demonstrates a difficult diagnosis of CCF and its potential relationship to spinal surgery. | Clinical case | clinical | en | 0.999997 |
PMC11699977 | Section title: Introduction Educational score: 3.994502305984497 Domain: biomedical Document type: Clinical case Language: en The increased incidence of second malignant neoplasms (SMNs) after Hodgkin lymphoma (HL) has been well-documented in the literature . In the head and neck region, surgeons encountering simultaneous primary malignancies like HL and squamous cell carcinoma (SCC) of the tongue are exceedingly rare, presenting unique diagnostic and treatment challenges . This case, a notable addition to the limited literature, underscores the intricacy of managing such dual malignancies in the head and neck region, emphasizing the importance of a comprehensive, multidisciplinary approach to these complex clinical scenarios. Section title: Case presentation Educational score: 2.7708747386932373 Domain: clinical Document type: Clinical case Language: en A 39-year-old smoker with a history of classical HL of the right cervical lymph node, clinically stage 1A, diagnosed in 2017 and, previously treated with chemotherapy, lost follow-up for six years and presented with left tongue ulcer, an enlarged and tender left level 2 lymph node, and a persistent left ear pain. Section title: Case presentation Educational score: 3.5185954570770264 Domain: clinical Document type: Clinical case Language: en Physical examination revealed a left lateral posterior tongue lesion and an enlarged, tender left level 2 lymph node. Further examination with a flexible scope showed a nasopharyngeal mass and the vocal cords were bilaterally movable with no masses. Otoscopy demonstrated a clear and intact tympanic membrane. Biopsies showed the existence of invasive, moderately differentiated SCC in the left lateral posterior tongue lesion, nonetheless, there was no evidence of malignancy infiltration in the nasopharynx. The imaging studies provided a comprehensive overview of the patient's condition. Section title: Case presentation Educational score: 2.478198766708374 Domain: biomedical Document type: Clinical case Language: en The CT scan of the head and neck showed increased fullness in the nasopharynx and mixed responses in the jugulodigastric lymphadenopathy, suggestive of disease progression . Section title: Case presentation Educational score: 3.537759304046631 Domain: biomedical Document type: Clinical case Language: en The PET-CT scan displayed prominent hypermetabolic activity in the oropharynx, specifically in the pre-epiglottic region and bilateral palatine tonsils, with significant metabolic activity in the left upper cervical lymph node, raising concerns for metastasis . Section title: Case presentation Educational score: 3.9148881435394287 Domain: biomedical Document type: Clinical case Language: en These imaging findings, collectively, indicated a complex scenario with both progressive and regressive elements in different areas. The notable hypermetabolic activity and lymph node involvement initially aimed towards metastasis from the SCC of the tongue. A left partial glossectomy, bilateral tonsillectomy, and left radical neck dissection with nasopharyngeal biopsy were determined following a discussion of the case on the tumor board. However, the intraoperative pathology revealed a dual pathology. Contrary to the metastatic expectations from the tongue SCC , the lymph nodes were predominantly affected by HL . Section title: Case presentation Educational score: 2.0069353580474854 Domain: clinical Document type: Clinical case Language: en Responded very well to the surgical approach, three months postoperatively, CT scans demonstrated negative findings and the patient regularly followed up every three months in our clinic. This unanticipated coexistence of SCC and HL within the lymph nodes significantly altered the clinical understanding and necessitated a revision in the treatment approach and prognosis. Section title: Discussion Educational score: 3.749234437942505 Domain: biomedical Document type: Clinical case Language: en HL predominantly manifests in lymphatic tissues but can occasionally present in extra-nodal sites, including the head and neck region . HL is commonly seen in late adulthood, the occurrence alongside SCC of the tongue, as presented in this case report, highlights a rare and intricate clinical scenario . The coexistence of these two distinct malignancies is not frequently documented, aligning with the literature that states multiple malignancies account for a small percentage (2-11%) of all head and neck malignancies . Section title: Discussion Educational score: 3.973726511001587 Domain: biomedical Document type: Study Language: en The increased occurrence of subsequent malignancies in HL survivors, as compared to the general populace, is partially a consequence of the long-term carcinogenic effects of chemotherapy and radiotherapy treatments. Furthermore, individuals who have overcome HL may possess a heightened predisposition towards the emergence of SMNs relative to survivors of other malignancies, a susceptibility that could be attributed to genetic factors inherent to HL . Section title: Discussion Educational score: 4.0654754638671875 Domain: biomedical Document type: Clinical case Language: en SCC of the tongue is a highly prevalent malignancy that is more common in the head and neck regions. However, the simultaneous development of HL and tongue carcinoma, which is similar to the infrequently documented cases of laryngeal cancer and lymphoma occurring simultaneously, adds complexity to the diagnostic, therapeutic, and prognostication processes . Similar to the case reported by Nigri and Khasgiwala where a patient with laryngeal SCC was later found to have HL, a synchronous lymphoma with laryngeal carcinoma in situ and a mucosa-associated lymphoid tissue (MALT)-type lymphoma with SCC of the larynx were reported as well . Our case underscores the importance of a thorough histopathological examination in cases of suspected or known malignancies in the head and neck region. Section title: Discussion Educational score: 4.051408767700195 Domain: biomedical Document type: Clinical case Language: en In the context of extra-laryngeal carcinoma coexisting with lymphoma, our case parallels the complexity seen in other reported cases where patients presented with multiple primary malignancies involving different regions of the head and neck. The development of a second malignancy in the presence of HL, while not as common as with well-differentiated lymphocytic lymphomas, remains a significant clinical consideration, especially in the head and neck region where lymphatic and mucosal tissues interact closely . While non-Hodgkin lymphoma (NHL) has a noted association with human immunodeficiency virus (HIV)-infected patients, this association is less clear with HL . Section title: Discussion Educational score: 3.6656951904296875 Domain: clinical Document type: Clinical case Language: en Our patient's clinical presentation, without HIV infection, aligns with the typical demographic profile for HL. The decision-making process in such cases is nuanced and requires a balance between aggressive treatment for local control and the careful management of potential systemic disease spread . Predisposing factors for the development of SCC, including chronic illness, smoking, and prior chemotherapy or radiotherapy, were considered in the management of our patient . However, our case featured the synchronous development of both tumors without a history of the common predisposing factors, presenting a unique clinical picture. Section title: Discussion Educational score: 3.7812423706054688 Domain: clinical Document type: Clinical case Language: en The diagnosis and management of our patient highlighted the necessity for comprehensive and thorough pre-operative examinations to ensure all potential primary sites and regions of tumor involvement are adequately assessed. This case contributes to the growing body of literature on the rare but clinically significant occurrence of concurrent HL and SCC of the tongue, emphasizing the need for meticulous diagnostic workup and individualized, multidisciplinary treatment approaches in managing such complex clinical scenarios. Section title: Conclusions Educational score: 3.616778612136841 Domain: clinical Document type: Clinical case Language: en We present an extremely rare case of a patient with classical HL who subsequently developed invasive SCC of the tongue. This case highlights the importance of ongoing surveillance of cancer survivors and the potential for the development of secondary malignancies. A multidisciplinary approach is crucial for the diagnosis, treatment, and follow-up of patients with coexisting primary diseases. | Clinical case | biomedical | en | 0.999999 |
PMC11699978 | Section title: Introduction Educational score: 3.8061327934265137 Domain: biomedical Document type: Other Language: en Penetrating thoracic injuries, especially those affecting cardiac structures, pose significant clinical challenges due to high mortality rates . Cardiac injuries involving the right atrium have a dire prognosis due to risks of rapid bleeding, cardiac tamponade, and hemodynamic instability. Timely identification and surgical intervention are crucial for improving survival rates, as even slight treatment delays can have severe outcomes . Section title: Introduction Educational score: 3.908686876296997 Domain: biomedical Document type: Other Language: en Although penetrating trauma is less common than blunt trauma as it accounts for only 0.1% of most trauma admissions, it results in higher fatality rates due to proximity to critical cardiac and vascular structures . Penetrating cardiac injuries, often from stabbings, are typically linked to severe assaults and accidents . The anatomical vulnerability of the right atrium may result in life-threatening bleeding. However, timely intervention remains problematic in resource-limited settings, with positive outcomes dependent on early diagnosis and skilled surgical personnel . Section title: Introduction Educational score: 3.5265910625457764 Domain: clinical Document type: Clinical case Language: en Penetrating injuries to the anterior chest wall can result in fatal damage to various thoracic structures, yet reports on these injuries and their management are scarce . Furthermore, documenting such cases is essential for enhancing surgical techniques, particularly in low-resource environments . This report discusses a case of penetrating chest trauma with a right atrium injury from a stab wound, effectively managed through rapid resuscitation and decisive surgical intervention. Section title: Case presentation Educational score: 3.5037994384765625 Domain: clinical Document type: Clinical case Language: en A 24-year-old male patient presented to the emergency department with a documented history of stab wounds located in the anterior precordial region, which penetrated the chest on the right side, approximately three hours post injury. Upon examination, the patient demonstrated a penetrating injury measuring 1.5x3 cm situated in the right fourth intercostal space within the parasternal region with an additional penetrating wound measuring 1x3 cm located at the fifth intercostal space along the midclavicular line . Section title: Case presentation Educational score: 3.616271734237671 Domain: clinical Document type: Clinical case Language: en The patient was assessed as semi-conscious and reported experiencing intense pain on the right side of the chest and respiratory distress while exhibiting signs of hemodynamic instability and an absence of air entry in the right thoracic cavity. The initial evaluation indicated a blood pressure of approximately 80/50 mmHg, a pulse rate of 150 beats per minute, a respiratory rate of 30 breaths per minute, jugular vein distention, and muffled heart sounds. Oxygen saturation was recorded at 90% while the patient received 2 liters of supplemental oxygen. After initial resuscitation efforts, the blood pressure improved to 90/50 mmHg, although the patient continued to experience respiratory difficulties and persistent hemodynamic instability. Immediately, a chest tube was inserted to address the right-sided hemothorax, evacuating 500 ml of blood upon insertion and continued to increase. Due to the patient's unstable condition, no radiologic investigations were undertaken. Section title: Case presentation Educational score: 3.6612956523895264 Domain: clinical Document type: Clinical case Language: en Within 15 minutes of hospital arrival, the patient was promptly transferred to the operating room after initial resuscitation with fluids and blood transfusion. A central venous catheter was inserted, and a broad-spectrum antibiotic was administered following induction. Firstly, a diagnostic laparoscopy was made to investigate the abdomen. The diaphragm, liver, spleen, and other abdominal organs were all examined and found intact. To treat the probable hemothorax and cardiac tamponade, the patient was positioned supine with the right side of the chest slightly elevated. Upon examination of the penetrating chest wounds, active bleeding from multiple intercostal vessels was identified and promptly addressed through suturing. The thoracic cavity was explored to palpate the diaphragm and assess for potential injuries. A defect in the pericardium was detected, necessitating lateral extension of the wound to evaluate the status of the underlying lungs and pericardium. Section title: Case presentation Educational score: 3.9720265865325928 Domain: clinical Document type: Clinical case Language: en During the surgical intervention, a pericardial tear measuring approximately 1.5×1.5 cm was observed, accompanied by clot formation and hemopericardium . Following the evacuation of the clot and blood from the pericardial space, a laceration and rupture of the right atrial wall were observed and briefly stabilized using the index finger during the thoracotomy . A tangential Satinsky clamp was applied to the lacerated area of the right atrial wall . The heart rate decreased from 160 to 115 beats per minute, and blood pressure improved to 90/60 mmHg. The clamped right atrial wall was repaired utilizing a 5-0 double-arm polypropylene suture in a double-layer technique until hemostasis was successfully achieved . The thoracic cavity was subsequently re-examined, the lung injury was addressed, and comprehensive hemostasis was ensured. The thoracic cavity was then rinsed with normal saline and closed in layers following the insertion of two chest drains-one positioned in the pericardium and the other within the chest cavity. The total blood transfusion during the operation was three packed cells, the total operative time was 120 minutes, and the total bleeding was 2300 cc. Section title: Case presentation Educational score: 2.2269558906555176 Domain: clinical Document type: Clinical case Language: en The patient was transferred to the intensive care unit for further evaluation, monitoring, and management. He was extubated on the first postoperative day and transferred to the general surgery ward on the third postoperative day. Chest X-rays showed no abnormalities . The chest tube was removed on the seventh day, and the patient was discharged on the ninth postoperative day . The patient was doing well after three and seven months of follow-up, and chest X-ray and echocardiography investigations revealed no abnormalities . Section title: Discussion Educational score: 4.104311943054199 Domain: clinical Document type: Clinical case Language: en Penetrating thoracic trauma resulting in right atrial rupture constitutes a rare yet critical injury that demands immediate assessment and surgical intervention . Due to its specific anatomical positioning, the heart is particularly vulnerable to penetrating injuries, with right atrial rupture posing a considerable mortality risk if not addressed expeditiously . The case reported by Valdés-Dupeyrón et al. exemplifies the imperative need for rapid resuscitation and suitable surgical management to secure patient survival, as was seen in our case . In the presented case, the patient sustained a penetrating stab wound to the right thoracic region, leading to hemopericardium and rupture of the right atrial wall. The successful application of the vascular clamp was followed by meticulous suturing of the atrial rupture, resulting in effective hemostatic control and stabilizing the patient's clinical status. Deploying double-arm polypropylene sutures in a layered fashion was instrumental in achieving hemostasis, a technique well-established in cardiac surgical practice for managing such catastrophic injuries . Section title: Discussion Educational score: 3.9855260848999023 Domain: biomedical Document type: Other Language: en Historically, cardiac injuries have been associated with dire prognoses and were deemed intractable; presently, approximately 90% of individuals succumb before arrival at the emergency department . Numerous researchers have identified correlations between mortality and the hemodynamic status of the patient upon admission, the type of weapon utilized, surgical findings, and the intricacy of the repair process . In our clinical scenario, the medical emergency team's intervention was indispensable for preserving these patients' lives. Cardiac injury should be anticipated in any patient presenting with penetrating injuries to the thoracic region, particularly on the anterior aspect of the thorax, predominantly on the left side, as well as in the upper abdomen and neck . Section title: Discussion Educational score: 4.033330917358398 Domain: biomedical Document type: Study Language: en Numerous researchers acknowledge that the survival rate following a penetrating cardiac injury is likely contingent upon the timeliness of medical intervention . Stranch et al. discovered a statistically significant correlation between the delay in hospital arrival, the clinical status upon admission, the mechanism of injury, and the implementation of aggressive surgical treatment with patient survival rates . The time between injury and operation for penetrating heart damage must be brief. Isaza et al. observed that the average was 60 minutes . Survival after a penetrating cardiac injury appears to be less likely in a patient five hours after the trauma. However, penetrating wounds in the anatomical area known as the "cardiac box" should raise the most concerns for penetrating heart damage . Focused and coordinated surveys, as well as resuscitation, are beneficial to any patient with penetrating thoracic trauma. In the present case, the time to surgical intervention was about three hours. In another report, Gucho et al. reported a delayed presentation of penetrating cardiac injury for five hours in a 21-year-old patient with a stab wound on his right chest's fourth intercostal space . Section title: Discussion Educational score: 4.206204891204834 Domain: biomedical Document type: Study Language: en Penetrating traumas to any of the cardiac chambers may precipitate acute cardiac tamponade and subsequent mortality rapidly. Hemorrhagic flow resulting from a lacerated pericardial injury will disseminate into the hemithorax, ultimately culminating in a fatality. Consequently, the pericardium serves a critical role in averting lethal exsanguination and enables patients to endure sufficiently long to attain medical intervention at a trauma center. Patients may exhibit various degrees of hemodynamic instability as a consequence of pericardial tamponade . Notably, the classical clinical manifestation of Beck’s triad (characterized by muffled heart sounds, jugular venous distension, and hypotension) or Kussmaul’s sign (indicative of jugular venous distension upon inspiration) is observed in merely 10% of patients presenting with pericardial tamponade . Section title: Discussion Educational score: 3.812248706817627 Domain: biomedical Document type: Other Language: en Penetrating trauma can result in a pneumothorax or haemothorax with significant blood loss. Some individuals see fast deterioration following chest injuries. However, with correct care, they can quickly improve. In patients with penetrating injuries, surgical operations are typically suggested, although diagnostic examinations are less necessary than in blunt trauma . Various surgical strategies exist for addressing penetrating cardiac trauma, including left anterior thoracotomy, right anterior thoracotomy, pericardial window, and median sternotomy. The latter approach is prevalent owing to its extensive exposure, albeit it is not as expeditious as other techniques . Section title: Discussion Educational score: 3.9584286212921143 Domain: clinical Document type: Clinical case Language: en In our case, we opted for an abdominal laparoscopic diagnostic procedure without prior radiological assessment due to the patient's declining condition. Consequently, we prioritized abdominal examination over thoracic assessment, believing the abdominal and hepatic injuries were of greater severity. Upon negative findings, the surgical strategy shifted to thoracotomy and pericardial exploration, resulting in the identification and repair of the atrial injury; a comparable case with certain deviations was documented by Alfraidy et al. . In a separate report by Valdés-Dupeyrón et al., the authors conducted a left thoracotomy combined with a transverse sternotomy in one patient, where a conventional sternotomy would have delayed vascular control . Section title: Discussion Educational score: 2.309018611907959 Domain: clinical Document type: Clinical case Language: en Despite the rarity of penetrating cardiac injuries in low-resource settings, favorable results are possible with appropriate resuscitation and surgical capabilities . The present case contributes to the existing literature on addressing similar injuries in economically weak healthcare systems with inadequate resources. Section title: Conclusions Educational score: 4.018948078155518 Domain: clinical Document type: Clinical case Language: en Penetrating thoracic trauma that affects the right atrium represents a rare yet potentially life-threatening condition that requires immediate assessment and intervention. This case exemplifies the critical nature of rapid diagnosis, proficient resuscitation, and thorough surgical repair in managing such injuries. The effective utilization of a tangential Satinsky clamp coupled with double-layer polypropylene sutures to control bleeding demonstrates that survival can be achieved even in severe cases of cardiac injury. Furthermore, this case further emphasizes the need to follow standard principles of resuscitation and guidelines for the management of thoracic trauma in places with a high incidence of penetrating chest trauma. Applying clinical diligence and the use of imaging like CT trauma series after stabilizing these patients could avoid potentially morbid negative explorations. Even in resource-poor settings, the use of emergency room/bedside ultrasound or diagnostic peritoneal lavage for abdominal injuries could avoid such aggressive exploratory attempts. | Other | biomedical | en | 0.999997 |
PMC11699980 | Section title: 1. Introduction Educational score: 3.7224247455596924 Domain: biomedical Document type: Other Language: en Head and neck cancer (HNC) refers to a group of heterogeneous tumors that originate, except for skin tumors, in the mucosa of the upper gastrointestinal tract, especially in the oral cavity, pharynx, and larynx . It ranks seventh in incidence and eighth in mortality estimates among the most prevalent cancers worldwide, excluding nonmelanoma skin cancer . Section title: 1. Introduction Educational score: 3.6191461086273193 Domain: biomedical Document type: Study Language: en HNC is a multifactorial disease whose risk factors arise from the interaction between environmental influences and genetic inheritance . Among environmental factors, smoking has emerged as the greatest risk factor, followed by alcohol consumption. When consumed simultaneously, risk increases from 10 to 100 times [ 4 – 6 ]. Section title: 1. Introduction Educational score: 3.9645156860351562 Domain: biomedical Document type: Study Language: en As HNC affects regions directly related to the digestive tract (reducing food intake) and induces a hypercatabolic state, carriers show significant weight loss from the time of diagnosis . Such nutritional decline is consistently associated with nutritional impact symptoms (NISs). NISs are physiological alterations caused by cancer that can compromise oral intake, including pain, dry mouth, difficulty swallowing, reduced appetite, and vomiting, among others . Approximately 96% of the population with HNC show one or more NISs before starting treatment . Section title: 1. Introduction Educational score: 3.891247034072876 Domain: biomedical Document type: Review Language: en Individuals with HNC and multiple NISs are more likely to experience reduced food intake, weight loss, functional capacity dysfunction, and decreased survival [ 12 – 14 ]. Systematic assessment and management of these symptoms before the start of treatment are crucial to prevent serious nutritional complications that often develop during antineoplastic therapy . An early approach allows for the implementation of appropriate nutritional and therapeutic strategies that can not only mitigate the progression of symptoms but also significantly improve the patients' quality of life. Furthermore, early treatment of NIS can prevent patients from reaching a state of severe malnutrition, which would further compromise the effectiveness of the treatment and survival . Section title: 1. Introduction Educational score: 3.697500228881836 Domain: biomedical Document type: Study Language: en Therefore, early recognition (pretreatment) of patients with a higher burden of NIS is crucial and provides insights that can assist in appropriate therapy before the development of malnutrition and associated factors. This study aimed to analyze the presence and severity of NISs and their associated factors in individuals with HNC before treatment. Section title: 2.1. Methodological Design and Study Population Educational score: 3.943770170211792 Domain: biomedical Document type: Study Language: en This is an epidemiological, analytical, and cross-sectional investigation of individuals with HNC who were treated at a reference oncology hospital in Greater Vitória, Espírito Santo, Brazil. The data represent the baseline of a longitudinal study titled “Nutritional Indicators, Mortality, and Associated Factors: A Hospital-Based Study in Individuals with HNC.” The target population consisted of patients diagnosed with squamous cell carcinoma (mouth, larynx, oropharynx, and hypopharynx) of all sexes who were aged 18 years and above. Section title: 2.2. Sampling and Selection Educational score: 2.8971774578094482 Domain: biomedical Document type: Study Language: en The study participants were invited to join after confirmation of their HNC diagnosis by the Head and Neck Surgery Medical Service at a cancer treatment reference hospital. The participants were patients receiving care through the Unified Health System (SUS), Brazil's public healthcare system. Following histopathological confirmation of the HNC diagnosis, all eligible individuals were invited by the researchers between September 2022 and January 2024. Participation was confirmed by signing informed consent forms that detailed the research. Data were collected by researchers who had undergone prior training in administering questionnaires and performing anthropometric measurements, including the standardization of measurement techniques to ensure data accuracy. Section title: 2.2. Sampling and Selection Educational score: 3.2425782680511475 Domain: biomedical Document type: Study Language: en To calculate the sample size, a sample universe of 200 patients was considered, corresponding to the average annual number of first consultation visits at the HNC outpatient clinic, a prevalence of 50% (to maximize the sample), a sampling error of 5%, and a confidence interval of 95%. The resulting minimum sample size totaled 132 individuals. The calculation was performed using Epi Info 7.2 software. Section title: 2.3. Inclusion and Exclusion Criteria Educational score: 3.9540343284606934 Domain: biomedical Document type: Study Language: en Participants of all sexes who were aged 18 years or above, diagnosed with squamous cell carcinoma of the oral cavity, larynx, oropharynx, and hypopharynx, and without previous treatment, were included. Cases were confirmed by histology and classified according to the ICD-10-03 into the following topographies: base of tongue (C01), other and unspecified parts of tongue (C02), gum (C03), floor of mouth (C04), palate (C05), other and unspecified parts of mouth (C06), tonsil (C09), oropharynx (C10), pyriform sinus (C12), hypopharynx (C13), and larynx (C32). Section title: 2.3. Inclusion and Exclusion Criteria Educational score: 3.5101749897003174 Domain: biomedical Document type: Study Language: en Subsequently, the ICD codes were grouped into four topographic categories: oral cavity (C01, C02, C03, C04, and C05), oropharynx (C05.1, C09, and C10), larynx (C32), and hypopharynx (C12 and C13). Patients were excluded from the study if they had a diagnosis of recurrent squamous cell carcinoma, had previously received cancer treatment, presented with multiple tumors, or lacked the clinical and mental capacity to respond to the administered questionnaire. Section title: 2.4. Data Collection Educational score: 4.042191505432129 Domain: biomedical Document type: Study Language: en A questionnaire was administered to evaluate patients' sociodemographic (sex, age, race/ethnicity, education, and income), lifestyle (smoking and alcohol consumption), and clinical data (tumor site and staging). It is worth noting that sociodemographic variables and lifestyle habits were self-reported. The Head and Neck Symptom Checklist (HNSC), a validated tool in individuals with HNC , was used to assess NIS. It consists of 17 symptoms assessed on a five-point Likert scale ranging from “1, not at all” to “5, very much.” For analysis, all 17 symptom scores were added, resulting in a total score that can range from 17 (no symptoms) to 85 (highest score for each symptom on the list) . Section title: 2.4. Data Collection Educational score: 4.111660957336426 Domain: biomedical Document type: Study Language: en The Nutritional Risk Screening tool was used to assess nutritional risk. It is a validated instrument and is based on data on dietary intake in the previous week, body mass index (BMI), weight loss in the last three months, age, and disease severity. Patients are scored according to malnutrition and the severity of the underlying disease, classified for each variable as absent (0), mild (1), or severe (2). Patients with a score of three or more are classified as at nutritional risk, while those with a score below three should be reassessed . In addition, anthropometric measurements (weight and height) were collected by the researchers, and body composition measurements (appendicular skeletal muscle mass) were obtained using a portable InBody 120 bioimpedance device. Section title: 2.5. Statistical Analysis Educational score: 3.985971689224243 Domain: biomedical Document type: Study Language: en Data were evaluated on Research Electronic Data Capture , uploaded to the cloud of the Federal University of Espírito Santo, and organized and analyzed on R (4.3.1) for Windows. Absolute and relative frequencies were used to describe the categorical variables. Measures of central tendency (means and medians) and dispersion (standard deviations and interquartile ranges) were used to describe continuous variables. NIS scores (17–85 points) were used as the dependent variable. A multiple linear regression was performed to quantify the participation of independent variables (sex, age, staging, education, income, topography, race/color, smoking, alcohol consumption, nutritional risk, BMI, and ASMM) in the outcome. The significance level for all tests was set at 5%. Section title: 2.6. Ethical Aspects Educational score: 1.0109602212905884 Domain: other Document type: Other Language: en This project was approved by the Research Ethics Committee of the Cassiano Antônio Moraes University Hospital under final opinion no. 6,241,277 and CAAE 56453322. 7.0000.5060. It complied with the ethical standards governing research involving human subjects. Section title: 3. Results Educational score: 3.6976475715637207 Domain: biomedical Document type: Study Language: en The sample consisted of 132 participants, with a higher proportion of older adults (61%)—with a mean age of 61 years, men (77%), mixed-race people (54%), with less than eight years of education (71%), and a family income of less than or equal to two minimum wages (76%). In terms of lifestyle habits, most participants reported being former smokers (52%) and former drinkers (65%). However, even after diagnosis, 19% continued to consume alcohol. The most prevalent topography was in the oral cavity (45%), with stage IV tumors (53%). Although 43% of participants were overweight (showing overweight and obesity), 45% of the population were at nutritional risk according to the NRS at the time of diagnosis ( Table 1 ). Section title: 3. Results Educational score: 3.794870138168335 Domain: biomedical Document type: Study Language: en Regarding NIS, 95% of participants showed one or more NISs ( n = 126), with the most recurrent symptoms being pain (68.93%), difficulty chewing (60.60%), thick saliva (59.84%), anxiety (58.33%), sore mouth (52.27%), and difficulty swallowing (50.0%) ( Table 2 ). Section title: 3. Results Educational score: 4.0500922203063965 Domain: biomedical Document type: Study Language: en Table 3 shows the data on the association between sociodemographic, lifestyle, and clinical variables and the total score of NISs. Topography, smoking, and nutritional risk showed an association. Cancer in the larynx ( p =0.031) had an estimated reduction of 6.67 points in NIS scores in comparison to that in the oral cavity. Regarding smoking, former smokers ( p =0.019) showed an estimated reduction of 5.87 points in NIS scores than smokers. Finally, participants with nutritional risk ( p =0.009) have an estimated increase of 6.15 points in total NIS scores when compared to those without nutritional risks (see Table 4 ). Section title: 4. Discussion Educational score: 4.095107078552246 Domain: biomedical Document type: Study Language: en This study evinced a high severity and quantity of NIS in its population. About 9 out of 10 participants reported one or more NISs before treatment, corroborating the findings by Farhangfar et al. , who observed that 94% of their HNC cohort had one or more NISs before antineoplastic therapy. NISs lead to a series of consequences for individuals, inducing an inflammatory response that limits energy and protein intake and contributes to increased stress and weight loss before and even during treatment . Additionally, NIS presence and severity tend to increase during treatment , which can impact treatment efficacy and sometimes necessitate treatment interruption until the specific symptom is reversed and the individual can resume therapy. Jin et al. showed that NIS worsened during radiotherapy early on, emphasizing the importance of managing these symptoms before starting therapy. Section title: 4. Discussion Educational score: 4.0867919921875 Domain: biomedical Document type: Study Language: en The most recurrent symptoms included pain (68.93%), difficulty chewing (60.60%), thick saliva (59.84%), anxiety (58.33%), sore mouth (52.27%), and difficulty swallowing (dysphagia) (50.0%). Results showed symptom clusters similar to previous studies. Kubrak, Olson, and Baracos found that the most common symptoms before treatment were pain (33%) and dysphagia (29%). Farhangfar et al. reported that the most prevalent symptoms were pain (63.6%), anxiety (62.9%), and lack of energy (58.5%). Furthermore, Granstrom et al. showed that the highest NIS scores at baseline occurred for pain (96%), anxiety (96%), and sore mouth (95%). Despite the discrepancies in symptoms across studies, pain is commonly the most reported symptom, typically ranking first in proportions. This study may explain this fact by the predominant location (the oral cavity) and the advanced stage of the disease (stage IV). Section title: 4. Discussion Educational score: 4.094824314117432 Domain: biomedical Document type: Study Language: en This study found an association between tumor location and NIS score before treatment. Individuals with HNC in the larynx had NIS lower quantity and/or severity than those with cancer in their oral cavity. These findings resemble those by Granstrom et al. , who found a higher NIS score in individuals with HNC in the oral cavity and oropharynx than in those with larynx cancer during and after treatment. The main signs and symptoms in laryngeal cancer include hoarseness, pain during swallowing, changes in voice quality, sensation of lumps in the throat, difficulty breathing, or even shortness of breath . Of these reported symptoms, the HNSC—the tool chosen for the study—includes only one: odynophagia (painful swallowing), with only 12% of the population reporting this type of NIS. Therefore, a partial explanation for a lower presence and/or severity of symptoms in laryngeal cancer may be due to the chosen tool ignoring the other characteristic symptoms of this type of cancer. Section title: 4. Discussion Educational score: 4.045578956604004 Domain: biomedical Document type: Study Language: en Individuals who quit smoking had lower NIS presence and/or severity than those who still smoked. Smoking is a well-established direct risk factor in the literature, considered the primary risk factor in HNC development . Tobacco acts as a factor for tumor growth, and cigarette smoke acts as a mutagenic and DNA-damaging agent that drives tumor initiation in normal epithelial cells . Thus, individuals who continue smoking after diagnosis contribute to this entire cascade driving the tumor, which is consistently related to a higher burden or severity of NIS. Section title: 4. Discussion Educational score: 4.0518999099731445 Domain: biomedical Document type: Study Language: en The presence of nutritional risk in individuals with HNC is relatively high from the time of diagnosis. Overall, 45% of the population in this study was at nutritional risk, agreeing with the findings by Wang et al. and by Bossi et al. , who found around 30.3% of the HNC population showing nutritional risk at the time of diagnosis. Furthermore, the presence of nutritional risk and total NIS scores showed an association. Individuals at nutritional risk had a higher presence or severity of NIS than those without nutritional risk. According to Wang et al. , NISs are positively correlated with nutritional risk in HNC patients in the perioperative period, resulting in higher scores according to the NRS-2002. Section title: 4. Discussion Educational score: 2.113905906677246 Domain: biomedical Document type: Study Language: en As a possibility of bias, information bias was minimized due to training of all staff in data collection and management. In addition, selection bias was also a possibility; however, all individuals who were in the CCP outpatient clinic during the research period were invited to participate in the research. The refusal rate was low, at 2.1%, out of a total of 143 individuals. Section title: 4. Discussion Educational score: 3.931769847869873 Domain: biomedical Document type: Study Language: en This study has limitations. Its cross-sectional design implies an analysis of NIS at a single point in time, which affects causality. Additionally, a multicenter evaluation with a larger number of patients could provide more accurate assumptions. However, the findings in this study complement the literature and show that health education and nutritional monitoring should be provided to patients from the moment of diagnosis to help them understand the importance of nutritional treatment, ensuring adequate nutritional intake and improving their nutritional status. Section title: 5. Conclusion Educational score: 2.4067351818084717 Domain: biomedical Document type: Other Language: en Individuals with HNC who smoke and have nutritional risk show higher estimates of NIS severity and quantity. However, those with tumors in their larynx have lower estimates of symptom severity than those with cancer in their oral cavity. | Other | biomedical | en | 0.999997 |
PMC11699984 | Section title: 1. Introduction Educational score: 1.711607813835144 Domain: other Document type: Other Language: en Approximately 112.5 million domestic Equidae are found worldwide, comprising 44.3 million donkeys, 58.5 million horses, and the remaining mules . The population of equines in Africa ranges from 17.6 million, including 11.6 million donkeys, 2.3 million mules, and 3.7 million horses . In Ethiopia, the total equine population has reached 2.03 million horses, 7.43 million donkeys, and 0.4 million mules in sedentary areas . Horses, donkeys, and mules are extensively utilized, particularly in rural socioeconomic activities. These animals serve various purposes across different sectors, including traction power, carting, recreation, festival participation, riding, and transportation . Section title: 1. Introduction Educational score: 1.8066681623458862 Domain: biomedical Document type: Other Language: en However, these huge populations of equines cannot provide the expected benefit to the national and local economy due to several infectious and noninfectious diseases, providing unsatisfactory care management and giving significantly less attention, especially to donkeys . Among the various factors contributing to the health deterioration of equine, parasites play a major role. Section title: 1. Introduction Educational score: 4.138810157775879 Domain: biomedical Document type: Study Language: en From parasitic health factors of equine, strongyle nematode parasites embrace the first place. The strongyles are nematode parasites found in the large intestine, specifically in the caecum and colon of equines. Strongyles of equines are categorized as large strangles and small strongyles (cytostome). The large strongyles comprise the three most important species found in equines Strongylus vulgaris ( S. vulgaris ), Strongylus edentatus ( S. edentatus ), and Strongylus equinus ( S. equinus ). S. vulgaris , S. edentates , and S. equinus are commonly known as the double-tooth strongyle, the toothless strongyle, and the triple-toothed strongyle, respectively. S. vulgaris is smaller than the other two large strongyle species . Among the gastrointestinal nematodes of horses, large strongyle infections, particularly S. vulgaris , have long been considered one of the most common and pathogenic parasites affecting horses . Strongylosis poses a serious problem in young horses raised on permanent horse pastures, although cases of severe disease may also occur in adult animals kept in suburban paddocks and subjected to overcrowding and poor management . Section title: 1. Introduction Educational score: 4.109757900238037 Domain: biomedical Document type: Study Language: en Strongylosis has been reported worldwide and affects nearly 90% of the horse population . In Ethiopia, equines make significant contributions to the national economy, yet parasitic helminths are among the most common factors hindering their maximum utilization. These parasites cause varying degrees of damage depending on the species, nutritional status, and immune system of the equines, resulting in decreased performance and productivity, as well as increased morbidity and mortality . The prevalence and types of internal parasites affecting equids are widespread, with equines being continually exposed throughout their lives . S. vulgaris and S. edentatus are among the most common equine health problems caused by strongyle species in Ethiopia, with S. equinus being less common . Although strongyle infections pose significant concerns for both the economy and equine health in Ethiopia, there is limited availability of recent data that shows the current status of strongyles, particularly in and around Bishoftu, Oromia, Ethiopia. Therefore, the objectives of this study were to determine the prevalence of strongyle parasites, to evaluate associated risk factors, and to identify the important genera of strongyles in horses and donkeys. Section title: 2.1. Study Area Educational score: 1.9520739316940308 Domain: biomedical Document type: Study Language: en The study was conducted in and around Bishoftu, located approximately 47 km southeast of Addis Ababa. Bishoftu is situated at 9°N latitude and 40°E longitude, with an altitude of 1850 m above the sea level in the central highlands of Ethiopia . The region experiences a bimodal pattern of rainfall, with the main rainy season extending from June to September (accounting for 84% of the total rainfall) and a short rainy season from March to May, resulting in an average annual rainfall of 875 mm. The mean annual minimum and maximum temperatures are 14°C and 26°C, respectively, with an overall average of 20°C. The mean relative humidity is 61.3% . Section title: 2.2. Study Animals Educational score: 3.7895095348358154 Domain: biomedical Document type: Study Language: en The studied animals were 364 equines (298 horses and 66 donkeys) in and around Bishoftu town managed under a traditional extensive management system that originated from five localities (Kality, Dambi, Dalota, Bambogaya, and G/Gorba). The study encompassed both clinically suspected and healthy groups of donkeys and horses. The study animals were primarily used for traction, transport, and cart pulling, from which samples were obtained. During sampling, the animal species, sex, age, and body condition were recorded. The ages of the equines were determined from birth records of owner history and dentition characteristics. The body condition scoring was based on the criteria of donkey sanctuary and was classified into poor, moderate, and good. Faecal samples were collected exclusively from animals that had not been treated or dewormed in the 3 months preceding the study to ensure unbiased results. Section title: 2.3. Study Design and Sampling Techniques Educational score: 4.094853401184082 Domain: biomedical Document type: Study Language: en A cross-sectional study was conducted from February 2022 to July 2022 to determine the prevalence of strongyle parasites in horses and donkeys, as well as to evaluate associated risk factors in and around Bishoftu. The study animal was conducted by using a simple random sampling method to examine the prevalence of equine (horses and donkeys) strongyles parasites. During the study, 364 equines (298 horses and 66 donkeys) were randomly selected based proportionality of their total numbers that were presented in and around Bishoftu town (Kality, Dambi, Dalota, Bambogaya, and G/Gorba). Five kebeles (peasant associations) were randomly selected using a sampling frame created from a complete list of kebeles in Bishoftu town. Similarly, the total population of each horses and donkeys in each kebele was determined, and the number of animals to be sampled was proportionally allocated based on their population in each kebele. Individual study animals were then randomly selected using a lottery system. The consent of their owners was obtained and conducted a brief interview. Faecal samples were collected directly from the rectum of the selected animals by the researchers to ensure sample quality and minimize bias. Section title: 2.4. Sample Size Determination Educational score: 4.04874324798584 Domain: biomedical Document type: Study Language: en The required sample size of the study was determined by using the formula given by , with a 75.8% expected prevalence that was previously done, a 95% confidence level, and 5% desired absolute precision. (1) n = 1.96 2 Pexp 1 − Pexp d 2 , where n is the sample size, Z (1.96) is the statistic corresponding to the level of confidence 95%, Pexp is the expected prevalence, and d is the desired absolute precision 5%. Therefore, based on the above formula, the desired sample size was 282 equines. However, the sample size was increased to 364, to increase the precision of the research and to obtain enough fecal egg outputs. Section title: 2.5.1. Sample Collection Procedure and Processing Educational score: 3.7960143089294434 Domain: biomedical Document type: Study Language: en With gloved hands, approximately 10 g of fecal samples were directly collected from the rectum of restrained donkeys or horses in the clean sampling plastic bottle. Following collection, each sample was uniquely labeled with the animal's identification parameters, including species, age, sex, origin/site, and body condition score, and kept in the ice box. The collected samples were then packed and transported to the Addis Ababa University Veterinary Parasitology Laboratory for immediate examination of strongyle parasite eggs. If immediate processing is not possible, the sample was stored at +4°C in the refrigerator. Section title: 2.5.2. Coprological Examination Educational score: 4.115627765655518 Domain: biomedical Document type: Study Language: en The fecal samples were subjected to coprological examination for the fecal flotation technique. Approximately 3 g of feces were measured and placed into a glass beaker, followed by the addition of 40 mL of flotation fluid (magnesium sulfate). The mixture was continuously stirred using a glass rod, and the dissolved suspension was strained through a tea strainer into another beaker. The suspension was then transferred to a test tube until a meniscus formed at the top of the tube, and a cover slip was gently placed over the meniscus, allowing it to stand for 20 min . The coverslip was removed and placed gently on a clean glass slide and examined under the microscope at low power magnification (10x) . Section title: 2.5.3. Identification of Strongyle at the Genus Level (Larvascopy) Educational score: 4.189375400543213 Domain: biomedical Document type: Study Language: en It is important to recognize that the eggs of all equine strongyles are nearly identical and cannot be distinguished based on egg morphology alone, whether at the subfamily, genus, or species level. To achieve differentiation, the eggs can be cultured to hatch and develop to the L3 stage. This process involves a coproculture (fecal culture), followed by the Baermann technique and microscopic larval identification . The modified Baermann technique was employed to isolate the third larval stage of nematode parasites from fecal cultures. First, a pooled fecal sample was collected from naturally infected horses that tested positive. The fecal culture of eggs to third-stage larvae was then carried out to differentiate the genus of equine strongyles. Ten g of fecal samples containing strongyle-type eggs were pooled, finely ground using a mortar and pestle, and cultured in a Petri dish with a small amount of water to moisten the sample. The samples were incubated at 27°C for 7 days and mixed periodically. The larvae were then recovered using the modified Baermann technique . After collection, the third-stage larvae were mounted on slides, killed with Lugol's iodine, and identified under a microscope to the genus level based on morphological characteristics as described by . Section title: 2.6. Data Management and Analysis Educational score: 3.85209584236145 Domain: biomedical Document type: Study Language: en The data collected from the study were coded and entered in a Microsoft Excel spreadsheet 2007, and the statistical analysis was performed using SPSS Version 16 software packages. The chi-square test was used to assess the difference in the prevalence of strongyles nematode parasites among different variables such as equine species, sex, age, body condition, and origin. The percentage was used to calculate the prevalence rate of strongyles parasites. In all cases, 95% confidence interval (CI) and p < 0.05 were considered for a statistically significant difference. In the current study, the data distribution was a non-normal distribution. Section title: 3.1.1. Overall Prevalence Educational score: 3.989945411682129 Domain: biomedical Document type: Study Language: en Based on the coprological analysis (egg morphology) (Supporting Information S1 ), the overall prevalence of strongyles nematode parasites of equine in the study area was found to be 56.6% (206/364) with a prevalence of 54.7% and 65.2% in horses and donkeys, respectively ( Table 1 ). Section title: 3.1.2. Risk Factors Analysis Educational score: 4.016171455383301 Domain: biomedical Document type: Study Language: en The analysis highlights significant associations between risk factors such as sex ( χ 2 = 16.10 and p ≤ 0.001), age ( χ 2 = 14.78 and p ≤ 0.001), and body condition ( χ 2 = 8.60 and p = 0.013) with the prevalence of stronglyles infection in horse. However, prevalence, while origin shows no significant variation ( χ 2 = 3.32 and p = 0.506) in ( Table 2 ). Section title: 3.1.2. Risk Factors Analysis Educational score: 4.131409168243408 Domain: biomedical Document type: Study Language: en The chi-square analysis revealed that there was no significant difference in the occurrence of strongyles parasitic infection among the equine species between horse and donkey ( χ 2 = 2.404 and p = 0.121) ( Table 3 ). Regarding body condition of the equines, the highest relative prevalence was observed in poor (64%) than those in medium (54%) and good (50%). Prevalence in female (70.7%) animals was higher than in male (52%). The prevalence of strangles nematode parasites according to age group was 59.3% and 42.1% in adult and young equines, respectively. The prevalence of strongyles parasite was insignificantly varied between sexes ( χ 2 = 2.644 and p = 0.266) and age of the equines ( χ 2 = 0.609 and p = 0.435) but it was significantly varied among body conditions ( χ 2 = 76.536 and p ≤ 0.001) ( Table 3 ). Section title: 3.1.2. Risk Factors Analysis Educational score: 3.7811758518218994 Domain: biomedical Document type: Study Language: en Among the five origins or sites study areas, the highest prevalence in horses and donkeys was revealed from Kality followed by Dalota, Bambogaya, Dambi, and G/Gorba, with the prevalence of 22%, 19.8%, 19.8%, 19.2%, and 19.2%, respectively. The study showed no statistically significant difference ( χ 2 = 3.205 and p = 0.524) between the study area and the prevalence of gastrointestinal parasites in both horses and mules . Section title: 3.2. Results of the Identified Genus of Equine Strongyles (Larascopy) Educational score: 4.077446460723877 Domain: biomedical Document type: Study Language: en Larvascopy examination analysis revealed that the two genera of strongyles were identified by the morphological characteristics of third-stage larvae stages. These two important identified genera were Cyathostomum and Strongylus . Cyathostomum spp. and were characterized by short, small, and thin larvae with a thin and long tail, and Strongylus was long, large, and broad larvae with a short esophagus and well-defined gut cells with a long tail as shown in Supporting Information S2 . Section title: 4. Discussion Educational score: 4.055983543395996 Domain: biomedical Document type: Study Language: en The coprological examination done for the current study using floatation techniques revealed an overall prevalence of strongyle nematode parasites in and around Bishoftu town to be 56.6%. This finding was closely aligned with previous reports by , who reported a prevalence of 47.4% in horses and donkeys in and around Kombolcha town, Ethiopia. The current study finding's prevalence was lower than the reports of , who documented prevalence rates of 64.61%, 100%, 99%, and 100% for Menz keya gerbil district, East Shewa, Adaa, and Akaki of East Shewa, respectively. The variation in the prevalence reported might be due to variations in sample size and sampling time as seasonality affects the occurrence of the parasites, feeding practice, and deworming habits . Section title: 4. Discussion Educational score: 4.0679779052734375 Domain: biomedical Document type: Study Language: en In the current finding, the prevalence of 65.2% and 54.7% were recorded in donkeys and horses, respectively, the difference being statically nonsignificant ( p > 0.05). The present study result is relatively in line with the works of , who reported a prevalence of 70.8% in donkeys and 58.5% in horses from South Wollo. This finding was higher than the reports of , who reported a prevalence of 44.55% in donkeys and 48.2% in horses. However, the current study's results were lower than the reports of , which reported a prevalence of 87.8% in donkeys and 66.7% in horses from Gondar, the reports of , which revealed 100% and 99% prevalence in donkeys and horses, respectively, from East Shewa-Adaa, and another study that reported a higher prevalence of 99.15% in Sudan . The higher prevalence in donkeys might be attributed to differences in feeding and deworming activities, while the lower prevalence in horses could be due to their predominantly being cart horses in the study area, which are less exposed and sometimes completely restricted from pasture and grazing. Furthermore, differences in prevalence in different areas might be attributed to variations in sampling areas, feeding systems, and accessibility to deworming and health services . Section title: 4. Discussion Educational score: 4.118624687194824 Domain: biomedical Document type: Study Language: en Regarding the body condition score of equines, the prevalence of strongyle parasites was 64%, 54%, and 50% in poor, medium, and good body-conditioned equines, respectively, and there was a statistically significant association ( p < 0.05) between the existence of strongyle infection and the body condition score of equines. This finding is in agreement with previous reports by , who reported a statistically significant association ( p < 0.05) between the occurrence of strongyle infection and the body condition score of equines. However, there was no significant difference in the prevalence of strongyle parasites in the equines from different origins of the study area. This is similar to the findings of . This may be due to the similarity in the agroecology of the study areas, the epidemiology of the parasites, and the management systems used for the equine species. In this study, there was no significant difference ( p > 0.05) observed in the prevalence of the parasites between young and adult equines. The highest prevalence of strongyle parasite infestation was observed in adults (53%) compared with young equines (3.6%). It may be attributed to the declining body condition and immunity of adult equines compared with young ones, as older equines are more frequently exposed to strongyle parasites due to extensive work overload and undernourished conditions . Section title: 4. Discussion Educational score: 4.167634010314941 Domain: biomedical Document type: Study Language: en In the present study, the equine strongyle parasites genera Srongylus and Cyathostomum were identified. These findings were conducted based on characteristics of strongyle nematode larva morphology similar to other studies conducted worldwide as well as in Ethiopia. This finding supports previous reports of in Cambridge , in Brazil , in the United States , and in Ethiopia. The current study revealed that equine strongyle infections are highly prevalent in the study area, indicating a significant health concern for equines. This highlights a critical gap in the control and prevention of parasitic diseases, suggesting that they are often overlooked or inadequately addressed. To reduce the prevalence of strongyle infections and improve the health and productivity of equines, it is essential to implement a strategic, programmatic approach to deworming. Moreover, this study has certain limitations, including the lack of an in-depth assessment of deworming practices, determination of infection levels, and evaluation of the efficacy of anthelmintic treatments. As such, it is recommended that future research builds upon this study as a baseline, conducting more comprehensive investigations to provide a more thorough, significant, and valuable contribution to equine owners, veterinarians, policymakers, and government agencies. These efforts will be crucial for developing effective interventions to control strongyle infections and improve overall equine health. Section title: 5. Conclusion and Recommendations Educational score: 3.9251530170440674 Domain: biomedical Document type: Study Language: en The current research underscores the vital role of equines as a means of transportation in rural and semiurban areas. However, strongyle parasites pose a significant threat to equine species and have a considerable economic impact. Equines often graze freely in pastures, exposing them to a high risk of parasitic diseases. Parasitized equines exhibit poor immunity and health status, leading to decreased working performance. Therefore, as a recommendation to control the burden of parasites, regular and strategic deworming programs using effective anthelmintics should be implemented consistently. Moreover, to obtain a clearer epidemiological picture, molecular characterization of parasites and assessment of anthelmintic resistance profiles should be conducted. | Other | other | en | 0.999996 |
PMC11699985 | Section title: 1. Introduction Educational score: 4.0828118324279785 Domain: biomedical Document type: Review Language: en Sudden cardiac death (SCD) is an unexpected lethal cessation of cardiac function that accounts for approximately half of the 17 million cardiovascular-related annual deceases that are estimated worldwide . One of the significant causes of SCD is dilated cardiomyopathy (DCM), which is the leading global indication for heart transplantation (HT) . DCM is defined as a condition in which the ventricles of the heart enlarge and the myocardium walls become thinner, compromising cardiac output and making it difficult to oxygenate the brain and body cells . The pathological manifestations of DCM can vary from mild symptoms of heart failure (HF) in adults to the onset of SCD at an early age . The prevalence of DCM in HF has been reported of approximately 1:250–400 and 5–7 new cases per 100,000 persons per year , although an accurate update estimation is difficult to assess. Section title: 1. Introduction Educational score: 3.9870617389678955 Domain: biomedical Document type: Review Language: en Around 40% of familial DCM cases can be clarified by genetic variants, and most of them were inherited as an autosomal-dominant disease in families . Diagnosis of SCD syndromes may be the most pressing challenge in cardiology due to its complex physiopathology, unknown molecular mechanisms and inadequacy of current genomic tools. Moreover, this challenge is compounded by difficulty in managing cardiac tissues. However, researchers continue to find new gene variants in many malignant cardiac cases such as DCM [ 6 – 8 ], that could be the cause of its etiology, which highlight the importance of genetic testing in certain individuals to implement an adequate preventive and personalized treatment plan if needed. Section title: 1. Introduction Educational score: 4.162952899932861 Domain: biomedical Document type: Study Language: en The current study describes the clinical features of a severe DCM in a 1-year-old infant and links this pathogenesis with the presence of two rare and presumptive deleterious genetic variants that change well evolutionary conserved regions in the ACTC1 and TTN structural genes. Additionally, the ACTC1 mutation appears to have a de novo origin, which highly supports its probable pathogenicity. Overall, the identification of these mutations broadens the base of pathogenic variants linked to SCD. Section title: 2.1. Subject and Methods Educational score: 2.6632182598114014 Domain: biomedical Document type: Study Language: en This study was approved by the Ethics, Biosafety, and Research Committees of the Children's Hospital of Mexico Federico Gómez, the Clinical Laboratory Evaluation Program from the New York State Department of Health, and by Main Line Health Institutional Review Board located at Lankenau Medical Center. Following the explanation of the study, the parents of the minor agreed to include him in the genetic screening, as well as themselves and the brother of the proband. The patient's clinical file, the laboratory database, and all the clinical images available in the hospital system were reviewed. The clinical information available includes familial and personal clinical history, all the clinical and surgical notes, physical examination notes, electrocardiogram results, thoracic roentgenography results, Doppler echocardiography results, along with the treatment indications and death certificate. Section title: 2.2. Genetic Study Educational score: 4.168163299560547 Domain: biomedical Document type: Study Language: en Genomic DNA was extracted from peripheral blood lymphocytes using a commercial kit (Gentra System, Puregene, Valencia, California, the United States of America). The Supporting Tables S2 and S3 describe the library of 48 structure-related and 84 electrical-related genes, respectively, that were amplified using the Ion AmpliSeq Library Preparation protocol (Thermo Fisher Scientific, Waltham, Massachusetts, the United States of America). Clonal fragments were loaded into an Ion 318 Chip Kit v2 BC with the Ion Chef system and analyzed by NGS using the Ion Torrent Personal Genome Machine. After variant calling from the Ion Reporter software, genetic variants were further selected for identification of candidate deleterious mutations, selecting only those with MAF under 0.03, by comparison to the reference genomic DNA sequences from the 1000 genomes' project , as well as other previously described criteria , which include the score evaluation of each mutation according to the in silico prediction tests PolyPhen-2, PROVEAN, and SIFT. Section title: 2.3. Variant Validation Educational score: 4.192946434020996 Domain: biomedical Document type: Study Language: en Genomic DNA (80–120 ng) was purified postamplification using the ExoSAP-IT kit (Applied Biosystems, Foster City, California, the United States of America), and sanger sequencing was performed with dideoxynucleotides using 0.8 μL of BigDye terminator v3.1 (Thermo Fisher Scientific, Waltham, Massachusetts, the United States of America) in the appropriate buffer, 0.5 μ M of each primer in separated reactions, 1 μL of PCR product, and 4 μL of HPLC grade water. Final PCR products were purified using Sephadex columns G-50 (Sigma-Aldrich, St. Louis, Missouri, the United States of America), and sequencing reactions were performed in an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, California, the United States of America). Electropherogram analysis of both DNA strands was achieved using sequencing analysis v.5.3 software (Applied Biosystems, Foster City, California, the United States of America). Section title: 2.4. Computational 3D Modeling Simulation and Protein Grade of Conservation Educational score: 4.187014579772949 Domain: biomedical Document type: Study Language: en Protein structural homology of wild-type cardiac actin and its found variant were performed using the online workspace of SWISS-MODEL . To generate the 3D protein structure, files from the protein data bank (PDB) were visualized in PyMOL 2.4.0 software (DeLano Scientific) , and the corresponding analysis of protein features was conducted. In parallel, the structure of wild-type cardiac titin and its variant were analyzed following the method of Cukier . We used the probability of occupancy for each pair of amino acids in dihedral angle space to generate an ensemble of conformations of the peptide encompassing five residues flanking each side of the mutant. Each eleven-residue peptide had 1300 allowed conformations generated after removing the conformations with high-energy overlap of atoms that violate the Ramachandran map. The range of conformations and the end-to-end distance for each peptide was analyzed. Section title: 2.4. Computational 3D Modeling Simulation and Protein Grade of Conservation Educational score: 3.9156274795532227 Domain: biomedical Document type: Study Language: en To assess the evolutionary importance of the amino acid change provoked by confirmed mutations, a comparison across different species was used to determine the amino acidic grade of conservation, wielding multiple protein alignment tools with the interest protein sequence from different species. Section title: 2.5. Heritability of Variants Educational score: 3.263882875442505 Domain: biomedical Document type: Study Language: en After performing Sanger sequencing of the available proband's relatives for the confirmed variants, a pedigree tree was constructed to elucidate the heritability pattern. Section title: 3.1. Clinical Case Educational score: 4.032872676849365 Domain: clinical Document type: Clinical case Language: en A one-year-old Mexican boy who presented severe HF and DCM was properly diagnosed based on the current classification . His parents and brother were apparently healthy individuals with no reported clinical history of SCD syndromes nor cardiac-related deaths within their family. The parents reported that the symptoms in the patient began during the first months of life but had rapidly intensified in the following weeks. The patient was diaphoretic, tachycardic, tachypneic, and hypotensive due to inadequate cardiac output. Palpation of the precordium demonstrated an apical impulse displaced downward and laterally a systolic murmur Grade III/VI and an S3-S4 gallop rhythm were audible in all the cardiac auscultatory focus. The twelve-lead electrocardiogram showed sinus tachycardia of 159 bpm, left increased precordial lead voltages secondary to ventricular dilation, an incomplete bundle branch left ventricle (BBLV) was apparent and abnormalities in the repolarization were noticed with inverted T waves in V3-V6 leads. Section title: 3.1. Clinical Case Educational score: 3.2689664363861084 Domain: biomedical Document type: Clinical case Language: en Chest roentgenography demonstrated marked cardiomegaly. The cardiothymic silhouette was enlarged mainly because of left atrial and ventricular dilation. Pulmonary venous congestion and pulmonary edema were evident as well. Section title: 3.1. Clinical Case Educational score: 4.21231746673584 Domain: biomedical Document type: Study Language: en Echocardiography confirmed the diagnosis of DCM with the left auricle and LV severely enlarged. End-diastolic and end-systolic LV diameters were increased (diameter at the end of LV diastole of 34 mm; z score +5.5, LV diameter at the end of systole of 31 mm; z score +7.1). The interventricular septum was bulging into the right ventricle, and the interventricular septum and free wall of the LV appeared diminished. Systolic function was decreased, and LV ejection fraction (LVEF) was calculated at 7%. The origin of both coronary arteries presented normally. Section title: 3.1. Clinical Case Educational score: 3.6245431900024414 Domain: clinical Document type: Clinical case Language: en The initial treatment of the patient was based on intravenous diuretics with combined inotropic and vasodilator support (levosimendan and milrinone) with incremental titration as the clinical situation dictated. Once the clinical situation improved and the patient was able to eat, the use of oral beta-blocking agent with bisoprolol was also initiated with incremental titration. In addition to optimal pharmacological management for the HF, with the function of the right ventricle still preserved, the patient underwent surgery for a pulmonary artery band as a bridge therapy for HT based on a recent worldwide experience report . Temporary improvement was obtained, and the patient was sent home with ongoing consultations; however, the child died suddenly at home 6 months later waiting for HT. Section title: 3.2. Genetic, Bioinformatic, and Conservative Analysis Identified Two Candidate Pathogenic Variants Related to DCM Educational score: 4.197793960571289 Domain: biomedical Document type: Study Language: en The NGS analysis in the patient suffering DCM yielded 243 Mb of read bases from which 95.61% obtained at least a Q20 score. 99.1% of the reads were on target, and 97.99% of them read at least 20x more with mean sequencing depth of 593 reads per amplicon. A summary of the coverage analysis data of the NGS is described in the Supporting Table S1 . Alignment and variant calling showed a total of 343 variants, derived only from the 48 structure-related gene panel. After the applied internal filtering criteria, two variant genes were discovered with a heterozygous single-nucleotide variation (SNV) each, whose minor allele frequency (MAF) was cero in the control cohort. Thus, we confirmed the coexpression of a TTN and ACTC1 mutants (Figures 4(a) and 4(b) ). Section title: 3.2. Genetic, Bioinformatic, and Conservative Analysis Identified Two Candidate Pathogenic Variants Related to DCM Educational score: 4.196275234222412 Domain: biomedical Document type: Study Language: en In silico predictors suggested the mutations to be deleterious to a certain extent ( Table 1 ). All of them classified the ACTC1 variant as disease-causing or damaging, while the TTN mutation was denoted by PolyPhen-2 as possibly damaging. Additionally, the conservative analysis revealed that both mutated amino-acid residues were situated in well-conserved domains among many orthologous proteins (Figures 4(c) and 4(d) ), suggesting the significance of the affected protein sites throughout evolution. Interestingly, no mutation in the 84 electrical-gene panel related to cardiac anomalies was found. Section title: 3.3. Hereditary Analysis Revealed a Segregated TTN Mutation and a De Novo ACTC1 Mutation Educational score: 4.109200954437256 Domain: biomedical Document type: Study Language: en Sanger test validation of the variants and further genealogy analysis conveyed that the TTN variant was transmitted from the father, which was also acquired by the proband's sibling. However, to our surprise, the ACTC1 variant was not present in the parents nor in the brother ( Figure 4(e) ). As the frequency of spontaneous mutations in human DNA is relatively low due to the DNA polymerase exonuclease activity, we decided to avoid false-positive results by performing Sanger sequencing of the same loci again in the four individuals, which resulted in no sequence changes, validating the presence of both mutations. Section title: 3.4. Computational 3D Modeling Analysis Supports Structural Changes in Actin p.Ala222Thr and Titin p.Glu11084Lys Variants Educational score: 4.25339412689209 Domain: biomedical Document type: Study Language: en Quality parameters declared a reliable template for protein 3D modeling (GMQE = 0.99; identity = 98.67%), and the structure of the monomeric actin was successfully constructed. Among several other observed structural shifts, distance changes between Ala222Thr neighboring residues in subdomain four were observed, as well as hydrogen bond rearrangements including a new steric contact between the mutant substitute 222Thr and 224D. The most relevant changes are depicted and described in Figure 5 . Section title: 3.4. Computational 3D Modeling Analysis Supports Structural Changes in Actin p.Ala222Thr and Titin p.Glu11084Lys Variants Educational score: 4.270552635192871 Domain: biomedical Document type: Study Language: en In the modeling of the region around the amino acid change in titin, this E11084K mutation appears to hinder the formation of compact states, leading to a larger average end-to-end distance for the peptide , which may impact the spatial distribution in the sarcomere. Section title: 4. Discussion Educational score: 4.0807719230651855 Domain: biomedical Document type: Study Language: en The presence of genetic deleterious variants expressed in cardiac tissues can lead to structural heart defects. Congenital heart defects compel people susceptible to an unexpected stoppage of the heart by triggering arrhythmias, the principal cause of SCD . These SCD syndromes are mainly explained by the alteration of two groups of cardiac proteins: (1) those that principally constitute the scaffolding of cardiomyocytes and give them support and structure and (2) those related to the action potential and electrical transduction. Section title: 4. Discussion Educational score: 3.9496381282806396 Domain: biomedical Document type: Study Language: en In a previous study carried out at the cardiomyopathies and arrhythmias research laboratory of The Children's Hospital of Mexico Federico Gómez, DCM was the most frequent variety of cardiomyopathy (62.5%) and the most common disease of all the hereditary SCD syndromes (including cardiomyopathies and hereditary electrical diseases; 42.37%) . The infant presented in this study corresponds to the case with the most malignant presentation (early diagnosis and death) within this cohort of Mexican children from 0 to 18 years with hereditary SCD syndromes, which give support to the clinical significance of the findings described here. Section title: 4. Discussion Educational score: 4.011852741241455 Domain: biomedical Document type: Study Language: en To date, variants in over 50 genes have been linked to familial DCM , unfortunately, many of these cases remain without a clear molecular etiology. Next generation sequencing studies performed by our research team showed that the infant case described here did not possess any genetic variations within a selected group of 87 ion channel genes related to cardiac anomalies (unpublished data). Nonetheless, we found two interesting germline variants within the structural cardiac genes ACTC1 and TTN , which aid in facilitating the identification of possible DCM-causing genes. Section title: 4. Discussion Educational score: 4.234701156616211 Domain: biomedical Document type: Study Language: en The quality of the data generated though NGS is an important parameter to be considered when used to correlate with clinical diagnosis because it is a mark of the experiment's reliability, also giving an insight about the quality of the technical performance of the experiment. Our results pertain to the recommended sequencing depth (120x) for the diagnosis of genetic variants with clinical significance and the detection of heterozygous SNV (35x) . Moreover, the obtained on-target reads percentage and mapped reads surpassed the threshold of a good-quality library (80% and 100,000, respectively) . Other studies searching for new genetic variants linked to cardiomyopathies by NGS showed similar or fewer quality data points in terms of the abovementioned parameters [ 8 , 9 , 22 – 24 ]. The first genetic mutation related to DCM was identified in ACTC1 (gene encoding for cardiac alpha actin protein) which was segregated in an autosomal-dominant pattern . Since then, several ACTC1 mutations have been identified from which up to 20% can cause DCM , some of them even overlap with different types of cardiac hereditary diseases [ 26 – 30 ]. Section title: 4. Discussion Educational score: 4.2717742919921875 Domain: biomedical Document type: Study Language: en Cardiac actin is a fundamental protein located in the sarcomere of cardiomyocytes, and it is crucial for myocardium contraction, force generation, and blood pumping. For this reason, it was very interesting to find that the ACTC1 mutation in the subject of study was not segregated by either of the parents, denoting its de novo origin, a very rare type of “spontaneous” acquisition. To the best of our knowledge, there is no peer-reviewed data regarding a phenotypic association of c.664G > A ACTC1 /p.Ala222Thr variant in the available literature or in databases such as PubMed, and it is absent in control cohorts as 1000 genomes, exome sequencing project, the exome aggregation consortium (ExAC), or the genome aggregation database (gnomAD). Notwithstanding, it is of important notice that there are five previous reports of this variant from different submitters in ClinVar database, two of them being classified as likely pathogenic in DCM cases also with a de novo origin. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines and the features we found, our research team also classified the c.664G > A ACTC1 variant as likely pathogenic in the present case (categories PM2, PM6, PP3, and PP5 are met). As nonrelated cases, these findings could remark the potential pathogenicity of the variant. Section title: 4. Discussion Educational score: 4.124600887298584 Domain: biomedical Document type: Study Language: en The origin of spontaneous SNV can happen during embryonic development through different mechanisms, and it is estimated that up to 74 SNV can be found in a single germinal genome . The detection of de novo variations is frequently initiated by genetic research, as there are DNA changes that do not come from family inheritance. These de novo mutations are generally very important in the etiology of diseases because they have been occurring outside of the usual parameters of natural selection, so they are more likely to cause undesirable functional consequences . This strongly suggests that ACTC1 mutation may play a pathological role in the studied DCM case. Section title: 4. Discussion Educational score: 4.5851640701293945 Domain: biomedical Document type: Study Language: en The amino acid change in cardiac actin occurred in a highly conserved region of actin in a variety of species ( Figure 4(c) ) which constitutes a hallmark that connote its possible pathogenic effect. This actin protein variant has a change from alanine to threonine in the 222nd position, in relation to the human genome reference GRCh37.p7. According to X-ray fiber diffraction studies, this position is located in an intra-subdomain four region ( Figure 4(f) ). Mutations in Subdomains 1 and 4 of actin have been recognized to likely affect protein-protein interactions . Subdomain 4 of actin maintains electrostatic interactions with proteins such as tropomyosin . Tropomyosin is a key protein in cardiomyocyte contraction because it acts together with troponin to sterically hamper the union of myosin heads when Ca ++ levels are low . On the other hand, it is scientifically recognized that hydrophilic amino acids of a given polypeptide chain are facing the surface of the molecule in protein folding, while the hydrophobic amino acids are located interiorly. The p.Ala222Thr actin variant provokes exactly the opposite, which could explain our in silico findings where structure and hydrogen bond arrangements change intra-subdomain four. That perhaps leads to defects of actin polymerization in vivo, hampering the formation of filamentous actin and in turn, and the contraction of cardiomyocytes as it was demonstrated for the p.Gly247Asp actin variant, located also in subdomain four . Either conformational or affinity changes between tropomyosin and actin or defects of actin polymerization would lead to cardiac remodeling such as DCM. However, further specific functional studies are needed to corroborate this hypothesis. Section title: 4. Discussion Educational score: 4.485856056213379 Domain: biomedical Document type: Study Language: en The index case described here also had a cosegregation of a heterozygous mutation in the TTN gene that encodes titin protein. For a matter of fact, its crucial role and its nucleotide length make TTN the major affected gene in DCM cases . Titin is located in the sarcomeres, between actin and myosin filaments, and it is the longest protein in humans. Its main purpose is to provide cell elasticity and structural network organization, preventing damage caused by cardiomyocyte resistance to contractions and providing passive tension to striated muscle. This ability is mainly attributed to an elastic structure enriched of proline, glutamate, valine, and lysine (PEVK), which constitutes an entropic biological spring and mimics a shock absorber . The conformations of the PEVK region appear to be sensitive to both pH and calcium concentration and, interestingly, the amino acid change in the titin variant that our research group found was a substitution of acidic and negatively charged glutamic acid by an alkaline and positive charged lysine in Position 11,084, which is also a conserved residue among species ( Figure 4(d) ). Specifically, this amino acid position is within the distal I band according to the spatial distribution of TTN in the sarcomere ( Figure 4(f) ) region where truncating variants have already been associated with DCM . Section title: 4. Discussion Educational score: 4.132242679595947 Domain: biomedical Document type: Study Language: en However, despite the efforts of the scientific community to assess the real implications of missense TTN variants, the majority of them are still considered ambiguous because it is hard (and sometimes contradictory) to determine their pathogenicity with current approaches, leaving them without both molecular and a precise clinical significance ; functional studies are also difficult to carry out due in part to the nature of titin, which is considered the largest coded protein in humans. In the present study, the c.33250G > A TTN allele is considered as a variant of uncertain significance (VUS) according to the ACMG guidelines because of the contradictory features we found when analyzing the data in the giving clinical and scientific context. Section title: 4. Discussion Educational score: 4.488372325897217 Domain: biomedical Document type: Study Language: en The mechanism by which the E11084K mutant contributes to cardiac disease may plausibly be one or more of several related effects. Charge pairs direct formation of compact configurations of the titin chain upon release of tension , so the insertion of a positive charge farther to the N -terminus of the PEVK region may disrupt the formation and stability of the compact or folded states that normally contribute to avoid mechanical damage. Disruption of the ability of titin to extend and collapse may inhibit its mechanosensory mechanism. On the other hand, it is possible that the poly- E motifs in titin interacts with F -actin , affecting the interactions that mediate the mechanical memory of the sarcomere. Section title: 4. Discussion Educational score: 4.215702533721924 Domain: biomedical Document type: Study Language: en Previous reports of this TTN variant are currently found in ClinVar database in the clinical context of DCM and other myopathies, six of them being classified as VUS, and two as benign. On the other hand, some observations explain the pathological implications that some missense TTN variants can represent in some individuals, as the one that demonstrated to cause partial protein unfolding and domain destabilization . Also, an in vitro study revealed that induced pluripotent stem cell cardiomyocytes carrying a missense mutation within the sarcomere Z/I junction portion in titin developed a DCM-like phenotype . Nonetheless, it is of noticeable importance that the missense c.33250G > A variant addressed in the present study was observed to be inherited by the father of the study infant subject, which was also acquired by his brother but who did not present any cardiac symptomatology at the time of sampling, suggesting a minor cardiac pathogenic effect when expressed alone. Section title: 4. Discussion Educational score: 4.330153465270996 Domain: biomedical Document type: Study Language: en Due to the suggestive pathogenic elements in all the previous examined data, one hypothesis to be considered in this work regarding the onset of DCM in our patient is an autosomal dominant behavior of ACTC1 mutation. Notwithstanding, due to the high complexity of cardiomyopathies, it is also suggestive that the cause of the malignant condition reported here may be derived from a polygenic synergistic effect of both ACTC1 and TTN variants, where this double hit is necessary to impair the phenotypic manifestation of an early and severe DCM. This type of polygenic effect has been also described and demonstrated in vitro and in vivo in another similar case of DCM , where two separate variants in VLN and TPM1 could not be associated with the DCM phenotype when evaluated separately but together, combined with disease-related stress factors. Section title: 4. Discussion Educational score: 4.089855194091797 Domain: biomedical Document type: Study Language: en A limitation in our study is that we may be missing other factors that can prompt DCM onset, such as environmental challenges, variants with epigenetic significance, genes in other networks that either directly or indirectly interact with the sequenced structural genes, or rare somatic mutations. Altogether, as many other VUS, it is necessary to go further with this study and to carry out functional experiments to determine whether these two rare variants can trigger DCM and the possible mechanisms involved. Finally, by comparing our results with the available literature, we can conclude there is a diverse genetic background in DCM patients. Our report of the germline variants in TTN and ACTC1 connote the complexity of the possible molecular causes of SCD syndromes and affirm that there are still pathogenic genetic factors we could be facing for the first time, which challenges the prescription of the more appropriate and beneficial treatment. | Review | biomedical | en | 0.999997 |
PMC11699986 | Section title: 1. Introduction Educational score: 4.045372009277344 Domain: biomedical Document type: Review Language: en Oral cancer (OC) ranks as the 16th most prevalent malignancy impacting the lips and oral cavity, encompassing the oropharynx, hypopharynx, and larynx . It ranks as the 15th most prevalent cause of mortality worldwide, with significant heterogeneity based on age, gender, race, ethnicity, and socioeconomic status . Despite the existence of several treatment options, the overall five-year survival rate for OC remains approximately 50% . OC, however rare in the Western world, is prevalent in high-risk nations . In 2017, South Asia recorded the highest incidence, death, and national burden of OC . OC ranks third among all malignancies in India and accounts for about 40% of total cancer-related mortality. Approximately 70,000 new cases and more than 48,000 deaths connected with OC occur each year. OC is quite prevalent in India, ranking as the second most common malignancy among males and the fourth among women. The prevalence escalates with age and is more prevalent in men . Section title: 1. Introduction Educational score: 3.9396986961364746 Domain: biomedical Document type: Study Language: en Oral squamous cell carcinoma (OSCC) was identified as the sixth most prevalent variety in 2016, accounting for 90% of head and neck malignancies . While OSCC can originate from anatomical sites in the head and neck, it predominantly manifests in the oral cavity . The etiology of cancer is mostly linked to detrimental behaviors, including betel nut consumption, smokeless tobacco use, and alcohol abuse, as well as factors such as hereditary predisposition and excessive sun exposure . The prevalence of OC among the younger demographic has lately increased due to filthy sexual practices associated with specific DNA viruses, including human papillomavirus and herpes simplex virus type I . Furthermore, due to the absence of a compulsion for cancer registration in India, the actual incidence and mortality rates are likely to be underestimated . Section title: 1. Introduction Educational score: 4.064037322998047 Domain: biomedical Document type: Study Language: en Recognizing the predisposing factors is crucial for facilitating early diagnosis and preventing cancer progression and subsequent mortality. OC can be discovered early by methods such as screening high-risk populations, opportunistic screening in primary healthcare settings, and the prompt identification and reporting of signs and symptoms by individuals and healthcare professionals to confirm a diagnosis . This strategy is highly beneficial, but a lack of information and social stigma may hinder patients from seeking care at an early stage. Limited awareness correlates with reduced survival rates in patients with OCs . Consequently, heightened knowledge of the disease and improved access to healthcare services will facilitate early diagnosis, mitigate disease development, and enhance prevention efforts. Encouraging regular dental exams is recommended to enable the opportunistic evaluation of potential indications and symptoms. Furthermore, this approach would need dentists and other primary healthcare workers to be well trained to recognize the early signs of OC. This study aimed to assess the knowledge and attitudes of dental and medical students regarding OC and to enhance their understanding of the associated hazards. Section title: 2. Materials and Methods Educational score: 3.773790121078491 Domain: biomedical Document type: Study Language: en A cross-sectional study was initiated on obtaining approval from the Institutional Ethics Committee at Manipal College of Dental Sciences, MAHE, Mangalore, bearing reference number 16117, from January 2022 to June 2022. A convenience sampling method was used to enroll the undergraduate students of II-year medical ( n = 144, Kasturba Medical College, Mangalore) and dental ( n = 112, Manipal College of Dental Sciences, Mangalore) fraternities. The rationale for selection of II-year participants was to evaluate their awareness prior to the exposure to theoretical and clinical knowledge and evaluation of cancer and related features. The participants were thoroughly instructed about the study objectives before handing out the printed questionnaire. Permissions to administer the questionnaire were obtained from the Heads of the respective institutions. Before administering the printed questionnaire to individual participants, written consent was documented from each participant to ensure their voluntary and active participation in the study. The participants were well-informed that their personal information would be held strictly confidential. Section title: 2. Materials and Methods Educational score: 2.561436176300049 Domain: biomedical Document type: Study Language: en The questionnaire employed was adapted from a published study by Carter and Ogden, to which permission was sought from the authors before use . The questionnaire was revalidated for content, scope, and appropriateness by three experts before being utilized in our study. It was agreed to use the questionnaire in its original format as prescribed by Carter and Ogden. The questionnaire collected confidential personal information such as name, gender, and institutional affiliation details. The questionnaire had 12 questions, with ten close-ended questions and two open-ended questions, in English. The data obtained were compiled and analyzed using SPSS (IBM SPSS 20.0, Chicago, USA) software. Gender and streams being categorical variables, the association of the students' awareness of gender and streams was correlated with the components of the questionnaire using the chi-square test. The data for each question were depicted using tests of proportion. The global significance was set at p < 0.05. Section title: 3. Results Educational score: 2.0880908966064453 Domain: biomedical Document type: Study Language: en Two hundred fifty-six participants were enrolled (144 medical and 112 dental) for the study. All participants had effectively filled out the questionnaire and returned the same within the allotted timeframe. In the medical sample, there were about 84 females and 60 males, while the dental sample had around 66 females and 46 males. All the participants involved in the study were aged between 18 and 21 years. The questionnaire data were analyzed and are reported in Table 1 . Section title: 3.1. Evaluation of Oral Mucosa Educational score: 4.044255256652832 Domain: biomedical Document type: Study Language: en The routine evaluation of the oral mucosa was significantly more frequent among medical graduates, with 50.40% reporting this practice compared to only 30.40% of dental graduates ( p =0.002). Some questions showed borderline significance, such as whether they screen the oral mucosa if patients are in high-risk categories ( χ 2 = 3.107, p =0.078), or the presence of ulcers ( χ 2 = 3.22, p =0.073); these results suggest trends that may warrant further investigation. Fewer dental students (44.40%) than medical students (58.80%) reported screening high-risk patients when they do not routinely examine the oral mucosa, and dental students related ulcer to be more related to malignancy than the medical students. Section title: 3.2. Awareness and Enquiry of Habits Educational score: 2.9327080249786377 Domain: biomedical Document type: Study Language: en There was no difference in awareness regarding tobacco chewing between the two groups. However, smoking habits were significantly more often included in history-taking by dental graduates (69.9%) than medical graduates (50%). Similarly, dental graduates were more likely to enquire about poor oral hygiene (23.3%) compared to medical graduates (8.2%) ( p =0.006). Section title: 3.3. Counseling on Risk Factors Educational score: 2.7962746620178223 Domain: biomedical Document type: Study Language: en Dental graduates were significantly more likely to advise patients on the risk factors of OC, with 99.1% doing so compared to 88.3% of medical graduates ( p =0.001). Section title: 3.4. Exposure to Oral Lesions Educational score: 2.7891974449157715 Domain: biomedical Document type: Study Language: en Ironically, medical graduates had significantly higher exposure to examining patients with oral lesions (33.6%) compared to dental graduates (9.6%) ( p < 0.001). This could be attributed to the fact that clinical postings start early on for the medical students as compared to dental students. Section title: 3.5. Awareness of Clinical Features of OC Educational score: 3.34269380569458 Domain: biomedical Document type: Study Language: en Both groups felt adequately informed about the clinical appearance of OC. However, dental graduates demonstrated greater awareness of clinical features such as white patches, proliferative growth, cauliflower-like growths, and pedunculated growths, compared to medical graduates. Medical graduates predominantly identified ulcers as a clinical feature but were less aware of other signs of OC. Section title: 3.6. Referral Patterns Educational score: 2.7027947902679443 Domain: biomedical Document type: Study Language: en Dental graduates were more likely to refer patients with suspected OC to oral and maxillofacial surgeons (55.7%) compared to medical graduates (39.8%). Medical graduates, however, were equally likely to refer patients to oral medicine departments (27.6%) as dental graduates (27.4%). Section title: 3.7. Need for Additional Training Educational score: 2.528475046157837 Domain: biomedical Document type: Study Language: en A significantly higher proportion of dental graduates (94.1%) expressed the need for additional teaching on OC compared to medical graduates (82%). Both groups preferred seminars and informational packets as their preferred modes of learning seminars over lectures. Overall gender-related differences were not significant in the present study, and both males and females exhibited equal understanding awareness and understanding related to all the abovementioned parameters. Section title: 4. Discussion Educational score: 4.068484306335449 Domain: biomedical Document type: Study Language: en This study aims to elucidate the behaviors and attitudes of dental and medical students regarding OC, a lethal oral disease, and to enhance knowledge of its related risk factors at an early age. Our study found that medical graduates examined the oral mucosa substantially more than their dentistry and medical student counterparts. This could be attributed to the fact that clinical exposure for medical students start from the II year itself. Approximately 49.6% of medical graduates and 69.6% of dentistry graduates do not assess the oral mucosa. Dental graduates must enhance their thoroughness in examining the oral mucosa and teeth. Early recognition of the significance of teeth and their related structures will enhance their comprehension of deviations from normalcy. Section title: 4. Discussion Educational score: 4.112182140350342 Domain: biomedical Document type: Study Language: en Our study noted that, within high-risk categories, oral cavity screening rose to 58.8% in medical groups and 44.4% in dental groups, compared to 50.4% and 30.4% for routine examinations conducted by both groups, respectively. The data indicate that medical students engage in a clinical program starting in their second year, but dental students are introduced to a clinical environment only in their third year. This may explain the results for parameters 1 and 2, wherein medical students might have assessed patients' oral cavities, a scenario that was improbable for dentistry undergraduates. Our findings are consistent with previously published literature . Dental students should be encouraged to periodically visit a clinical setting to enhance their understanding of normal and pathological conditions of the oral cavity. Furthermore, this exposure can be expanded for spontaneous screening of high-risk patients during routine oral examinations, even at the community level. Section title: 4. Discussion Educational score: 4.135503768920898 Domain: biomedical Document type: Study Language: en When comparing information about the etiology of OC, both medical and dental students demonstrated sufficient understanding of risk factors such as tobacco use and smoking. Medical graduates predominantly screened for chewing tobacco as the most prevalent high-risk category, whereas dental graduates focused more on smoking. Poor oral hygiene prompted more dentists to assess the oral mucosa than medical graduates. Nonetheless, the students had a limited understanding of additional risk factors related to alcohol usage and viral infections . Dental students had a greater enthusiasm for informing their patients about the risk factors associated with OC than their medical counterparts. This is likely due to the fact that a dental graduate's area of specialization is the oral cavity, in contrast to that of medical students. A study indicates that medical students exhibit a deficient attitude toward OC, attributed to little exposure during their educational experience . To foster awareness, broader comprehension must be imparted to the undergraduate students. The majority of dental students had negative responses regarding the opportunity to evaluate patients with oral lesions, in contrast to medical students. This disparity may be attributed to the variations in curricula among the fraternities. Section title: 4. Discussion Educational score: 4.068459987640381 Domain: biomedical Document type: Study Language: en The dentistry group had much greater awareness of the clinical signs and symptoms of OC compared to the medical students. Nonetheless, both medical and dental graduates perceived themselves as sufficiently versed about their clinical presentation. Both student groups concurred that the ulcerative lesions are symptomatic of cancer. Nevertheless, dental graduates exhibited superior awareness of the malignancy of white patches (74.7%), proliferative growth (74.7%), verrucous development (35.6%), and pedunculated growth (27.6%). Nonetheless, literature reports exhibit inconsistencies contingent upon geographical location and several study characteristics . This may indicate enhanced awareness among dental students. The importance of early clinical indicators should be underscored to facilitate prompt diagnosis. Section title: 4. Discussion Educational score: 4.148326873779297 Domain: biomedical Document type: Study Language: en In our study, medical graduates predominantly recommend referring patients to a physician when oral lesions are identified, in contrast to dentistry graduates. Enhancing understanding of the dentist's function in OC detection and prevention within the medical curriculum may elevate knowledge of streamlined referral mechanisms. Our findings indicated that, upon graduation, medical and dental students would preferentially refer patients to specialists in oral and maxillofacial surgery or oral medicine. Nonetheless, 12.2% of the medical graduates will refer patients to ENT specialists. Medical graduates possess greater awareness of ENT, a subject that is undervalued in dentistry, potentially explaining the unfamiliarity observed among dental students. Both groups concurred that the optimal recommendation for an OC patient is to an oral and maxillofacial surgeon. The outcome concurred with the extant literature . A knowledgeable and proficient healthcare team is essential to reduce the disparities between patient screening and referral to a specialist. This knowledge will facilitate an efficient referral system to avert unnecessary diagnostic and preventive delays. Medical and dentistry students similarly lack adequate understanding concerning the prevention and detection of mouth cancer and require additional information on the subject. The dentistry graduates would desire substantially more information about OC compared to the medical graduates. The information pack was the predominant request among medical graduates in contrast to seminars requested by dental graduates. Numerous findings have been documented by numerous authors within the pertinent literature. Section title: 4. Discussion Educational score: 4.063320159912109 Domain: biomedical Document type: Review Language: en Soares et al. concluded that students had a good knowledge of the etiology of OC and were alert in their examinations to the possibility of detecting malignant lesions. However, they noted that the clinical features of the lesion were not sufficiently clear to the students. They suggested that knowledge of OC, in particular its clinical presentation, needed to be reinforced throughout the undergraduate dental course to enable raising suspicions and making an early diagnosis of lesions . Anushya, Dhanraj, and Keerthi also opined the necessity for an improvement of the teaching program regarding oral examination . Rai et al. and Chan et al. reported general lack of awareness about OC and its associated risk factors among student cohorts (dental and medical) of the Singaporean population and Malaysian population, respectively . They too emphasized upon the need for targeted education and to provide training to improve diagnostic skills for every dental undergraduate. Similar sentiments were echoed in studies conducted by Fotedar et al. , Kumar and Ak, Keser and Pekiner, and Saleem, Mahmoud and Joseph. Given considerable morbidity and effect on quality of life, initiatives to prevent the condition and enhance early detection are essential [ 23 – 26 ]. Nazar, Ariga, and Shyama studied knowledge, attitudes, and practices regarding OC among recently graduated dentists in Kuwait and found that majority of dentists were knowledgeable and aware of the numerous aspects of OC. They also concluded that the training programs in OC education must be prioritized and reinforced, with a particular emphasis on early detection and prevention. They strongly advised that dentists participate in continuing education programs and seminars to increase their understanding of the risk factors and diagnosis of OC . Section title: 4. Discussion Educational score: 3.993347644805908 Domain: biomedical Document type: Study Language: en The findings of the present study indicated a disparity in knowledge and awareness of OC between the two study groups. The current curricular design does not allow dental students to see patients during their second year of training, in contrast to medical students, who begin patient interactions at the start of their second year. Engaging second-year dental students as observers in dental screening camps may address this gap. Medeiros et al. assessed knowledge about OC among dental students and Primary Health Care Dentists in Brazil and concluded the need for proper training to examine and identify signs of OC . Algudaibi et al. studied the difference of knowledge of OC between dental and medical practitioners and found that dental practitioners were found to be more knowledgeable about the high OC risk sites and predisposing factors than medical practitioners . As the adage goes “nipping the evil in the bud,” understanding risk factors along with diagnostic instruments to identify early symptoms can help alleviate the burden of OC. There were certain limitations in the study. A convenience sampling was performed; hence, the entire student community of both the institutes was not subjected to the questionnaire. A larger community was not provided with the questionnaire due to the higher risk of dropouts. Only the Mangalore campus of Manipal Academy of Higher Education was enrolled in the study for strategic access to the geographical location. Section title: 5. Conclusion Educational score: 4.081768989562988 Domain: biomedical Document type: Study Language: en Within the study limitations, it was concluded that the medical students had more knowledge about OC in terms of causes. In contrast, the dental students had more awareness of the signs and symptoms related to OC. It was also seen that dental students sought more information related to OC than medical students. Through this study, an attempt to elicit knowledge and awareness was made, and an introduction of an important step was attempted. The information gained through this questionnaire helps the students change perspectives regarding OC. An educational intervention or early exposure to dental/oral health screening camps should be implemented promptly to sensitize the students and familiarize them with the initial signs and symptoms of OC. The study's extended scope involves conducting further analyses involving multiple institutions and larger sample size to investigate factors affecting OC awareness among medical and dental students, including demographic variables, curriculum content, and clinical exposure, to gain a deeper understanding of the underlying reasons for the observed disparities in knowledge and practices between these groups. | Review | biomedical | en | 0.999996 |
PMC11699989 | Section title: 1. Introduction Educational score: 3.9806079864501953 Domain: biomedical Document type: Review Language: en Inflammatory bowel disease (IBD) is a chronic nonspecific disease that includes Crohn's disease (CD) and ulcerative colitis (UC). Common clinical symptoms include recurrent abdominal pain, diarrhea, and posterior tenesmus . A primary aim in treating IBD is to enhance the quality of life, given its chronic nature involving alternating between the active and remission phases, requiring lifelong management. The incidence rate of IBD is high in developed countries such as North America and Western Europe. The annual incidence rate can reach 29.3/100,000 people . However, in the past 20 years, the incidence of IBD in Western countries has remained relatively stable or even decreased, while the incidence in Eastern Europe and Asia has shown a continuous increasing trend . More specifically, the incidence of IBD has increased drastically in East Asian countries such as China , South Korea , and Japan . Section title: 1. Introduction Educational score: 3.9397213459014893 Domain: biomedical Document type: Review Language: en The course of IBD is characterized by alternating active and remission phases, protracted recovery, and the need for lifelong treatment, all of which seriously affect the patient's quality of life. The etiology and pathogenesis of this disease remain unclear but are generally believed to be closely related to genetics, the environment, etc. Currently, drug therapy is the most crucial treatment approach; however, it encounters numerous challenges, such as low response rates and potential side effects. Increasing research attention is directed toward investigating the potential advantages of nonpharmacological interventions, such as physical activity (PA), for patients with IBD. Section title: 1. Introduction Educational score: 4.124856948852539 Domain: biomedical Document type: Review Language: en Some evidence based on the population shows that the implementation of an exercise intervention can reduce the risk of various chronic diseases, including cancer and cardiovascular disease . Furthermore, exercise interventions can improve patient outcomes with depression . The impact of exercise and PA on the gastrointestinal tract is an emerging research field. Several studies have shown an inverse relationship between PA and the risk of gastrointestinal related diseases such as colon cancer , diverticular disease , gallstones , or constipation . However, some studies have also shown the harm of exercise to gastrointestinal symptoms such as nausea, heartburn, diarrhea, and gastrointestinal bleeding. In recent years, studies have found that exercise may have multiple health benefits for IBD [ 16 – 18 ]. From the perspective of pathological and physiological mechanisms, regular exercise can reduce visceral fat content, inhibit the release of inflammatory factors, and induce an anti-inflammatory microenvironment, providing theoretical support for exercise assisted treatment of IBD . Recent studies have confirmed the existence of the “gut-muscle” axis , indicating that exercise has a regulatory effect on gut microbiota, which is crucial in the development of IBD . Section title: 1. Introduction Educational score: 3.781038522720337 Domain: biomedical Document type: Review Language: en According to the WHO, engaging in PA can lower the risk of disease progression or premature death while also enhancing physical function and overall quality of life in adults with chronic diseases . Nevertheless, research on PA in patients with IBD is still in its preliminary stages . Given the crucial role of PA, this study reviews the current status of PA in patients with IBD with the aim of providing a reference for PA practice. Section title: 2.1. Concepts Educational score: 4.106471061706543 Domain: biomedical Document type: Review Language: en PA involves increased energy expenditure of the body resulting from skeletal muscle contraction, including all types of sports or exercise, such as leisure activities and commuting. PA can be categorized into various types, such as static exercise and dynamic exercise, based on the form of muscle contraction. In addition, PA can be classified into anaerobic exercise and aerobic exercise according to the metabolic law of energy supply during exercise. Based on the background of daily life sources, PA can be categorized into occupational, family (household chores), transportation (such as walking and cycling), and leisure (various sports) . The intensity of these activities directly affects the effectiveness and safety of PA for patients with IBD. Key indicators of activity intensity include reserve oxygen uptake, reserve heart rate, oxygen uptake, heart rate, and metabolic equivalent (MET) . In particular, MET is a globally recognized and widely used approach for quantifying the energy expenditure of PA. One MET is the resting metabolic rate, indicating the amount of oxygen consumed while resting quietly in a chair, approximately 3.5 mL/kg/min. The resting state is 0–1.5 METs, low-intensity PA (such as walking, laundry, and cooking) is 1.5–2.9 METs, moderate-intensity PA (such as brisk walking, heavy cleaning work, and dancing) is 3–5.9 METs, and high-intensity PA (such as running, doing heavy agricultural work, and kicking) is 6 METs or higher . We summarized the exercise modes corresponding to PA of different intensities ( Table 1 ). Patients with IBD should perform PA according to their condition and appropriate intensity levels. Studies have indicated that walking for more than 1.5 h per day for patients with IBD does not lead to further improvements in quality-of-life scores. Thus, additional research is needed to determine the optimal intensity of PA that maximizes benefits for patients with IBD. Currently, consideration can be given to adhering to the 2020 WHO guidelines for PA and sedentary behavior . The guide recommended that adults and patients with chronic disease engage in 150–300 min of moderate-intensity or 75–150 min of high-intensity aerobic exercise per week or an equivalent combination of the two. Section title: 2.2. Assessment Tools Educational score: 4.07493257522583 Domain: biomedical Document type: Study Language: en PA can be measured using two primary approaches: objective and subjective. Objective approaches involve the use of wearable devices such as pedometers, accelerometers, and heart rate monitors . Subjective approaches primarily assess the type, frequency, and duration of PA through self-reported questionnaires, scales, or logs. Examples include the International Physical Activity Questionnaire (IPAQ) , the Global Physical Activity Questionnaire , the Short Questionnaire to Assess Health Enhancing Physical Activity , and the Gordon Leisure Time Questionnaire . Among them, the IPAQ has high reliability and validity and has been extensively employed in numerous countries . The IPAQ can be divided into two versions: long and short. The long version comprises five sections: occupation, household chores, transportation, leisure PA, and sitting. Regarding the frequency and time of PA inquiries in the PA project, each PA intensity was truncated at 180 min. The short test comprises seven questions that assess the total time individuals spend each week in four types of PA: intense PA, moderate PA, walking, and sitting. The IPAQ recommends computing an individual's weekly PA level (MET minute per week). This is done by multiplying the MET value of a specific intensity of PA by the average daily activity time (minute per day) and the number of days per week the activity is performed. The total weekly PA level was then determined by summing the PA levels at different intensities. Section title: 3. PA and IBD Incidence Risk Educational score: 3.812621831893921 Domain: biomedical Document type: Review Language: en Epidemiological data indicate that the incidence rate of IBD is relatively high in Europe (505 per 100,000 people), Canada (248 per 100,000 people), and the United States (214 per 100,000 people), among other developed regions . In addition, the incidence rate of IBD annually increases in developing countries and regions, such as Asia, the Middle East, and South America . The etiology of IBD remains unclear, and increasing research is examining the influence of lifestyle factors on IBD onset. The correlation between PA as a lifestyle and IBD onset remains a topic of debate. Section title: 3. PA and IBD Incidence Risk Educational score: 3.998537302017212 Domain: biomedical Document type: Study Language: en First, some studies revealed a correlation between the incidence of IBD and occupational characteristics, possibly because of the varying PA levels among different occupational groups. Sonnenberg analyzed the occupational distribution of 12,014 patients with IBD using German social security statistics and discovered that outdoor work and high PA level work were protective factors for IBD onset. Conversely, occupations involving working in air-conditioned environments or irregular shifts for extended periods were associated with an increased risk of IBD. In a prospective study in Denmark, researchers conducted a 5-year or 10-year follow-up survey involving over 2.2 million people, and the findings indicated that sedentary work may increase the risk of developing IBD . Section title: 3. PA and IBD Incidence Risk Educational score: 4.161386489868164 Domain: biomedical Document type: Study Language: en Second, there is a relationship between IBD risk and PA levels. Persson et al. conducted a case–control study examining various risk indicators associated with IBD. In an analysis involving 152 patients with CD, 145 patients with UC, and 305 control populations, regular weekly relative ratio (RR) = 0.6, 95% confidence interval (CI): 0.4–0.9) and daily (RR = 0.5) PAs (95% CI: 0.3–0.9) were found to potentially reduce the risk of developing CD. In a case–control study conducted in Slovakia involving 338 adult patients with IBD (190 with CD and 148 with UC), insufficient PA during childhood was associated with an increased incidence of CD (odds ratio (OR) = 2.7, 95% CI: 1.5–5.0) and UC (OR = 2.0, 95% CI: 1.1–3.5). Insufficient PA level was identified as an independent risk factor for IBD onset . In a national health interview survey conducted in the United States with 60,155 participants, 786 individuals (1.3%) had IBD. The study revealed a positive correlation between IBD incidence and PA deficiency (OR = 1.38, 95% CI: 1.16–1.66) . Klein et al. compared environmental factors between newly diagnosed patients with IBD and healthy controls and found that the control group tended to engage in moderate or severe PA before bedtime, whereas patients with IBD reported less PA before bedtime. Furthermore, one extensive prospective cohort study focusing on American women identified a negative relationship between PA and the incidence of CD, but not UC. Section title: 3. PA and IBD Incidence Risk Educational score: 3.9987857341766357 Domain: biomedical Document type: Study Language: en However, some studies have reported that the incidence of IBD is not correlated with PA levels. For instance, the European Cancer and Nutrition Prospective Study cohort assessed and monitored 300,724 participants, identifying 177 confirmed cases of UC and 75 cases of CD . No significant correlation was observed between PA and UC ( p = 0.97) or CD ( p = 0.42). Furthermore, Halfvarson et al. examined the potential exposure factors for IBD in a genetically identical Swedish–Danish twin cohort and effectively mitigated the potential influence of genetic factors. Their findings revealed no significant difference in PA between the patients and their twins before the diagnosis of IBD . Section title: 3. PA and IBD Incidence Risk Educational score: 3.708146333694458 Domain: biomedical Document type: Review Language: en In summary, research findings on the causal relationship between IBD incidence and PA levels are inconsistent ( Table 2 ). This variability could stem from different confounding factors and retrospective biases. Future large-scale studies are necessary to provide more definitive evidence and confirm this causal relationship. Section title: 4. Promoting and Limiting Factors of PA in Patients With IBD Educational score: 4.03541898727417 Domain: biomedical Document type: Review Language: en Although many patients with IBD recognize the positive impact of PA on their condition and are willing to engage in it, their PA levels are generally lower due to symptoms such as abdominal discomfort, fatigue, pain, negative emotions, and other related factors. Based on a survey conducted by Guthold et al. across 51 countries, 17.7% of healthy adults exhibit insufficient levels of PA . The proportion of patients with IBD with insufficient PA is significantly higher than that of healthy adults. Moreover, there is no substantial difference in PA levels between active and remission patients with CD . Tew, Jones, and Mikocka-Walus reported that 33.3% of patients with IBD do not meet the recommended PA levels . Conversely, Fagan, Osborne, and Schult found that only 66% of patients with IBD in remission or with mild-to-moderate activity met the WHO recommendation of at least 150 min of moderate-intensity aerobic PA per week . Furthermore, patients with IBD typically engage in low-intensity PA. In a survey involving 859 adult patients with IBD conducted by Tew, Jones, and Mikocka-Walus , 57% reported walking as their most common activity, 34% expressed a preference to avoid running or jogging, and only 17% reported participating in high-intensity PA. Thus, when advising patients with IBD about physical activities, it is crucial to consider their preferences and prioritize low-intensity activities to encourage their full participation and enthusiasm. Section title: 4. Promoting and Limiting Factors of PA in Patients With IBD Educational score: 4.089181900024414 Domain: biomedical Document type: Study Language: en Various factors limit patients with IBD from participating in PA, and effectively identifying these factors can aid in developing targeted strategies to encourage and support their engagement in activities. According to Tew, Jones, and Mikocka-Walus, as the course of IBD progresses, fatigue, anxiety, and depression tend to worsen, leading to a corresponding decrease in the PA levels of patients . In addition, most participants (677/859) in the study indicated that IBD limited their ability to engage in PA, with influencing factors such as abdominal/joint pain (70%), fatigue/fatigue (69%), disease onset (63%), and increased urgency to use the toilet (61%). Furthermore, the PA levels of patients with CD were independently associated with depression, disease activity, and motor dysfunction. An online survey conducted by the Crohn's Disease and Colitis Association in the United Kingdom was used to assess exercise habits among the 918 participants with IBD . The findings indicated that patients with IBD generally accepted PA, with a majority (72%) reporting positive feelings toward PA. However, 80% of the survey respondents indicated that they had to temporarily or permanently stop exercising at some point due to severe disease symptoms. IBD significantly hindered their ability to participate, enjoy, and derive benefits from PA. A semistructured interview study conducted in the Netherlands examined the role of PA in patients with IBD. The findings indicated that 86% of patients experienced positive effects from PA, such as improvement in overall health, quality of life, and self-image. However, symptoms experienced during the active phase of IBD often lead to postponement or discontinuation of PA . Section title: 5. Impact of PA on the Clinical Outcomes of Patients With IBD Educational score: 3.742863416671753 Domain: biomedical Document type: Review Language: en According to population-based evidence, PA can lower the risk of several chronic diseases, such as cancer and cardiovascular disease, and enhance patient prognosis . Nevertheless, the safety and potential health benefits of PA for patients with IBD remain unclear. Before recommending specific PA practices, it is essential to understand their impact on the clinical outcomes of patients with IBD, including disease activity, quality of life, and mortality. There are many benefits of PA for people with IBD . We also summarized the impact of PA on the clinical outcomes of IBD ( Table 3 ). Section title: 5.1. PA and Disease Activity Educational score: 4.132654190063477 Domain: biomedical Document type: Review Language: en Several studies have investigated the correlation between PA and disease activity, but the findings have been inconsistent. PA level may be associated with disease activity in patients with CD compared with those with UC. A cross-sectional survey conducted in the Netherlands revealed a negative relationship between PA levels and disease activity in patients with CD. Patients with higher PA levels had lower disease activity. In contrast, no correlation was observed between PA levels and disease activity in patients with UC. A prospective cohort study conducted in the United States examined the relationship between PA levels and disease activity over 6 months in patients with IBD . The findings revealed that patients with remission-stage CD with higher PA levels experienced a significant decrease in disease activity after 6 months (OR = 0.72, 95% CI: 0.55–0.94, p = 0.02). However, remission-stage UC patients with higher PA levels did not exhibit a significant decrease in disease activity after 6 months (OR = 0.78, 95% CI: 0.54–1.13, p = 0.18). Watanabe et al. investigated the correlation between four types of PA (sedentary, standing, walking, and vigorous activity) and clinical remission in 327 patients with UC. No significant correlation was observed between mucosal healing and any type of PA and clinical remission in patients with UC. However, high vigorous activity levels and total daily METs may be negatively correlated with mucosal healing but not with clinical remission. In a randomized controlled study conducted by Seeger et al., 45 patients with CD in remission or mild activity were randomly assigned to a control group, endurance group, or muscle training group for 3 months, with PA intervention thrice weekly . The findings indicated no significant change in disease activity between the two groups; however, both intervention groups reported improved quality of life. In summary, PA might help mitigate disease activity in patients with IBD, particularly patients with CD. Although the findings from various studies may vary, no negative effect of PA on disease activity has been found. Section title: 5.2. PA and Mortality Rate Educational score: 4.031694412231445 Domain: biomedical Document type: Study Language: en Cucino and Sonnenberg examined the mortality data of patients with IBD from 1991 to 1996 at the National Center for Health Statistics in the United States. They observed that after adjusting for gender and race, patients with IBD in occupations involving physical labor and agriculture (such as farmers and workers) had lower mortality rates compared with those in sedentary occupations involving indoor work (such as salespeople and secretaries). Lo et al. used the Cox proportional hazards model to examine three large cohort data: Nurse Health Study , Nurse Health Study II , and Health Professional Follow-up Study . They observed that among 828 patients with IBD (363 CD and 465 UC), patients with UC with high PA levels had the lowest risk of all-cause mortality (HR = 0.14–0.41, p = 0.002). However, no significant correlation was observed in patients with CD. Section title: 5.3. PA and Quality of Life Educational score: 4.188290596008301 Domain: biomedical Document type: Study Language: en Engaging in PA can enhance the quality of life for patients with IBD. A cross-sectional study conducted in South Korea investigated the correlation between PA levels and quality of life in patients with IBD . The study included 158 patients with IBD and evaluated their PA levels using the IPAQ, the European Qol Five-Dimensional Questionnaire, and the European Quality of Life Visual Analog Scale. The findings indicated that higher PA levels were associated with improved quality of life, and improvement in leisure activities and nonsweating exercise was particularly associated with improved quality of life. In an intervention study, Loudon et al. developed a 12-week walking plan for patients with sedentary remission or mild active CD. The exercise plan involved walking thrice weekly, and 12 patients completed it. The study revealed that after the intervention, patients exhibited significant improvements in the IBD stress index, quality of life score, BMI, and other outcome indicators. Similarly, Elsenbruch et al. conducted an intervention for patients with UC in remission using a 10-week, 60-h structured training program. This program incorporated stress management training, moderate exercise, Mediterranean diet, behavioral skills, and self-care strategies. Quality of life was better in the intervention group than in the control group. However, no significant impact on clinical or physiological parameters was observed. Ng et al. implemented a low-intensity walking program for 32 patients with CD in remission or mild activity. This program involved walking three times a week, with each session lasting 30 min for 3 months. Their findings indicated that patients who participated in the exercise program experienced a significant improvement in their quality of life, with no adverse effects observed on disease activity. Van Erp et al. developed a 12-week intensive exercise program for patients with IBD in remission. This program included three sessions per week, each consisting of personalized high-intensity aerobic and progressive resistance training lasting for 1 h. Their study demonstrated that a personalized high-intensity exercise program can substantially improve fatigue, quality of life, and cardiopulmonary function in patients with IBD in remission. Recent studies have emphasized the bidirectional relationship between PA and IBD, advocating for the inclusion of PA in IBD management to improve patients' quality of life and reduce IBD-related complications. Our mixed study also showed that recreational PA was associated with improved patient-reported outcomes . In summary, PA can potentially improve the quality of life of patients with IBD, although the PA schemes implemented in current studies vary widely. The approaches and safety considerations of PA are crucial for patients with IBD . Future research can summarize the evidence from relevant studies to develop the best activity plan to guide the widespread adoption of PA. Section title: 5.4. PA and Body Composition Educational score: 4.043442726135254 Domain: biomedical Document type: Study Language: en PA can cause changes in the body composition of patients with IBD, such as reductions in the levels of inflammatory biomarkers . A previous study used real-time detection of biochemical indicators in patients with IBD before and after PA to investigate the effects of PA on body composition. D'incà et al. assessed the intestinal motility, permeability, and antioxidant levels of six remission-stage patients with CD and a healthy control population after PA. The participants were asked to engage in continuous activity for 1 h at a maximum oxygen consumption of 60%. The findings revealed that patients with CD had an increased cecal transit time after exercise compared with the control group. However, the difference was not significant, and the amount of urinary zinc was substantially increased. Another study investigated the safety of moderate-intensity continuous and high-intensity intermittent activities in youth with CD. The study involved 30-min cycles with six cycles of 4 × 15 s at 50% and 100% peak mechanical power, respectively. Blood samples were collected at rest, midpoint during exercise, end of exercise, and 30 and 60 min after recovery to assess changes in inflammatory cells, cytokines, and growth factors. The findings indicated that neither activity led to exacerbation of acute inflammation. Seeger et al. conducted moderate-intensity autologous weight and endurance training on patients with CD during the remission and mild activity phases, revealing that PA can enhance upper and lower limb strength as well as grip strength. Section title: 5.4. PA and Body Composition Educational score: 4.044689178466797 Domain: biomedical Document type: Study Language: en Furthermore, several studies have examined the long-term effects of PA in patients with IBD. In a randomized controlled trial conducted by Robinson et al. , 117 patients with CD were randomly assigned to either a control group or a low-intensity exercise program. Bone density of the hip joint and spine was measured using a dual-energy x-ray absorptiometer at baseline and after 12 months of exercise. After 12 months of follow-up, a significant relationship was observed between patient bone density and exercise frequency. Low-intensity exercise can enhance bone density in patients with CD, thereby reducing the risk of osteoporotic fractures. In a randomized controlled trial conducted by Jones et al. , 47 adults with CD in remission were randomly assigned to either the exercise or the control group. The exercise group underwent a 6-month resistance training program. At the end of the 6th month, the control group demonstrated substantial improvements in lumbar spine bone density, muscle strength, and physical performance compared with the exercise group. Section title: 6. Prospects for PA in IBD Educational score: 3.934335947036743 Domain: biomedical Document type: Study Language: en The intensity and duration of exercise programs carried out vary from study to study. In a study conducted by Jones et al. , the exercise group received a 6-month combined impact and resistance training program, involving three, 60-min sessions per week and a gradual tapering of supervision to self-management. Moderate endurance exercise training and muscle training were performed for 30 min three times per week for 12 weeks in the study conducted by Seeger et al. . In Ng's study , patients performed low-intensity walking at an interval of three times per week for a duration of 3 months, and each walking session lasted for 30 min. We can find that the exercise was mainly medium or low intensity, and the frequency was mostly three times a week, and each time was not more than 1 h. Section title: 6. Prospects for PA in IBD Educational score: 3.897151231765747 Domain: biomedical Document type: Study Language: en In addition, the independent relationship between results and exercise frequency confirms the “training effect” of fully compliant patients . However, some studies have unclear definitions of the duration and intensity of exercise interventions, as well as a lack of effective exercise supervision, which may lead to poor compliance among participants and dilute exercise effectiveness. Although current studies suggest the potential benefits of PA for IBD, the measurement of PA mostly relies on questionnaires, which may lead to recall bias. Wearable device–based accelerometers can more accurately measure PA. Section title: 6. Prospects for PA in IBD Educational score: 2.224020004272461 Domain: biomedical Document type: Other Language: en With the aging population, the number of elderly people suffering from IBD is gradually increasing . Elderly IBD patients face greater risks due to advanced age, more underlying diseases (such as hypertension and diabetes), and the use of hormones and immunosuppressants . Some traditional Chinese medicine exercise rehabilitation programs, such as Tai Chi and Ba Duan Jin , may be more suitable for elderly IBD patients, but there are currently no relevant reports among IBD patients. Resolving the above issues will help unlock greater power for PA in IBD. Section title: 6. Prospects for PA in IBD Educational score: 3.0431177616119385 Domain: biomedical Document type: Other Language: en Future study needs to explore the best type of exercise and the most appropriate exercise time from an evidence-based perspective, build an exercise program suitable for IBD patients of different ages and disease degrees, and finally verify the effectiveness of the exercise program through randomized controlled trials. Section title: 7. Limitations Educational score: 3.858872652053833 Domain: biomedical Document type: Review Language: en This review has several limitations that deserve transparent discussion. Some studies have revealed the potential benefits of exercise for people with IBD; however, exercise programs vary from study to study, and our review focused on exercise effects without summarizing the characteristics of these exercise intervention programs. It is still necessary to sort out these exercise programs and explore the optimal type of exercise for people with IBD in the future. In addition, our review did not summarize the differences in the impact of exercise on different age groups, which is not conducive to developing personalized exercise prescriptions. Section title: 8. Conclusion Educational score: 3.860147476196289 Domain: biomedical Document type: Review Language: en Patients with IBD have insufficient PA levels, and the correlation between PA and the incidence and disease activity of IBD remains controversial. Nevertheless, PA has demonstrated beneficial effects on clinical outcomes, particularly in reducing mortality, enhancing quality of life, and improving body composition. Future research should investigate various approaches, intensities, and volumes to determine the optimal activity plan for patients with IBD. In clinical practice, patients must undergo exercise health screenings and risk evaluations, considering patient goals, disease status, living conditions, and other relevant factors to tailor personalized exercise prescriptions for each patient. | Review | biomedical | en | 0.999996 |
PMC11699990 | Section title: 1. Introduction Educational score: 4.316180229187012 Domain: biomedical Document type: Study Language: en Muscle stem cells, also called satellite cells (SCs) and defined by the transcription factor paired box 7 (Pax7), are responsible for skeletal muscle maintenance and repair. Once activated after injury, SCs enter the cell cycle, commit to myogenic progenitor cells (MPCs), differentiate into myoblast cells, and finally fuse into multinucleated myotubes. A subset of SCs undergoes asymmetric division, renewing the SC pool . However, endogenous SCs may be inefficient in responding to severe trauma or chronic degenerative diseases such as malnutrition, neuromuscular diseases, and sleep apnea [ 2 – 4 ]. When more than 20% of the muscle volume is missing, as in volumetric muscle loss (VML), endogenous self-repair is hindered . Moreover, SCs are found in very limited numbers in adult muscles and exhibit undesirable amplification activity in vitro ; thus, these SCs are not the appropriate stem cell pool for muscle repair. Section title: 1. Introduction Educational score: 4.110976219177246 Domain: biomedical Document type: Study Language: en Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells (MSCs) that exhibit therapeutic potential for tissue regeneration . These cells are easily established from dental pulp and have several advantages compared to other types of MSCs: better proliferative potential and migratory capabilities , a simpler primary isolation method, a higher success rate in long-term in vitro culture , and long-term storage without losing their stemness and ability to differentiate . Kerkis et al. found the therapeutic effect of human DPSCs on Golden Retrievers with muscular dystrophy. Recently, DPSCs were shown to undergo myogenic differentiation (MyoD), and these myogenic lineage cells could treat the dystrophic muscles of mdx/SCID mice . However, the current myogenic efficiency is still quite low . Here, we searched for novel factors to develop appropriate MyoD systems for DPSCs. Section title: 1. Introduction Educational score: 4.510252952575684 Domain: biomedical Document type: Study Language: en MyoD is regulated by genes such as MyoD1, myogenic factor 5 (Myf5), myogenic regulatory factor 4 (MRF4), Desmin, and myosin heavy chain (MyHC) to determine cell fate, including myoblast differentiation into myocytes, fusion into myotubes, and maintenance of myofibers . Factors, molecules, or stimuli can be applied to affect MSCs' fate and commitment , and biocompatible scaffolds are provided for the natural environment in the use of MSCs . A recent RNA-seq analysis identified that repressed bone morphogenetic protein (BMP) and activated Wnt signaling pathways are required for nascent somites during embryonic myogenesis . Wnt activation alone can induce pluripotent stem cells (PSCs) committed to myogenic progenitors. It further requires BMP inhibition to maintain progenitor fate, followed by the myogenic program to generate myofibers and associated Pax7 + cells . These results suggest that Wnt activation or BMP antagonism might be important mechanisms in myogenic regulation. Section title: 1. Introduction Educational score: 4.2896528244018555 Domain: biomedical Document type: Study Language: en Noggin, a secreted homodimeric glycoprotein, is promoted by Wnt-1 in medial somites and is required for embryonic somite and skeletal patterning . Then, Noggin was found to inhibit the actions of BMP-2, -4, -5, -7, -13, and -14, thus blocking Smad-dependent signaling . BMP signaling has been shown to play a role in controlling SC lineage progression during embryonic myogenesis , and these SC progeny secrete BMP antagonists, such as Noggin, for the differentiation of muscle progenitor cells . Noggin-null mice display defective skeletal muscle fibers . However, the effect of Noggin on the MyoD of DPSCs has not yet been reported. Section title: 1. Introduction Educational score: 3.8319504261016846 Domain: biomedical Document type: Study Language: en In the present study, Noggin was applied to MyoD systems for DPSCs. Then, we implanted these pretreated DPSCs combined with Matrigel as scaffolds into the mouse tibialis anterior muscle with VML. Here, we explore the DPSCs repair capacity in muscle injury. Section title: 2.1. Cell Culture and Identification Educational score: 4.134244918823242 Domain: biomedical Document type: Study Language: en DPSCs were acquired as previously described and cultured in α -MEM (Gibco, NY, USA) with 10% fetal bovine serum (FBS, Gibco, NY, USA) and 1% penicillin-streptomycin (Gibco, NY, USA) at 37°C in the presence of 5% CO 2 and 95% air. Flow cytometric analysis was used to determine the cell surface markers present on DPSCs. DPSCs were identified with phycoerythrin (PE)-conjugated antibodies against human CD29 and FITC-conjugated antibodies against human CD90 from BD Biosciences (CA, USA), fluorescein isothiocyanate (FITC)-conjugated antibodies against human CD44 from eBioscience (CA, USA), and PE-conjugated antibodies against human CD34 and FITC-conjugated antibodies against human CD45 from BioLegend (CA, USA) and analyzed using FlowJo software (FlowJo, OR, USA). Cell cycle analysis was also performed after staining for 24 h with propidium iodide (PI) (Beyotime, Shanghai, China) according to the manufacturer's protocol using flow cytometry (ACEA NovoCyte, CA, USA). Section title: 2.2. MyoD Assay Educational score: 4.1577348709106445 Domain: biomedical Document type: Study Language: en DPSCs were seeded on 6-well plates at a cell density of 4000 cells/cm 2 in an expansion medium consisting of α -MEM with 10% FBS for adherence. When the confluence reached 80%, the culture medium was replaced first with a myogenic induction medium containing the following for 24 h: IMDM (Gibco, NY, USA) + 2% FBS + 1 μM 5-Aza-2′-deoxycytidine (5-Aza) (an important trigger for myogenic commitment differentiation ) (Sigma-Aldrich, CA, USA) . IMDM + 2% FBS without 5-Aza served as the base control medium. Subsequently, cells were rinsed three times in phosphate-buffered saline (PBS, HyClone, UT, USA) and then transferred to the following differentiation medium: IMDM with 10% FBS (defined as Day 0) . The differentiation medium was supplemented with or without Noggin (100 ng/mL = 100N or 200 ng/mL = 200N) or 50 ng/mL BMP4. Noggin from Sino Biological (Beijing, China) and BMP4 from HumanZyme (IL, USA) were used. Cells were cultured for 3 weeks, the differentiation medium was changed every 3 days, and the cells were observed under a microscope to confirm the formation of myotubes. Section title: 2.3. VML Defect Modeling and Stem Cell Transplantation Educational score: 4.103085517883301 Domain: biomedical Document type: Study Language: en Animal protocols were approved by Cyagen IACUC (No. ACU20-A034). Male Balb/C mice (8 weeks old) were purchased from Shanghai Bikai Biotechnology. All animals underwent surgery to create a muscle defect by unilaterally resecting the tibialis anterior muscle under 2% pentobarbital sodium anesthesia. The thin layer of fascia covering the tibialis anterior muscle was dissected away, and a 2 × 5 mm area was stained with hematoxylin. After a blunt separation, a 2 mm-deep cut was made around the stained hematine, creating a 2 × 5 × 2 mm defect. This VML defect accounted for a loss of ~40% of the tibialis anterior muscle. DPSCs were cultured and differentiated by 5-aza and Noggin treatment, and Matrigel (Corning, NY, USA) was used to provide a scaffold for stem cell transplantation. Matrigel alone ( n = 6) or Matrigel combined with Noggin-treated ( n = 6) or untreated DPSCs ( n = 6) was used to fill the dissected area. If no immediate transplantation was performed ( n = 6), the incision was sutured closed. Mice were allowed to heal for 30 days. Section title: 2.4. Hematoxylin and Eosin Staining Educational score: 4.079771041870117 Domain: biomedical Document type: Study Language: en The tibialis anterior muscles were excised from mice in the four groups and fixed in ice-cold 4% PFA. After dehydration in a graded alcohol series, the tissue was embedded in paraffin and cut into 5 µm-thick sections by using a Leica RM2255 rotary microtome (Leica Microsystems, Mannheim, Germany). After deparaffinization in xylene and hydration through a graded alcohol series to ddH 2 O, hematoxylin, and eosin (Servicebio, Wuhan, China), staining was performed according to the manufacturer's protocols. Section title: 2.5. RNA Extraction and Real-Time Polymerase Chain Reaction (PCR) Educational score: 4.125436782836914 Domain: biomedical Document type: Study Language: en Total RNA was extracted from the cells using TRIzol (Life Technologies, CA, USA). The RNA concentration was quantified using a spectrophotometer by measuring the OD260/280 ratio (1.80–1.95). DNA was removed by gDNase at 42°C for 3 min. cDNA was reverse transcribed using a FastQuant RT Kit (Tiangen, Beijing, China) at 42°C for 15 min and at 95°C for 3 min. Subsequently, SYBR Green Premix (Tiangen, Beijing, China) was used for the amplification of cDNA on a Light Cycler 96 system (Roche, Basel, Switzerland) with each primer at 0.6 mM and 150 ng of cDNA template. The thermocycling conditions were as follows: 95°C for 15 min, followed by 40 cycles at 95°C for 10 s and at 60°C for 32 s. Transcript levels were normalized using β -actin as a housekeeping gene and analyzed with the 2 −∆∆Ct method. The primer sequences can be found in Table S2 . Section title: 2.6. Western Blot Analysis Educational score: 4.121078014373779 Domain: biomedical Document type: Study Language: en Total protein was extracted in RIPA lysis buffer (Thermo Fisher Scientific, MA, USA) plus protease/phosphatase inhibitor cocktail . Lysate proteins (25 mg) were loaded onto SDS-polyacrylamide gels (7.5% or 10%) and subsequently transferred onto PVDF membranes. Target proteins on the membranes were incubated overnight at 4°C with primary antibodies diluted 1:1000–1:200 in 1 × TBS-Tween (TBS: 0.05 M Tris, 0.15 M NaCl [pH 7.5]; with 0.2% Tween-20). Antibodies against β -actin , Pax7 , and Myf5 from Abcam; MyHC IIB (BF-F3) and MyHC IIA (SC-71) from Developmental Studies Hybridoma Bank (DSHB, ID, USA); p-Smad 1/5/9 and Smad 1 from Cell Signaling Technology (CST, MA, USA); and MyoD , Desmin , MRF4 , Pax3 , Six1 , Eya2 , ID1 , and MSX1 from Proteintech (IL, USA) were used. After intensive washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1–2 h. The membranes were visualized using Super Signal West Dura substrate (Thermo Scientific, MA, USA), and the bands were detected with an AI600 imager (GE Healthcare, IL, USA). Section title: 2.7. Immunofluorescence Microscopy Educational score: 4.146756172180176 Domain: biomedical Document type: Study Language: en Cells, myofibers, and sections were fixed with 4% paraformaldehyde and placed in PBS containing 0.25% Triton X-100 for 10 min. After the samples were blocked with 3% bovine serum albumin (BSA, Solarbio, Beijing, China) in PBS for 30 min at room temperature, they were then incubated with the primary antibody in PBS containing 2.5% BSA at 4°C overnight. In addition to the primary antibodies listed above, antibodies against Pax7 (PAX7, DSHB), CD34 , human Lamin A/C , human nucleoli (hNu) , and Laminin were used. After washing, the samples were incubated with anti-mouse Alexa-594 or Alexa-488, or antirabbit Alexa-594 or Alexa-488 (Jackson ImmunoResearch, PA, USA), and then with 4′,6-diamidino-2-phenylindole (DAPI). Images of the cells were captured using a microscope (Leica Microsystems, Mannheim, Germany) and analyzed with ImageJ. Section title: 2.8. Cell Cycle Analysis Educational score: 4.054045677185059 Domain: biomedical Document type: Study Language: en The cells were harvested and fixed in 70% precooled ethanol overnight at 4°C. After washing with PBS the next day, the cells were incubated with a solution containing RNase A and 50 µg/mL of PI for 30 min at 37°C. Then, the cell cycle analysis was examined using a Novocyte 2040R flow cytometer (ACEA Biosciences, Hangzhou, China) with a cell count of ~0.5 × 10 4 ~ 2.0 × 10 4 frequency of G1, S, and G2 phases using NovoExpress software version 1.4.1 (ACEA Novocyte). The results were graphed using GraphPad Prism software. Section title: 2.9. Statistical Analysis Educational score: 3.422464609146118 Domain: biomedical Document type: Study Language: en We performed statistical analyses using GraphPad Prism 5.0. Data are expressed as the mean ± standard deviation. The protein quantities were analyzed using semi-quantitative analysis. Comparisons between groups were performed using the Mann‒Whitney U test or Kruskal‒Wallis H test with multiple comparisons. Statistical significance was defined as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Section title: 3.1. Noggin Promotes the Formation of Myotubes in DPSCs Educational score: 4.126744270324707 Domain: biomedical Document type: Study Language: en We cultured and identified DPSCs and found that 5-Aza can induce MyoD in DPSCs by increasing myogenic markers but with low efficiency. To examine whether Noggin could affect the MyoD of DPSCs, we treated DPSCs with Noggin after 5-Aza induction . First, we found that Noggin had no significant effect on the proliferation of DPSCs but increased myotube formation in DPSCs . A more pronounced myotube morphology was observed after treatment with the Noggin protein for 21 days, and the differential index of multinuclear myotubes increased to ~20% . The protein expression levels of MyHC , such as MyHCIIA and MyHCIIB , were significantly increased compared to those in the control group. This finding implies that Noggin might facilitate the formation of myotubes in DPSCs. Section title: 3.2. Noggin Accelerates the Progression of MyoD in DPSCs Educational score: 4.133330345153809 Domain: biomedical Document type: Study Language: en To evaluate the effect of Noggin treatment on the MyoD of DPSCs, we assessed the expression of myogenic genes such as MyoD, MRF4, and Desmin on Days 7, 14, and 21. Noggin increased the expression of MyoD and Desmin but had no effect on the mRNA expression of MRF4 on Day 7 . Immunofluorescence staining for MyoD and Desmin revealed elevated levels of MyoD and an increased quantity of long, spindle-shaped myotube-like cells in the Noggin-treated groups. Moreover, the protein expression levels of Desmin and MRF4 significantly increased upon treatment with Noggin on Day 14 . In our study, Noggin was shown to play an important regulatory role in accelerating the MyoD of DPSCs, which might be a novel factor in myogenesis. Section title: 3.3. Noggin Increases the Generation of Pax7 + Satellite-Like Cells in DPSCs Educational score: 4.1158857345581055 Domain: biomedical Document type: Study Language: en SCs are characterized by the expression of the paired box (Pax)3/7. Six1 and eya2 have been shown to activate SCs (Pax7 + ) and promote myoblast differentiation. Notably, we found that Noggin increased the protein levels of Pax7 in DPSCs cultured in a proliferation medium . To determine SCs generation through MyoD of DPSCs with Noggin, we measured the relative mRNA levels on Days 1, 3, and 7, as well as the protein expression levels of Six1, Pax3, Pax7, and Eya2 on Days 7, 14, and 21. Section title: 3.3. Noggin Increases the Generation of Pax7 + Satellite-Like Cells in DPSCs Educational score: 4.262757301330566 Domain: biomedical Document type: Study Language: en The mRNA expression of Pax7 increased in a concentration-dependent manner under Noggin treatment, reaching its peak on Day 3 and remaining higher than that of the control on Day 7 . Consistent with the expression of Pax7, the mRNA expression of Pax3 increased on Day 3 and remained elevated on Day 7 . Additionally, the mRNA expression of Six1 and Eya2 increased on Day 7. Western blotting and immunofluorescence staining for Pax7 revealed that Noggin upregulated the protein expression of Pax7 on Days 7, 14, and 21 . The protein expression levels of Pax3 , Six1 , and Eya2 were also increased in the Noggin-treated groups on Day 14 and remained high on Day 21 . These results suggest that Noggin promotes the myogenic process in DPSCs by upregulating the expression of Six1/Eya2 in addition to Pax3/Pax7. Section title: 3.4. Noggin Antagonizes BMP by Downregulating p-Smad 1/5/9, Thus Facilitating the MyoD of DPSCs Educational score: 4.118348121643066 Domain: biomedical Document type: Study Language: en To determine whether Noggin regulates the MyoD of DPSCs by regulating Smad signaling, we first simulated 3D protein structures using SWISS-MODEL and visualized them with PyMOL software, which showed that Noggin competitively inhibits the binding of BMP4 to BMP-receptor I A (BMPRIA) . Then, we blocked the effect of Noggin by adding the BMP protein. The protein levels of Pax7, Pax3, Eya2, and Desmin were observed . Compared with that of the Noggin treatment group (200N), their expression decreased in the Noggin + BMP treatment group. Among them, Pax7 was most prominent when compared with that in the 5-Aza or control groups . Section title: 3.4. Noggin Antagonizes BMP by Downregulating p-Smad 1/5/9, Thus Facilitating the MyoD of DPSCs Educational score: 4.185900688171387 Domain: biomedical Document type: Study Language: en To further explore the downstream regulation of BMP signaling by Noggin, we determined the phosphorylation levels of members of the BMP/p-Smad pathway . The protein levels of p-Smad 1/5/9 increased following the activation of the BMP pathway by adding BMP4 . Noggin persistently eliminated the phosphorylation levels even after subsequent stimulation with BMP4 . Downstream effectors of the BMP/p-Smad pathway, such as inhibitors of DNA binding 1 (ID1) and msh homeobox 1 (MSX1), were also downregulated by Noggin treatment . Therefore, our results indicate that Noggin facilitates the MyoD of DPSCs by inhibiting BMP/Smad activation. Section title: 3.5. Noggin-Pretreated DPSCs Combined With Matrigel Can Effectively Repair Muscle Injury in VML Educational score: 4.124567985534668 Domain: biomedical Document type: Study Language: en To test the utility of DPSCs in repairing muscle injuries, we established a mouse VML injury model that exhibited a loss of ~40% of the tibialis anterior muscle . In the untreated VML-injured group, the volumetric defect remained . Shrinkage occurred due to the defect area. The muscle defect deformity exhibited the deposition of a thin layer of disorganized collagenous scar tissue . The injury defect exceeded the threshold and could not be restored by the endogenous regenerative potential of the skeletal muscle. This result indicates the success of VML modeling. Section title: 3.5. Noggin-Pretreated DPSCs Combined With Matrigel Can Effectively Repair Muscle Injury in VML Educational score: 4.052475452423096 Domain: biomedical Document type: Study Language: en The utilization of scaffolds can provide temporary mechanical support and the necessary growth environment for seed cell adhesion, growth, proliferation, and differentiation. Therefore, we used the thermosensitive hydrogel Matrigel to provide a scaffold for stem cell transplantation . We observed that the Matrigel group exhibited a reduced shrinkage area and decreased muscle fibrosis . In addition, we found some mononuclear cells infiltrating in the defect . Section title: 3.5. Noggin-Pretreated DPSCs Combined With Matrigel Can Effectively Repair Muscle Injury in VML Educational score: 4.10866117477417 Domain: biomedical Document type: Study Language: en To examine whether Noggin-pretreated DPSCs could better repair muscle injury than the controls, we transplanted DPSC bioconstructs into the defects of VML muscles . We implanted untreated DPSCs or Noggin-pretreated DPSCs reconstituted in Matrigel into the defects. Morphometric analysis of muscle cross-sections revealed that the Noggin-treated DPSCs showed a 73% decrease in the size of the defect and a 69% decrease in scar tissue . In contrast to the defects in the above two groups, which exhibited irreversible and robust fibrotic scars, the defects treated with DPSCs showed markedly reduced fibrotic tissue surrounded by cells of varying morphologies, such as fibrotic, inflammatory, and vessel-like cells, indicating the tissue repair process . In contrast, Noggin-treated DPSCs might have accelerated this process and facilitated the improved formation of muscle tissue, leaving little cell infiltration . Section title: 3.6. Noggin-Pretreated DPSCs Bioconstructs Can Benefit the Muscle SC Population and Promote Myogenic Repair Educational score: 4.225616455078125 Domain: biomedical Document type: Study Language: en To explore the contribution of grafted cells to muscle injury, we immunostained muscle cross-sections for Pax7 (SC marker), MyoD (activated SCs/myoblasts), hNu, and human LaminA/C (specific antibody to trace human cells), and Laminin (to identify position within the sarcolemma). Pax7/MyoD costaining revealed that stem cell transplantation increased the proportion of activated SCs compared to the sham or Matrigel groups . We also observed that hNu was integrated into the nucleus of regenerated tissue and was located on the Laminin-stained muscle sarcolemma , a phenomenon more readily discernible in the Noggin-treated DPSCs groups than in the DPSCs groups. Increased numbers of hLaminAC + /Pax7 + and hNu + /MyoD + cells were also found in the Noggin-treated DPSCs groups compared with the DPSCs groups, indicating an increase in donor-derived SCs . These results suggest that Noggin-treated DPSCs partially benefited the SC population. This benefit might provide support for muscle regeneration in VML injury. Section title: 4. Discussion Educational score: 4.113343238830566 Domain: biomedical Document type: Study Language: en In our study, we found that Noggin could promote the MyoD of DPSCs and increase the generation of satellite-like cells by regulating the BMP/Smad signaling pathway. Then, we treated VML model mice with Noggin-pretreated DPSCs bioconstructs and found that Noggin pretreatment improved the repair of muscle injury. Our work implies that Noggin can promote the MyoD of DPSCs, rendering DPSCs an important source of muscle stem cells for muscle repair. Section title: 4. Discussion Educational score: 4.52818489074707 Domain: biomedical Document type: Study Language: en Noggin, a secreted BMP antagonist, was promoted by Wnt-1 in medial somites and found to promote the expression of MyoD in embryonic tissues . Our results showed that Noggin could promote the MyoD of DPSCs and increase the number of Pax3 + /Pax7 + cells . Previous studies have concluded that Pax3-mediated myogenesis requires an environment where Six1 synergizes with Eya2 to activate the expression of MyoD . Our results showed that Noggin promoted the expression of Six1 and Eya2, suggesting that these Pax3 + /Pax7 + cells were in an environment conducive to subsequent MyoD. We also found that Noggin eliminated Smad phosphorylation, accompanied by decreased levels of MSX1 and ID1 . Consistent with our results, Noggin inhibited p-Smad1/5/8 and repressed ID1 and MSX1 expression, impeding myoblast differentiation [ 29 – 31 ]. Sustained release of BMP4 significantly decreased Pax7 + and MyoD + SC densities . When BMP4 was added to antagonize Noggin, we observed inhibition of the MyoD of DPSCs. Our data revealed that Pax3/7 might be related to the BMP/Smad pathway in the context of Noggin effects. Previous research has shown that activated Pax7 + cells coexpressed p-Smad1/5/8 in injured mouse skeletal muscle sections . In the competition for MSX1, the expression of downstream effectors of the BMP/Smad pathway, Pax3 or Pax7, was increased towards the myogenic fate . Knockout of Noggin leads to increased levels of p-Smad1/5/8, ID1, and MSX1, and a decreased population of mesenchymal Pax7 + muscle precursor cells . Section title: 4. Discussion Educational score: 4.144454002380371 Domain: biomedical Document type: Study Language: en Muscle regeneration is ensured by Pax7 + SCs; when activated, SCs are able to divide asymmetrically, enabling self-renewal or myoblast differentiation . ESCs-derived Pax7 + cells can give rise to both muscle fibers and Pax7 + satellite-like cells when grafted in vivo, suggesting that these cells might behave like SCs . In our study, we found that Noggin expanded Pax7 + cells in DPSCs. After 21 days of differentiation in vitro, the expression levels of late myogenic marker genes (MyHC) and SC marker (Pax7) were both higher with Noggin treatment . These results revealed that Noggin could promote the generation of Pax7 + satellite-like cells from DPSCs . Section title: 4. Discussion Educational score: 4.110494136810303 Domain: biomedical Document type: Study Language: en We then transplanted Noggin-pretreated DPSCs combined with Matrigel into defects of a VML injury model. In our study, Noggin-pretreated DPSCs bioconstructs decreased the size of the defect and increased the number of donor-derived hLaminAC + /Pax7 + and hNu + /MyoD + cells in mouse muscles . Some scholars transplanted ESC- or PSC-derived muscle progenitor cells into NSG-mdx4cv mice and found that donor-derived hLaminAC + /Pax7 + SCs contributed to the muscle stem cell pool in tissues . The successful use of Pax7 + satellite-like cells derived from ESCs in muscle injury may be attributed to their self-renewal capacity within the SCs pool . Section title: 4. Discussion Educational score: 4.222661972045898 Domain: biomedical Document type: Study Language: en DPSCs are a type of MSCs from dental pulp, with easy access, long-term storage, and high proliferative and differentiation potential compared to other types of MSCs [ 9 – 12 ] and can serve as a proper stem cell source for muscle regeneration. SCs derived from MSCs were shown to give rise to differentiated muscle fibers . However, the contribution of 5-Aza-induced DPSCs engraftment might only be a paracrine effect instead of MyoD . In contrast to muscle-derived cells or ESCs, the transplant efficiency of MSCs was relatively low, with ~1%–2% as hybrid myofibers . We speculate that the low myogenic efficiency may be attributed to fewer SCs. Our results indicated that Noggin expanded Pax7 + satellite-like cells in DPSCs, which increased the percentage of donor-derived cells to ~15%–20% . This finding suggests that Noggin-treated DPSCs can partially benefit the muscle SC pool and promote myogenic repair in VML injury. Section title: 5. Conclusions Educational score: 4.224750995635986 Domain: biomedical Document type: Study Language: en In this study, we demonstrate that Noggin, a secreted BMP antagonist, is an important protein that promotes MyoD and increases the generation of Pax7 + satellite-like cells by modulating BMP/Smad signaling. These satellite-like cells combined with Matrigel can effectively repair mouse tibialis anterior muscle injury in a VML model, leading to increased repair size and decreased scar tissue. Noggin-treated DPSCs bioconstructs can benefit the Pax7 + SC pool and promote muscle regeneration. This finding suggests the importance of producing Pax7 + SCs and highlights the potential of these SCs for improved muscle regeneration. Noggin might enhance the generation of satellite-like cells from DPSCs to provide potential transplantable stem cells for muscle regeneration. Further investigations are needed to study the relationship between Pax7 and Smad/its downstreams in order to improve myogenic induction conditions and enhance the transplantation of DPSCs into damaged muscle tissue. | Study | biomedical | en | 0.999995 |
PMC11699991 | Section title: 1. Introduction Educational score: 3.1652307510375977 Domain: biomedical Document type: Study Language: en Atlantic salmon ( Salmo salar ) is the most important aquaculture species in Norway. In order to reduce feed costs and develop sustainable feed, high-energy diets and the replacement of marine-based ingredients with plant-based ingredients have been widely applied in Norwegian salmon feed in recent years . These strategies have resulted in an increase in the fat level, a decrease in the n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) level, and decreases in the zinc (Zn) level and availability in feed . Section title: 1. Introduction Educational score: 4.089594841003418 Domain: biomedical Document type: Study Language: en Zn is crucial for multiple physiological functions in fish, such as forming Zn-containing enzymes and promoting skeletal development . Feeding fish with Zn-deficient diets induced Zn-deficiency diseases, such as lens cataracts, skin erosion, and skeletal malformation [ 5 – 7 ]. Considering reductions in Zn level and availability in plant-based feeds, additional Zn must be provided to meet the Zn requirement in S. salar . However, Zn digestibility is lower than 40%, and Zn retention is less than 30% in S. salar , indicating that a high level of Zn in the feed would have a negative environmental impact. In this regard, the EU limited the upper Zn level in salmon feed to 180 mg kg −1 , and the EFSA has suggested further reducing it to 150 mg Zn kg −1 . Therefore, it is necessary to increase knowledge on enhancing Zn availability and utilization in salmon feeds. Section title: 1. Introduction Educational score: 4.245183944702148 Domain: biomedical Document type: Study Language: en Even though fish can osmotically absorb aqueous minerals, feed remains the predominant resource to maintain body minerals homeostasis . In the case of Zn, the absorptive site is primarily the intestine . Similar to mammals, fish have two families of cellular Zn transporters: zips (Zrt/Irt-like proteins, transport Zn into cytosol) and znts (Zn transporters, transport Zn out of the cell) . In addition, because zip4 and znt1 are located at the apical and basolateral epithelium, respectively, they are regarded as two key proteins in intestinal Zn uptake and transport . After Zn enters the cell, metallothionein (mt) would bind with Zn to prevent Zn toxicity . If cellular Zn levels exceed the mt binding capacity, transcription factors such as metal regulatory transcription factor 1 (mtf1) would be upregulated, thus increasing the expression of znt1 and promoting the Zn efflux . However, very little is known about the regulation of Zn transporters in Atlantic salmon. Section title: 1. Introduction Educational score: 4.189953804016113 Domain: biomedical Document type: Study Language: en Intestinal Zn uptake and transport are affected by several factors, including anti-nutrient factors (such as phytate), mineral interactions (with iron [Fe] and manganese [Mn]), chemical form of Zn (chelated vs. inorganic) . Furthermore, dietary component interaction might be another factor impacting intestinal Zn uptake and transport, such as fat level and n-3 LC-PUFA. In mammals, higher plasma and femur Zn levels were observed in rats fed with 3% fat diets compared to those in the 26% fat diet group . Also, rats fed with fish oil-based diet (rich in n-3 PUFA) obtained higher hepatic Zn levels than those fed with a lard-based diet (rich in saturated fatty acids [SFA]), indicating that n-3 PUFA may have a positive effect on Zn absorption . A recent study in Atlantic salmon also reported that fish fed with low-fat/high n-3 LC-PUFA diets obtained the higher whole-body Zn level compared to those fed with high-fat/low n-3 LC-PUFA diets . Due to the crucial physiological role and the upper legal limitations in feed, understanding the Zn uptake as affected by dietary components is important to the aquaculture industry. Section title: 1. Introduction Educational score: 3.4651551246643066 Domain: biomedical Document type: Study Language: en Furthermore, dietary Zn may impact intestinal fat transport. According to a previous study, rats fed Zn-deficient diets obtained lower triacylglycerol (TAG) absorption and abundant intestinal lipid droplets, demonstrating the importance of Zn on intestinal fat transport . However, the effect of dietary Zn on intestinal fat transport in fish is still unclear. Section title: 1. Introduction Educational score: 4.075004577636719 Domain: biomedical Document type: Study Language: en In previous fish nutrition studies, samples were generally collected at fasting status or 24 h after the final meal. Worth mentioning, different postprandial sampling time points may obtain different plasma minerals and TAG levels, indicating the importance of the sampling time selected when investigating plasma parameters. However, reports on postprandial plasma profiles related to lipids and minerals in S. salar are still scarce. Therefore, the aim of this study was to investigate the combined effect of dietary fat level and Zn, and the combined effect of dietary n-3 LC-PUFA and Zn on postprandial plasma profiles in S. salar . In addition, intestinal gene expression related to Zn and lipid uptake and transport in selected postprandial time points were also analyzed. Section title: 2.1. Experimental Diets Educational score: 4.103348731994629 Domain: biomedical Document type: Study Language: en Diet formulations and analyzed nutritional compositions are shown in Table 1 . In trial 1, four experimental diets containing one of two different levels of fat (%) and Zn (mg kg −1 ) were formulated and referred to as follows (fat/Zn): HFHZ, 35/200; HFLZ, 35/120; LFHZ, 32/200; LFLZ, 32/120. In comparison, Norwegian S. salar commercial feeds for growing out contain 35%–39% fat level . Additionally, the EU restricts the maximum Zn in S. salar feed to 180 mg kg −1 , whereas 120 mg Zn kg −1 is a suboptimal level in S. salar plant-based feed . Fat levels in the feed were adjusted by varying the levels of fish oil and rapeseed oil, with wheat gluten, guar meal, and wheat meal used to balance the composition. Zn levels in the diet were modified by adding Zn sulfate at different doses. Section title: 2.1. Experimental Diets Educational score: 4.108852386474609 Domain: biomedical Document type: Study Language: en In trial 2, three experimental diets were used. The first two experimental diets (HFHZ and HFLZ) were the same as trial 1, while the third diet contained higher n-3 LC-PUFA (with high fat and Zn levels). Three diets were named as follows (eicosapentaenoic acid [EPA] + docosahexaenoic acid [DHA], % of total feed/Zn, mg kg −1 ): HFHZ, 2.0/200; HFLZ, 2.0/120; HPUHZ, 3.2/200 (average level of DHA + EPA in Norwegian commercial salmon feed is 2.2% . Different dietary n-3 LC-PUFA levels between HFHZ and HPUHZ were achieved by adjusting the ratio of rapeseed oil and fish oil. In addition, due to the lack of cholesterol (CHO) in rapeseed oil, additional CHO was provided in HFHZ, HFLZ, LFHZ, and LFLZ to obtain a similar CHO level as the HPUHZ diet. Section title: 2.2. Fish Feeding and Management Educational score: 1.5241917371749878 Domain: biomedical Document type: Other Language: en Two feeding trials were performed at the Matre Research Station of the Institute of Marine Research in Norway. All the sampling procedures were performed on euthanized fish. The study was evaluated by the animal experimentation administration of IMR (Forsksdyrforvaltningen) and approved as a noninvasive animal study conducted in accordance with Norwegian regulations on the use of animals in research in line with the EU directive 2010/63/EU. This trial was exempt from an animal ethics approval (FOTS application) to the Norwegian Food Safety Authority, according to the regulation “FOR-2015-06-18-761 Regulation concerning the use of animals for scientific purposes, § 6. Godkjenning av forsøk.” Section title: 2.2. Fish Feeding and Management Educational score: 3.982903242111206 Domain: biomedical Document type: Study Language: en In both feeding trials, S. salar were fed a commercial feed (Skretting Norway) for 2 weeks to acclimate them to experimental conditions. In trial 1 , 280 S. salar with IW of 718 ± 58 g were randomly distributed to eight tanks , with 35 fish in each. Four experimental diets (HFHZ, HFLZ, LFHZ, and LFLZ) were randomly assigned to the tanks in duplicates. Fish were provided experimental diets twice per day until apparent satiation (9:00 and 16:00) for 4 weeks. During the nutritional intervention, the fish were held in full-strength seawater with the water temperature ranged from 8 to 9°C. Section title: 2.2. Fish Feeding and Management Educational score: 3.913088083267212 Domain: biomedical Document type: Study Language: en In trial 2 , 225 fish with a weight of 667 ± 20 g were randomly distributed to nine tanks with 25 fish in each. Three experimental diets (HFHZ, HFLZ, and HPUHZ) were randomly assigned to nine tanks in triplicates. Further, fish were fed with one of three experimental diets twice daily until apparent satiety (9:00 and 16:00) for 4 weeks. During the nutritional intervention, the fish were held in full-strength seawater, and the water temperature ranged from 10 to 12°C. Section title: 2.3. Postprandial Sampling Educational score: 4.108030319213867 Domain: biomedical Document type: Study Language: en As shown in Figure 1 , at the termination of the feeding trial, fish were fasted for 48 h to completely empty their gastrointestinal tract, which was considered their preprandial status (0 h), and their blood and intestine were sampled. Next, fish were fed with respective diets for 2 h, and the postprandial time was calculated after this feeding. Similar samples (blood and mid-intestine) were collected at different postprandial time points. In trial 1, five fish were sampled from each tank (total 10 fish from each dietary treatment group) at 2, 4, 8, 14, 24, and 36 h after feeding. In trial 2, three fish from each tank (total nine fish from each dietary treatment group) were sampled at 4, 6, 10, 14, 26, 32, and 38 h after the feeding. Section title: 2.3. Postprandial Sampling Educational score: 4.091835975646973 Domain: biomedical Document type: Study Language: en During the sampling, fish were randomly and carefully removed from the tank to reduce stress and then euthanized with an overdose of tricaine mesylate (MS-222, Tricaine Pharmaq). The euthanized fish were measured for weight and length, following which blood was drawn from the caudal vein using a lithium heparin-coated vacutainer. The blood samples were centrifuged (14,200 g, 2 min, 4°C) to obtain supernatant (plasma), which was then frozen on dry ice for plasma parameters analysis. Afterward, the mid intestine (just behind the pyloric ceca) was collected after emptying the intestinal contents and immediately frozen in liquid nitrogen for further analysis. During the sampling, the state of feed remnants in the stomach was inspected and recorded, which served as the indicator of fish ingestion. The number of fish without pellets in their stomachs is presented in Table S1 . If the fish hadn't eaten, the sample was excluded from further analysis. Section title: 2.4. Chemical Composition Analysis Educational score: 3.958801031112671 Domain: biomedical Document type: Study Language: en The chemical composition in the feed was analyzed after homogenization. Moisture, protein, and ash were analyzed by drying to constant weight (105°C), the Kjeldahl method (N x 6.25), and muffle furnace (550°C), respectively; fat level in feeds was analyzed by acid hydrolysis and extraction with diethyl ether. Section title: 2.4. Chemical Composition Analysis Educational score: 4.132692813873291 Domain: biomedical Document type: Study Language: en FAs composition in feed was analyzed as described in . Briefly, after isolation of lipids from feed (Folch solution) and completely dried chloroform–methanol phase (using N 2 gas), residual lipids were trans-methylated overnight with 2′-2′-dimethoxy propane, methanolic HCl, and benzene at room temperature. Methyl esters were isolated and analyzed using a gas chromatograph equipped with a split injector, an SGE BPX70 capillary column (SGE Analytical Science), and a flame ionization detector. He 2 served as the carrier gas, with the injector and detector temperatures both maintained at 280°C. The oven temperature was initially increased from 50 to 180°C at a rate of 10°C per minute, then further increased to 240°C at a rate of 0.7°C per minute. Individual FA methyl esters were identified by comparison with previously characterized standards. Results were further analyzed using HP ChemStation software. FAs results were presented as a percentage of the total fatty acids in the feed, and the absolute amount of EPA + DHA per gram of feed was calculated using C23:0 methyl ester as the internal standard. Section title: 2.4. Chemical Composition Analysis Educational score: 4.132390022277832 Domain: biomedical Document type: Study Language: en Mineral contents (Zn, copper [Cu], Fe, Mn, and selenium [Se]) in diet and plasma were measured using inductively coupled plasma mass spectrometry (ICP-MS), as described in . Specifically, samples (0.2 g dry feed with 0.5 mL deionized water or 0.5 mL plasma) and 2 mL concentrated HNO 3 were added into test tubes and digested in a Milestone UltraWave Microwave Digestion System (Milestone Inc., USA). Further, digested samples were diluted to 25 mL by deionized water. Next, minerals were analyzed in ICP-MS (Thermo Scientific, USA) equipped with an autosampler (FAST SC-4Q DX, Elemental Scientific, USA). The eluate was introduced into the nebulizer tube of the ICP-MS, and minerals were analyzed in the KED reaction mode. To correct for instrumental drift during the analysis, a solution of germanium and rhodium was added online. The ICP-MS was tuned prior to analysis using a 1 ppb tuning solution B (Thermo Fisher, in 2% HNO3 and 0.5% HCl). Data collection and processing were carried out using Qtegra software (Thermo Scientific). An external calibration curve ranging from 10 to 500 ng mL −1 was utilized to quantify the minerals. Section title: 2.4. Chemical Composition Analysis Educational score: 3.6966493129730225 Domain: biomedical Document type: Study Language: en Plasma lipids, including TAG, CHO, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and total protein (TP), were measured using Pentra C400 (HORIBA; Montpellier, France), following the instruction of the manufacturer. Section title: 2.5. Gene Expression Educational score: 3.8759796619415283 Domain: biomedical Document type: Study Language: en Intestinal samples for gene expression analysis were selected at three postprandial time points based on the plasma TAG profile: exogenous lipid had not entered circular system (trial 12 h; trial 2:4 h), exogenous lipid entered circular system (trial 1 and trial 2:14 h) and exogenous lipid abundantly entered circular system (trial 1:24 h; trial 2:26 h). Section title: 2.5. Gene Expression Educational score: 4.1146240234375 Domain: biomedical Document type: Study Language: en Candidate genes for qPCR included two house-keeping genes ( β -actin and elongation factor 1 alpha [ef-1 α ]), five genes related to Zn transport (solute carrier family 39 member 4 [zip4], metallothionein A [mta], metallothionein B [mtb], solute carrier family 30 member 1 [znt1], mtf1) and 11 genes related to lipid transport and metabolism (Niemann-Pick C1-like 1 [npc1l1], cluster of differentiation 36 [cd36], fatty acid transporter protein 4 [fatp4], fatty acid transporter protein 6 [fatp6], fatty acid-binding protein 2 [fabp2], diglyceride acyltransferase 1 [dgat1], microsomal triglyceride transfer protein [mtp], apolipoprotein B [apob], carnitine O-palmitoyltransferase 1 [cpt1], monoacylglycerol O-acyltransferase 2 [mgat2], apolipoprotein A-IV [apoa4]). Section title: 2.5. Gene Expression Educational score: 4.146834373474121 Domain: biomedical Document type: Study Language: en Total RNA from intestine samples was isolated using the Maxwell HT simplyRNA Kit (Promega, USA) and the Biomek 4000 automated liquid handler (Beckman Coulter, USA), following the instructions of the manufacturer. In addition, the quality and quantity of isolated RNA were measured in the Bioanalyzer and the spectrophotometer , respectively. Further, an inverse transcription kit (Thermo Fisher Scientific, USA) was used to synthesize cDNA, following the instructions of the manufacturer. Afterward, RT-PCR for target genes was quantified on the qPCR instrument (Bio-Rad, USA) with SYBR GREEN PCR Master Mix (Roche-Norge, Norway) , with the following program: 10 min preincubation at 95°C, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a melting curve. The expression level of the target gene was normalized on the CFX Maestro (Bio-Rad, USA) based on two house-keeping genes. The associated information of candidate genes is listed in Table 2 . Except for β -actin and ef-1 α , other primers were newly designed through NCBI online primer designing tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? ). One-step RT-PCR (QIAGEN one-step RT-PCR kit) was used to evaluate the primer specificity, according to the instructions of the manufacturer. Section title: 2.6. Statistical Analysis Educational score: 4.0802226066589355 Domain: biomedical Document type: Study Language: en The normality and variance of the data homogeneity were analyzed under the Shapiro–Wilk test and Levene's test ( p > 0.05), respectively. In trial 1, the weight gain (WG) was analyzed by two-way ANOVA, with fat level and Zn as independent variables ( n = 2). In addition, results from other growth performance parameters (initial weight [IW], initial condition factor [ICF], final weight [FW], final condition factor [FCF]) were analyzed by “two-way nested ANOVA,” with fat level and Zn as independent variables (tank as a random factor). Furthermore, results from plasma minerals, plasma lipids, and intestinal gene expression were analyzed by three-way nested ANOVA, with fat level, Zn, and time as independent variables (tank as a random factor), and the significant results between “0h” and “selected time point” were analyzed by multiple comparisons with “Dunnett's test” (5 samples from each tank, total 10 samples from each dietary treatment). In trial 2, the WG was analyzed by t -test between the HFHZ and HFLZ (or HFHZ vs. HPUHZ) groups ( n = 3). In addition, results from other growth performance parameters (IW, ICF, FW, FCF) were analyzed by t -test nested between the HFHZ and HFLZ (or HFHZ vs. HPUHZ) groups (tank as a random factor). Furthermore, results from plasma minerals, plasma lipids, and intestinal gene expression were analyzed by two-way nested ANOVA, with diet (Zn or n-3 LC-PUFA) and time as independent variables (tank as a random factor), and the significant results between “0h” and “selected time point” were also analyzed by multiple comparisons with “Dunnett's test” (three samples from each tank, total nine samples from each dietary treatment). If the p -value was <0.05, results between groups were regarded as significant. All experimental results were presented as the mean ± standard deviation (SD). Statistical analysis was performed in the software GraphPad Prism 8 (Insightful Science, USA) and R , respectively. Section title: 3.1. Growth Performance Educational score: 3.963216781616211 Domain: biomedical Document type: Study Language: en The growth performance of S. salar fed the different diets from trial 1 and trial 2 are presented in Table 3 . In trial 1, the WG of the fish ranged from 18.5% to 22.2%, while in trial 2, it ranged from 39.6% to 44.9%. In addition, the survival rates (SRs) of fish fed with different diets were 100% in trial 1 and 94%–98% in trial 2. However, 4-week dietary treatments did not impact the fish growth among all experimental groups in trial 1 and trial 2 ( p > 0.05). Section title: 3.2. Postprandial Plasma Minerals Educational score: 4.11253023147583 Domain: biomedical Document type: Study Language: en In trial 1, reduced Zn level in feed significantly reduced the postprandial plasma Zn level . Additionally, postprandial plasma Fe ( p < 0.01) and Cu ( p < 0.05) profiles were significantly impacted by the time change. Peak values of plasma Fe (14–24 h, p < 0.01) and Cu (2−4 h and 36 h, p < 0.05) appeared at specific postprandial time points. Further, the postprandial plasma Cu profile was significantly affected by Time x Fat ( p < 0.05). Compared to low-fat diet treatment, high-fat diet treatments reduced the plasma Cu level at postprandial 4 h but increased the plasma Cu level at postprandial 36 h. On the contrary, postprandial plasma profiles of Zn, Mn, Fe, and Se were not affected by dietary fat level ( p > 0.05). Section title: 3.2. Postprandial Plasma Minerals Educational score: 4.105372428894043 Domain: biomedical Document type: Study Language: en In trial 2, the postprandial plasma Zn profile was not affected by dietary Zn ( p =0.20) . In addition, HFHZ treatment significantly reduced the postprandial plasma Mn level compared to HFLZ treatment ( p < 0.05). In addition, fish fed with the high n-3 LC-PUFA diet obtained significantly higher postprandial plasma Mn level compared to those fed with the low n-3 LC-PUFA diet ( p < 0.05), which is attributed to different Mn levels in diets ( Table 1 ). However, postprandial plasma profiles of Zn, Fe, Cu, and Se were not affected by dietary n-3 LC-PUFA ( p > 0.05). Section title: 3.3. Postprandial Plasma Lipids Educational score: 4.113565921783447 Domain: biomedical Document type: Study Language: en The postprandial plasma TAG profile was significantly impacted by the time changes in trial 1 . After 14 h of feeding, the plasma TAG level had risen significantly ( p < 0.01) compared to that at preprandial (0 h). Further, the peak value of plasma TAG appeared at postprandial 24 h and was statistically elevated to 36 h ( p < 0.01). However, dietary fat level and Zn did not impact the postprandial plasma TAG profile ( p > 0.05). Section title: 3.3. Postprandial Plasma Lipids Educational score: 4.125705242156982 Domain: biomedical Document type: Study Language: en In trial 2, the postprandial plasma TAG profile was significantly affected by the time change , which agrees with the result in trial 1. Compared to preprandial status (0 h), plasma TAG level was significantly higher between 10 and 38 h after the meal ( p < 0.05). Also, plasma CHO was affected by postprandial time ( p < 0.05), and the result at postprandial 32 h was significantly higher than that at postprandial 6 h. However, the postprandial plasma TAG profile was not affected by dietary n-3 LC-PUFA and Zn ( p > 0.05). Section title: 3.4. Postprandial Intestinal Gene Expression Related to Zn Uptake and Transport Educational score: 4.115528106689453 Domain: biomedical Document type: Study Language: en As shown in Figure 6 (trial 1, all results presented in Table S4 ), intestinal mRNA expression related to Zn uptake and transport was not affected by dietary Zn and fat level ( p > 0.05). However, gene expression related to Zn uptake and transport, including zip4 ( p < 0.01), znt1 ( p < 0.05), and mtf1 ( p < 0.01), were significantly affected by postprandial timing. Compared to the 2 h postprandial mark, zip4 , znt1 , and mtf1 were upregulated at 24 h. Section title: 3.4. Postprandial Intestinal Gene Expression Related to Zn Uptake and Transport Educational score: 4.112766742706299 Domain: biomedical Document type: Study Language: en In trial 2, reduced Zn level in feed significantly downregulated the intestinal mRNA expression of mta and mtb postprandially . In addition, postprandial timing significantly impacted intestinal mRNA expression related to Zn uptake and transport, including zip4 ( p < 0.05), znt1 ( p < 0.01), and mtf1 ( p < 0.01). At 26 h postprandially, zip4 was upregulated, while znt1 and mtf1 were downregulated compared to the 4 h mark. Section title: 3.5. Postprandial Intestinal Gene Expression Related to Lipid Uptake and Transport Educational score: 4.121796607971191 Domain: biomedical Document type: Study Language: en In trial 1, intestinal mRNA expression related to lipid uptake and transport was not significantly affected by dietary Zn . However, the expression levels of dgat1 , mtp , and cpt1 were significantly influenced by the interaction between time and fat levels ( p < 0.05), with reduced fat level downregulating the expression of these genes at 14 h postprandially. Additionally, most mRNA expressions related to lipid uptake and transport were significantly impacted by postprandial timing ( p < 0.05). Compared to postprandial 2 h, CHO uptake gene ( npc1l1 ) was downregulated at 14 h, and genes related to TAG uptake and transport ( cd36 , fatp4 , fatp6 , mgat2 , dgat1 , mtp , apob ) and fatty acid β -oxidation ( cpt1 ) were downregulated at 24 h. Section title: 3.5. Postprandial Intestinal Gene Expression Related to Lipid Uptake and Transport Educational score: 4.092777252197266 Domain: biomedical Document type: Study Language: en In trial 2, dietary Zn did not impact intestinal mRNA expression related to lipid uptake and transport , consistent with the results observed in trial 1. However, postprandial timing had a significant impact on most of these gene expressions ( p < 0.01). At 26 h postprandially, genes related to lipid transport and metabolism, including npc1l1 , cd36 , fatp4 , fatp6 , fabp2 , dgat1 , mtp , apob , and cpt1 , were downregulated compared to the 4 hr mark. Section title: 4.1. Postprandial Kinetics of Zn and Other Minerals Educational score: 4.385165214538574 Domain: biomedical Document type: Study Language: en Increasing knowledge on the postprandial absorption kinetics of Zn and its dietary impact could improve the understanding on dietary Zn availability. Different from reports in rainbow trout and hybrid striped bass , there did not appear a significant absorptive peak in the postprandial plasma Zn profile in the present two trials. In the above-mentioned studies, one or more of the diets contained dietary Zn levels that were deficient or very low, resulting in very low basal plasma Zn concentration and hence inducing a visible peak in plasma Zn immediately after a meal. In trial 1, although postprandial plasma Zn concentration was correlated with dietary Zn level, as reported in other fish at fasting status [ 29 – 31 ], mRNA expression related to intestinal Zn uptake ( zip 4 ) and transport ( znt 1 ) was not affected by dietary Zn. These results are due to the passive uptake mechanism in the intestine. It is well known that active uptake mechanisms in the intestine, primarily DMTs, ZIPs, and ZnTs, are upregulated at limiting dietary Zn concentrations, whereas at optimal or high dietary Zn levels, passive uptake mechanisms are dominating . Apart from intestinal Zn absorption, other Zn metabolic processes, such as storage and excretion, are also attributed to the regulation of plasma Zn status in vertebrates . In contrast with trial 1 (8–9°C), the postprandial plasma Zn was not affected by dietary Zn in trial 2 (10–12°C), which is due to the different water temperatures. As reported by Bervoets, Blust, and Verheyen , increased temperature would increase metal diffusion and reaction rates, resulting in increased metal accumulation in tissues. Also, increased temperature and dietary Zn level interactively increased hepatic Zn accumulation in Pelteobagrus fulvidraco . In trial 2, increased Zn level in feed increased the intestinal mt ( mta and mtb ) expression, indicating that more exogenous Zn is temporarily stored in the intestinal epithelium by binding with mt before transport into plasma , and thus the postprandial plasma Zn level was not affected by dietary Zn. Temperature-dependent intestinal Zn transport was also reported in other fish studies [ 37 – 39 ]. These results suggest the importance of the regulatory effect of the intestine on plasma Zn . Therefore, a mismatch between Zn absorptive markers in the intestine and plasma Zn status at the different time-points can be attributed to the differential regulative mechanisms. Section title: 4.1. Postprandial Kinetics of Zn and Other Minerals Educational score: 4.1956915855407715 Domain: biomedical Document type: Study Language: en Besides the differential Zn regulation of absorptive epithelia and systemic regulation, marine fish present different intestinal Zn adaptations when subjected to different dietary Zn levels . In the two present trials, intestinal mRNA expression related to Zn influx ( zip 4 ) and efflux ( mtf 1 and znt 1 ) were not affected by dietary Zn, indicating the dynamic balance between intestinal Zn influx and efflux. In comparison, Sparus aurata (marine fish) fed with Zn-deficient diets (7.9 mg Zn kg −1 ) increased intestinal Zn influx but reduced intestinal Zn efflux to maintain intestinal Zn homeostasis, whereas fish fed optimum Zn diet (64.7 mg Zn kg −1 ) maintained the dynamic balance between intestinal Zn influx and efflux . These results indicate that suboptimal Zn supplementation in S. salar feed (120 mg Zn kg −1 ) does not impair the dynamic balance between intestinal Zn influx and efflux after 1-month feeding trial. In addition, increased n-3 LC-PUFA or reduced fat level in feed increased whole-body Zn content in S. salar . However, intestinal Zn uptake and transport were not affected by dietary n-3 LC-PUFA and fat level in the present study. Also, the postprandial plasma Zn profile was not affected by fat level or n-3 LC-PUFA. These results suggest that dietary fat level and n-3 LC-PUFA might impact other Zn metabolism processes (such as storage and excretion) rather than intestinal absorption, resulting in impacting whole-body Zn status. Section title: 4.1. Postprandial Kinetics of Zn and Other Minerals Educational score: 4.233063697814941 Domain: biomedical Document type: Study Language: en Apart from Zn, postprandial plasma levels of other divalent trace minerals were also affected in the studies. In trial 1 (8–9°C), the postprandial plasma peak values of Fe and Cu appeared later than the report in Oncorhynchus mykiss (17.5°C) , which is due to different experimental temperatures. Increased water temperature in an appropriate range is generally associated with an increased passage rate of chyme through the gastrointestinal tract of fish . In addition, two discontinuous peak values of postprandial plasma Cu were observed in trial 1, which is similar to the report in O. mykiss . It can be explained that the Cu absorption site in S. salar occurs in the stomach, mid-intestine, and posterior intestine, similar to the report in O. mykiss . Furthermore, increased fat level in feed reduced plasma Cu level at postprandial 4 h but increased plasma Cu level at postprandial 36 h, indicating increased dietary fat level depressed Cu absorption in the stomach but promoted Cu absorption in the intestine. Increased fat level in feed significantly increased the Cu utilization in rats (2% vs. 8% fat level) . However, knowledge on the effect of fat level on Cu availability in animals is still scarce. In addition, mineral interaction is another important factor impacting mineral status in fish . In trial 2, postprandial plasma Mn level was negatively correlated with dietary Zn level, which may be due to the competitive effect between Zn and Mn during absorption. In the case of Mn, its active transport during absorption is mediated by the divalent metal transporter 1 (DMT1), which also transports other divalent metals, such as Zn, Fe, and Cu . Also, previous studies reported that high Zn treatment significantly reduced intestinal Fe and Cu absorption in O. mykiss . In the present study, postprandial plasma Fe and Cu were not affected by dietary Zn, indicating that S. salar fed with diets ranging from 120 to 200 mg Zn kg −1 did not impact Fe and Cu absorption. Section title: 4.2. Postprandial Lipids Kinetics Educational score: 4.131181240081787 Domain: biomedical Document type: Study Language: en The pyloric ceca and mid-intestine are the two sites for lipid digestion and absorption in S. salar . In the present study, the plasma TAG began to significantly increase at postprandial 14 h (trial 1, 8−9°C) /10 h (trial 2, 10−12°C), indicating the time point when chyme started to enter the pyloric ceca and the exogenous lipid enters the circulation system . Although a small proportion of plasma TAG may be attributed to hepatic VLDL secretion, lipid metabolism in the liver was not analyzed in the current study. In addition, the peak value of plasma TAG appeared at postprandial 24−36 h (trial 1, 8–9°C) and 32 h (trial 2, 10 −12°C), respectively, indicating the majority of chyme had reached the pyloric ceca and mid-intestine. Section title: 4.2. Postprandial Lipids Kinetics Educational score: 4.115785121917725 Domain: biomedical Document type: Study Language: en FAs are taken up and transported by the intestine through membrane-associated proteins, although a small proportion is performed by passive diffusion . In the two present trials, intestinal mRNA expression related to lipid uptake and transport was not affected by dietary Zn. Additionally, postprandial plasma TAG level was not affected by dietary Zn. In previous studies, Zn-deficient diet treatment increased intestinal fat accumulation in rats , and it is correlated with inhibition of intestinal lipoprotein formation . Also, Zn-deficiency impaired intestinal lipid transport in S. aurata . Therefore, these results suggest that the 120 mg Zn kg −1 in S. salar feed is not low enough to impact intestinal lipid transport (compared to the 200 mg Zn kg −1 diet). Section title: 4.2. Postprandial Lipids Kinetics Educational score: 4.159226894378662 Domain: biomedical Document type: Study Language: en In trial 1, the postprandial plasma TAG was not affected by dietary fat level, suggesting that dietary fat level (32%–35%) did not alter the intestinal TAG transport. Similarly, a recent S. salar study reported that increased fat level in the feed from 16% to 25% did not impact the mRNA expression of the key lipoprotein assembly gene ( mtp ) . However, in trial 1, reduced fat level in feed upregulated intestinal mRNA expression related to TAG re-esterification ( dgat1 ), lipoprotein assembly ( mtp ), and β -oxidation ( cpt1 ) at postprandial 14 h, when the chyme entered the pyloric intestine. These results may be linked to the higher wheat gluten content in the low-fat diet compared to the high-fat diet (17.5% vs. 10.2%). A previous study showed that increasing dietary wheat gluten from 15% to 30% elevated the intestinal gene expression associated with lipid metabolism and transport in S. salar . However, it is important to note that mRNA expression may not directly correspond to protein synthesis levels due to regulatory processes such as mRNA editing, modification, and degradation . In trial 1, the downregulation of dgat1 , mtp , and cpt1 genes at postprandial 24 h compared to 14 h suggests possible mRNA degradation. Future studies could provide more comprehensive insights by simultaneously quantifying mRNA and protein expression. Section title: 4.2. Postprandial Lipids Kinetics Educational score: 4.083452224731445 Domain: biomedical Document type: Study Language: en In the two present trials, the postprandial plasma CHO level was not affected by the dietary Zn, fat level, and n-3 LC PUFA, which is attributed to the similar CHO level among different experimental diets, as reported in other fish studies . Similarly, changing the dietary fat level (31% vs. 38% fat level) or fatty acid profiles did not impact the plasma CHO level in S. salar . Also, suboptimal supplementation of Zn in feed did not alter the plasma CHO level in other fish [ 63 – 66 ]. Section title: 5. Conclusion Educational score: 4.130048751831055 Domain: biomedical Document type: Study Language: en To sum up, increased Zn level in feed significantly increased the postprandial plasma Zn level in S. salar (8 −9°C). Analyzed markers related to intestinal Zn uptake and transport were not affected by dietary fat level (32%–35% of feed) and n-3 LC-PUFA (2%−3.2% EPA + DHA of feed). Since dietary fat and n-3 LC-PUFA affect whole-body Zn content in Atlantic salmon, further study is needed to explore how these components influence other Zn metabolism processes, such as retention and excretion. Additionally, markers related to intestinal fat transport were unaffected by Zn levels ranging from 120 to 200 mg kg −1 in the feed. | Study | biomedical | en | 0.999996 |
PMC11700017 | Section title: Introduction Educational score: 3.842710256576538 Domain: biomedical Document type: Study Language: en In numerous countries, individuals with brain injuries are a primary focus of rehabilitation, and driving assessments play an important role in occupational therapy. Post-stroke evaluations encompass off-road tests, such as neuropsychological examinations and driving simulators, and on-road tests involving actual driving alongside physical function assessments . The on-road test is considered the gold standard for examining real-world driving skills among these assessments . Because various studies have aimed to determine the predictability of on-road test results , it is an essential factor to ensure the reliability and validity of on-road tests. Section title: Introduction Educational score: 3.734384536743164 Domain: biomedical Document type: Review Language: en While some on-road tests for brain-injured individuals have been developed globally, only a few have been validated with a high degree of reliability and validity. A systematic review identified a scarcity of on-road tests demonstrating general reliability and validity . This scarcity suggests that a few on-road tests may partially exhibit reliability and validity. A subsequent study by Bellagamba et al. reinforced these findings, concluding that currently available on-road tests lack sufficient reliability and validity. Consequently, the reliability and validity of on-road tests remain inadequately established. Section title: Introduction Educational score: 3.714927911758423 Domain: other Document type: Study Language: en Despite uncertainties regarding the reliability and validity of the on-road tests themselves, past efforts have focused on predicting driving skill assessment outcomes using off-road tests. Studies have attempted to formulate prediction equations using multiple off-road tests to predict actual on-road evaluation outcomes . However, this methodology cannot inherently verify the validity of the on-road test, leaving the authenticity of the results unverified. Consequently, these results are utilized as predictive tools without confirming sufficient validity. Alternatively, exploring alternative conceptual frameworks that involve both on-road and off-road tests introduces the crucial aspect of discriminant validity. Discriminant validity, a statistical concept, assesses whether a measurement captures unique attributes distinct from other related measurements . Several previous studies have used discriminant validity to demonstrate the validity of driving evaluations, supporting the validity of these assessments . Instead of relying on outcome predictions derived from past on-road test results as observed in previous studies, it is more beneficial to authenticate whether the on-road test effectively encapsulates the essence of driving skills, embodying discriminant validity. This methodology enhances the assurance of on-road test validity. Section title: Introduction Educational score: 3.918588876724243 Domain: biomedical Document type: Study Language: en Taking an alternative perspective, the legal and road conditions in different countries contribute to the complexity of on-road testing. On-road tests evaluate several key components of driving, such as cognitive abilities (e.g., attention, decision-making, and visual perception), motor skills (e.g., pedal and steering control), and environmental awareness (e.g., responding to vehicles and pedestrians, adherence to traffic rules) . Eligibility criteria for these assessments often include a confirmed diagnosis of brain injury, sufficient physical ability to operate a vehicle, and adequate cognitive functioning to manage driving-related tasks. However, the specific criteria for determining eligibility for driving assessments vary across countries or institutes, reflecting differences in legal regulations, cultural contexts, and healthcare systems. Existing on-road tests are primarily conducted on actual roads, exposing brain-injured patients to various hazards such as other vehicles and road users. In contrast, Japan conducts some on-road tests in a closed course at driving schools under instructor guidance. Legal regulations in Japan may restrict brain-injured survivors from driving on public roads until they pass an aptitude test from the local public safety commission . These legal constraints necessitate the assessment of on-road tests for brain-injured individuals solely on closed courses within driving schools . In other situations, other countries have difficulty conducting on-road tests under actual road conditions due to legal regulations . Section title: Introduction Educational score: 1.231791615486145 Domain: other Document type: Other Language: en Considering scenarios where specific countries are restricted from public on-road tests, a selective and comprehensive assessment approach incorporating both closed-course (driving school) and public-course on-road tests becomes preferable. However, most existing on-road tests have omitted closed courses, and when included, they often involve simple driving in a parking lot . While public on-road tests in real driving settings are valuable, regulatory frameworks in some countries may limit their feasibility. In such cases, on-road tests within a driving school setting offer a valuable alternative to assess critical driving skills, including operational proficiency and attentiveness. Section title: Introduction Educational score: 1.8834961652755737 Domain: other Document type: Study Language: en In response to these problems, we developed the Standardized On-Road Assessment for Drivers (SOAD), incorporating both closed-course and public-course on-road tests, and demonstrated its content validity . Given that no on-road tests with established reliability and validity exist, the investigation into the discriminant validity of SOAD becomes significant. The presence of a valid on-road test for a closed course could prove advantageous, especially in countries facing legal constraints such as Japan. Therefore, the primary objective of this study is to scrutinize the discriminant validity of the closed course version, contributing to the further validation of SOAD. Section title: Materials and methods Educational score: 1.7163642644882202 Domain: biomedical Document type: Study Language: no Design and setting Section title: Materials and methods Educational score: 2.1189522743225098 Domain: biomedical Document type: Study Language: en This study used a cross-sectional design. We conducted this study at five rehabilitation hospitals and their affiliated driving schools in Japan. This study was approved by the Ethics Committee of the Tokyo University of Technology (No. E21HS-019), and written informed consent was obtained from all participants in the study. Section title: Materials and methods Educational score: 1.3569051027297974 Domain: biomedical Document type: Other Language: hr Object Section title: Materials and methods Educational score: 3.3505852222442627 Domain: biomedical Document type: Study Language: en The inclusion criteria were as follows. 1) Patients referred by a physician for driving assessment at the collaborating hospital, 2) patients who were primarily diagnosed with a stroke, and 3) patients who will undergo an on-road assessment. The exclusion criteria were 1) patients with severe cognitive impairments that prevent understanding of the research content, 2) patients with aphasia that would interfere with the assessments, and 3) patients who do not provide consent for participation in this study. Although vision is a factor that influences driving, it was not included as a criterion in the present study. Instead, the focus was placed on examining correlations with the off-road tests utilized in previous driving studies. Section title: Materials and methods Educational score: 1.0496245622634888 Domain: other Document type: Other Language: de SOAD Section title: Materials and methods Educational score: 2.115222215652466 Domain: other Document type: Study Language: en SOAD was developed through the Delphi method, incorporating items from previous on-road assessments and insights gathered from experts. The content validity of SOAD has been substantiated through this process . The SOAD on the closed course (in driving school) involves scoring a total of 40 items, comprising eight categories related to driving (physical function, cognitive functions, mechanical operation, driving attitude, vehicle position, following the traffic rule, basic driving, safety confirmation) and four items related to dangerous action (Table 1 ). SOAD items cover detailed aspects of driving performance, such as operational proficiency, attentiveness, and adherence to traffic rules. Their comprehensive descriptions are provided in the SOAD manual. Section title: Materials and methods Educational score: 1.8348294496536255 Domain: other Document type: Other Language: en SOAD assessments were conducted using a training vehicle equipped with auxiliary brakes. SOAD on the closed course utilizes the course outlined in the provisional license test as specified by Japanese regulations, which includes maneuvers such as changing the vehicle's direction and navigating through narrow passages. Participants drive the course twice, receiving brief feedback from the assessor after the first attempt and then proceeding to the second attempt. Scoring is based on whether there were mistakes on the items during the first and second attempts. Items are scored on a three-point scale (zero to two points), while dangerous actions are scored on a binary scale (presence or absence). A high score means good driving skills. Section title: Materials and methods Educational score: 1.073561668395996 Domain: biomedical Document type: Other Language: it Data collection Section title: Materials and methods Educational score: 4.02493143081665 Domain: biomedical Document type: Study Language: en In this study, several off-road tests were adopted as alternative conceptual frameworks for discriminant validity. The off-road tests utilized in this study are the Mini-Mental State Examination Japanese Version (MMSE-J), Trail Making Test Japanese Version (TMT-J), Kohs Block Design Test (KBDT), Rey-Osterrieth Complex Figure Test (ROCF), Frontal Assessment Battery (FAB), and Stroke Drivers’ Screening Assessment Japanese Version (J-SDSA), which have been commonly employed in previous research for guessing driving skills in individuals with brain injuries . However, these assessments are conducted to evaluate the cognitive functions (higher brain functions) of stroke patients and do not measure the actual driving skills. Since on-road tests are often compared with evaluations by driving instructors, their correlation with off-road tests can help establish discriminant validity. Section title: Materials and methods Educational score: 1.9770560264587402 Domain: biomedical Document type: Other Language: en For the on-road assessment using SOAD, driving courses at authorized Japanese driving schools affiliated with each hospital were utilized. The assessment was conducted with a vehicle equipped with auxiliary brakes, with the presence of an occupational therapist and a driving instructor. The SOAD assessment was scored by a trained occupational therapist, while the off-road tests were administered by an occupational therapist in the collaborating hospital. 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: 2.940751314163208 Domain: biomedical Document type: Study Language: en Statistical analyses of the discriminant validity were conducted by the research team (TS, KS, JK, YH) independent of the evaluators. Data were managed in the cloud, and the research team carried out the analysis via online meetings. In this study, the score for each item and each category score from the 40 usual items, as well as the scores from each off-road test, were used as variables. Following confirmation of normality, the correlation coefficient was employed for statistical analysis by the Spearman Ranked test. All statistical analyses were performed with IBM SPSS Statistics for Windows, Version 24 with a significance level of 5%. Section title: Results Educational score: 3.9224636554718018 Domain: biomedical Document type: Study Language: en The study group consisted of 108 stroke patients, 83 men and 25 women, with ages ranging from 34 to 80 years (mean: 50.0 years, SD: 10.2). The participants' diagnoses were as follows: 62 individuals were diagnosed with cerebral infarction, 26 with cerebral hemorrhage, 10 with subarachnoid hemorrhage, three each with traumatic brain injury, encephalitis, and brain tumor, and one individual with hydrocephalus. The average driving experience of the participants was 36.3 (SD: 11.0) years. The means and standard deviations of the off-road test are shown in Table 2 . There were two missing values in the FAB, and the optional items of SOAD have some missing values (60 for item 9, 102 for item 12, 87 for item 14, and 26 for item 23). Section title: Results Educational score: 3.776872396469116 Domain: biomedical Document type: Study Language: en Several correlations were observed between the items of the SOAD and various off-road tests, though most of these correlations were not statistically significant (Table 3 ). Among the off-road tests, the J-SDSA dot time showed the highest number of significant correlations with six items (maintain the appropriate posture for safe driving, operate a gear change appropriately, position in the lane appropriately, maintain appropriate distance, slow down appropriately, check safety when turning left), followed by the MMSE-J with four items (operate brakes appropriately, reverse park appropriately, check rearview, check safety using head/body movement), and the TMT-J with three significant items (TMT-A: operate a gear change appropriately, follow yield situation appropriately, check safety using head/body movement, TMT-B: operate indicators appropriately, slow down appropriately, check safety when starting to drive). Section title: Results Educational score: 2.5048506259918213 Domain: biomedical Document type: Study Language: en Conversely, in terms of SOAD items, the greatest number of significant correlations with off-road tests was observed in items 34 and 35, both showing correlations with four tests, followed by item 13 with three tests. The highest correlation coefficient was found between J-SDSA dot error and item 36 (r = -0.37), followed by item 20 and dot time (r = -0.32) and item 34 and the KBDT (r = 0.30). All other significant correlations had correlation coefficients below 0.30. Section title: Discussion Educational score: 4.127656936645508 Domain: biomedical Document type: Study Language: en The results of this study revealed generally weak correlations between SOAD and the various off-road assessments, particularly in evaluations of practical driving-related skills. This pattern supports the discriminant validity of SOAD by demonstrating its ability to evaluate unique driving capabilities that are not assessed by off-road tests. Discriminant validity refers to the degree to which a test or measurement distinguishes between the construct it aims to measure and other, potentially related constructs. It indicates that the assessment effectively captures distinct attributes, ensuring the measured skills are distinct from those in similar evaluations . In the field of rehabilitation, discriminant validity is frequently established by calculating the correlations between assessments of distinct conceptual constructs . In this study, we examined the discriminant validity by calculating correlations between off-road and on-road tests. The on-road test evaluates actual, multifaceted driving skills, while the off-road test assesses specific cognitive functions, each targeting fundamentally different constructs. Therefore, this methodology is appropriate, as it enables a clearer distinction between the specific capacities assessed by each test. Section title: Discussion Educational score: 3.3608486652374268 Domain: biomedical Document type: Study Language: en Correlated items suggest areas where SOAD aligns well with existing neuropsychological assessments (e.g., TMT and SDSA) , while uncorrelated items highlight the unique aspects of SOAD that are not assessed by conventional off-road tests. These findings underscore the complementary role of SOAD in driving evaluations and emphasize the importance of integrating both on-road and off-road assessments for a comprehensive evaluation of driving skills. Section title: Discussion Educational score: 4.143033027648926 Domain: biomedical Document type: Study Language: en This finding aligns with prior studies, which suggest that off-road assessments primarily focus on cognitive domains such as visual processing and attention but fail to fully represent the complex skills required for safe driving . For instance, some off-road tests measure isolated skills (e.g., visual scanning or attention switching), which may not encompass real-world driving demands, such as maneuvering in traffic, which requires continuous integration of sensory input, executive functioning, and motor response . This fact suggests that items 34 and 35, which require advanced visual confirmation, had more significant correlations with off-road tests. Consequently, off-road tests may miss critical components of driving skills that SOAD captures, emphasizing the unique relevance of the on-road test within rehabilitation settings. The wide age range of participants (34-80 years) could be considered a potential source of variability in the study. However, age-related declines in both neuropsychological performance and driving skills are demonstrated, and these changes likely occur in parallel . Therefore, the influence of age on the correlation between neuropsychological tests and driving skills may not significantly impact the validity of the discriminative analysis. Section title: Discussion Educational score: 1.707565188407898 Domain: other Document type: Other Language: en In Japan, where legal regulations restrict individuals with brain injuries from undergoing public on-road tests until they pass specific evaluations , the use of closed-course SOAD tests becomes especially significant. This regulation by the National Police Agency ensures that assessments are performed in a safer, controlled environment, where patients can demonstrate critical driving abilities, such as attentiveness and obstacle avoidance, without exposing themselves or others to risk on public roads. This closed-course setting aligns with local legal requirements and supports a thorough evaluation of practical driving competencies. Section title: Discussion Educational score: 1.8267004489898682 Domain: other Document type: Other Language: en Furthermore, this scenario is not unique to Japan. Similar restrictions exist in several other countries, where legal policies or road infrastructure constraints limit on-road testing for individuals with cognitive impairments to closed courses . In these cases, closed courses offer an effective alternative for assessing driving skills under realistic but safe conditions, though they differ from public road conditions. Despite being a simulation, closed-course testing allows for a comprehensive evaluation of essential driving skills. It provides a feasible and valid alternative to direct road testing in countries where legal frameworks limit public on-road access for specific populations. Section title: Discussion Educational score: 2.542933702468872 Domain: biomedical Document type: Study Language: en The results of our study support the use of SOAD in such restricted settings, where it can assess distinct and essential driving skills not covered by off-road tests. The limited number of statistically significant correlations observed between SOAD and off-road assessments, combined with low correlation values, emphasizes the unique contributions of closed-course tests like SOAD in identifying practical driving skills essential for real-world scenarios. Section title: Discussion Educational score: 2.123539686203003 Domain: other Document type: Study Language: en While this study supports the discriminant validity of SOAD, its design is limited by the controlled setting of a closed course. To fully verify SOAD’s utility, future research could compare closed-course results with public-road driving assessments in countries where legal frameworks permit such comparisons. Additional studies that incorporate a public course would also contribute to a deeper understanding of the reliability and validity of SOAD. Section title: Conclusions Educational score: 4.050651550292969 Domain: biomedical Document type: Study Language: en This study showed that there was a slight correlation between the off-road test and the SOAD in stroke individuals. This result indicates that the SOAD has minimal overlap with off-road cognitive assessments and highlights its discriminative validity. It demonstrates that the SOAD provides a valuable tool for assessing driving skills in a controlled environment. This study emphasizes its role as a reliable tool for evaluating the driving abilities of stroke patients, particularly in settings with legal constraints, such as Japan. | Study | biomedical | en | 0.999995 |
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