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txp vs. Nsr.

PMC10034431

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b4e791959f2a4b3382bd0280344aadaf

Membership Functions, IDP.

PMC10034431

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Normal Distributions, IDP.

PMC10034431

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dfc2dbab28654855a1880c300fb7908a

MOSY framework.

PMC10034431

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Generation of Cfes from Nso vectors A.

PMC10034431

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Rationale of MOSY with example agreements between Cfes and Cso.

PMC10034431

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Nhe distributions, IDP.

PMC10034431

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ϕ vs. Generation, IDP.

PMC10034431

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ψ vs. Nsr, IDP.

PMC10034431

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A, PCP.

PMC10034431

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Nhe distributions, PCP.

PMC10034431

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a0a7d1a5822b4f0aaeefbb44de202a04

The PRISMA flow diagram of the study selection process.

PMC10035935

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Forest plots depicting studies using LL-BFRT compared to studies using LL-RT in muscle strength.Forest plot comparing low-load resistance training with blood flow restriction (LL-BFR) and low-load resistance training alone (LL-RT) on muscle strength of a) chest press (1RM) b) shoulder flexion (dynamometry in kgs). Abbreviations: CI, confidence interval; IV, inverse variance; Random, random effects model; SE, standard error.

PMC10035935

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271be8b5c2434719bd79452e444c6318

Forest plots depicting studies using LL-BFRT compared to studies using HL-RT in muscle strength.Forest plots comparing low-load resistance training with blood flow restriction (LL-BFRT) and high load resistance training alone HL-RT in strength of: a) chest press (1RM), and b) sensitivity analysis by removing one study (Thiebaud et al., 2013) that included substantially older participants. Abbreviations: CI, confidence interval; IV, inverse variance; Random, random effects model; SD, standard deviation.

PMC10035935

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c95fddb6620b4327aa5cdf61b84a4aa7

Four morphologic characteristics of colorectal cancers. a Concentric or annular appearance of a tumor that encompasses the colon lumen with bowel wall thickening and luminal narrowing (arrow). b Asymmetric bowel wall thickening from a tumor arising from the left side of the rectum (arrow). c Polypoid tumor filling the lumen of the ascending colon. d Exophytic tumor extending predominantly beyond the colon wall

PMC10036972

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Distribution of tumor size according to confidence in retrospective diagnosis of colorectal tumors. Box plots show median tumor size according to radiologist-rated confidence level in the retrospective diagnosis of prospectively and retrospectively detected tumors

PMC10036972

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Retrospectively detected tumor near the hepatic flexure. Concentric mass (arrows) narrows the caliber of the colon lumen

PMC10036972

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Retrospectively detected polypoid mass in the ascending colon. A soft-tissue mass (arrow) fills the lumen of the ascending colon

PMC10036972

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a18e8ea7a27440a1a8237a45b346daa6

An initially undetected tumor in the transverse colon that was retrospectively detected after consensus review. Asymmetric soft-tissue thickening is present along the inferior border of the mid transverse colon (arrow). This tumor was initially undetected on retrospective review by one reviewer but was detected by a second independent reviewer, and it was agreed by consensus to be present

PMC10036972

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62a055f553ed4ed1afe3ca984ca0983f

Retrospectively detected polypoid tumor in the ascending colon. a Coronal view of the ascending colon showing a round, polypoid mass (arrow) in the ascending colon. b Axial view of the same mass (arrow). Note that the soft-tissue tumor attenuation can be easily distinguished from typical stool that contains air

PMC10036972

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Effect of MF on concentration and dose dependencies of H2O2 formation in 1 mM phosphate buffer, pH 7.4 (PB), and in 1 mM phosphate buffer, pH 7.4, with 150 mM NaCl (PBS) upon X-ray-irradiation: a Drug concentration dependence, irradiation with 10 Gy; b Dose dependence. * – significantly different from control (p < 0.05, n = 10)

PMC10036983

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a Influence of MF on the generation of hydroxyl radicals in 1 mM phosphate buffer, pH 7.4 (PB), irradiated with 5 Gy of X-rays. b Impact of 10 mM MF on the decay of H2O2 (initial concentration is 1.5 µM) in PB. * – significantly different from control (p < 0.05, n = 10)

PMC10036983

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665633ab2fa14f06b67e0294b2826369

The effect of MF on the formation of long-lived reactive protein species (LRPS) in BSA and BGG under irradiation and on their decay with the formation of ROS: a Formation of H2O2 in a solution of BSA 10 mg/ml in 1 mM PBS upon irradiation with 15 Gy depending on the time after irradiation and the effect of MF (10 μM, 250 μM, and 1 mM) on this process; b Estimation of 6-h area under the “concentration of hydrogen peroxide–time” curve (AUC) for PBS, BSA 10 mg/ml, and BSA 10 mg/ml with MF 250 μM after irradiation with 15 Gy; c Formation of H2O2 in PB upon X-ray-irradiation at a dose of 10 Gy. Impact on this process of sodium sulfate (Na+) or copper sulfate (Cu2+) and the addition of 250 μM MF; d Time dependence of the formation of H2O2 in a solution of BGG 5 mg/ml in PBS after irradiation with 15 Gy and the effect of MF (10 μM, 250 μM, and 1 mM) on this process. Except for b, n = 10 for all the plots

PMC10036983

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Influence of MF on the formation of 8-oxoG in the solution of salmon sperm DNA in PBS under X-ray-irradiation at a dose of 10 Gy. * – significantly different from control (p < 0.05, n = 10); ** – significantly different from 250 μM MF (p < 0.05, n = 10)

PMC10036983

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(A) SERS measurement for different concentrations of imidacloprid on 10 layers of AgNs (B) Log relationship graph for imidacloprid characteristic peaks versus concentrations.

PMC10040700

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(A) Reusability performance of 10 layers of AgNs towards imidacloprid detection. (B) UV–Vis of AgNs substrate before wash to 5th cycle of washing (C) Photos of the surface of the AgNs substrate before wash (a) to 5th cycle (f) of washing (D) SEM image for AgNs surface after 5th cycle washing.

PMC10040700

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(A) Chest CT on day 43 revealed infiltrative shadows with contraction tendency in both lung lobes. (B) Chest CT on day 87 revealed extensive consolidation and ground-glass opacity in both lung lobes. (C) The bronchial lumen at the junction of the right upper lobe branch and the middle bronchial trunk was filled with yellow fluid. (D) Chest radiograph on day 87 (time of admission) revealed consolidation in both lung fields. (E) Chest radiograph on day 87 (8 hours after admission) revealed rapid deterioration of consolidation in both lung fields. (F) Chest radiograph on day 87 (16 hours after admission) revealed a complete loss of permeability in both lung fields. CT, computed tomography.

PMC10040893

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Transesophageal echocardiography, midesophageal window, 40° short-axis view of aortic valve in semi-open position in early systole showing quadricuspid pattern of aortic valve

PMC10041404

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Short-axis view of aortic valve in midesophageal window, 47° view of TEE. The appearance of AV mimics a normal tricuspid valve. AV = Aortic valve, TEE = Transesophageal echocardiography

PMC10041404

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Transesophageal echocardiography, midesophageal window, 40° short-axis view with better focus on aortic valve in open position showing incomplete partitioning of noncoronary cusp of AV or possible fusion and raphe between supernumerary and noncoronary cusps of an originally quadricuspid AV. AV = Aortic valve

PMC10041404

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e93b3d93292a47ccacee7a1e2586435a

Transesophageal echocardiography, midesophageal window, 140° long-axis view of aortic valve. Color Doppler image shows severe aortic regurgitation

PMC10041404

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4485c2cb58f44a3e9d22bff599d0b778

Trends of under-five mortality rate in Ethiopia from 2000–2019.

PMC10042344

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Geographical locations of data points and under-five mortality in Ethiopia: 2000(a), 2005(b), 2011(c), 2016(d), 2019(e) and 2000–2019 (f).

PMC10042344

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The predicted geospatial map for under-five mortality in Ethiopia: 2000(a), 2005(b), 2011(c), 2016(d), 2019(e) and 2000–2019(f).

PMC10042344

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Student ratings of learning experiences.CV = curriculum vitae.

PMC10042501

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Student responses to “this activity should be incorporated into the curriculum.”

PMC10042501

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Calcium spike precedes nuclear expulsion during apoptosis-induced nuclear expulsion regardless of Padi4.a. Percentage of E0771-LMB cells undergoing nuclear expulsion measured using an expulsion algorithm following treatment of staurosporin with Q-VD-OPh. (n = 6304 of Padi4WT cells, n = 3987 of Padi4WT cells with Qvd-Oph and n = 7550 of Padi4KO cells examined over 3 independent experiments) b. Percent cell death in iCaspase9 cells treated with AP1903. n = 3 biologically independent experiments. c. Individual traces showing chromatin expansion for 200 Padi4WT and Padi4KO H2B-GFP+ iCaspase9 4T1 cells treated with AP1903. d. Western blot of Padi4, caspase-3 (Casp3) in Padi4WT and Padi4KO 4T1 cells. e - f. Dynamics of chromatin expansion (e) and calcium influx (f) during nuclear expulsion in Padi4WT or Padi4KO 4T1 cells treated with navitoclax with caspase inhibitors. g - i. Western blot of caspase-3, MLKL, Gasdermin D (Gsdm D) and H3Cit (g), LDH release (h) and Cell viability (i) in Padi4WT and Padi4KO 4T1 cells upon indicated drug treatments. Cells were primed by TNF-a 4 hours before navitoclax treatment. QVD-OPh (QVD) was added 1 hour before navitoclax treatment; n = 3 biologically independent experiments. j-m. Western blot of caspase 1, −3 and gasdermin D (Gsdm D) and H3Cit in 4T1 and RAW264.7 (j), LDH release (k) and cell viability (l) of 4T1 and LDH release in RAW264.7 (m); n = 3 biologically independent experiments. n. Individual 4T1 cell traces showing chromatin expansion (green) and calcium levels (red) for cells treated with raptinal. o. Dynamics of calcium influx and chromatin expansion during nuclear expulsion in Padi4WT 4T1 cells treated with staurosporine. p. Individual 4T1 cell traces showing chromatin expansion (green) and calcium level (red) for cells treated with A23187 ionophore (left) or PAF (right). q. Dynamics of calcium influx and chromatin expansion during nuclear expulsion in Padi4WT 4T1 cells treated with A23187 ionophore (left), PAF (right). r. Dynamics of calcium influx during nuclear expulsion in Padi4KO 4T1 cells treated with A23187 ionophore and PAF. All data are represented as mean ± s.e.m., and p values are based on two-tailed student’s t-test. Western blotting and IF were repeated at least twice, and representative data are shown. Source data

PMC10042736

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Tumor cell NEPs are found in mouse models of metastasis.a. Quantification of overlapping CitH3 and H2B-mCherry in the lungs of mice that received TVI of Padi4WT or Padi4KO 4T1 cells. n = mice. Error bars represent s.e.m., and p value is based on two-tailed student’s t-test. (*) P<0.05. b. CitH3 and H2B-mCherry immunostaining of NEPs in the lungs of mice that received TVI injection of 4T1 cells for 4 days. Right 4 panels are higher magnified images. Red: Tumor cells (H2B-mCherry), Green: citH3, Blue: DAPI. c. Super-resolution images showing overlap of CitH3 and H2B-GFP staining of metastatic tumor cells of EO771-LMB 28 days after MFP injection. Green: H2B-GFP only in tumor cells; Red: CitH3; Blue: DAPI. d. Representative IF images of tumor NEPs or NETs in lung metastases from mice bearing EO771-LMB tumors 35 days after MFP injection. Bottom panels are higher magnified images of citrullinated chromatin in tumor cells (most left) or NETs (right 5 panels): Cyan: Neutrophil elastase (NE), Green: Myeloperoxidase (MPO), Red: citH3, Blue: DAPI. e. IF images for CitH3 in tumor cells as well as NETs in necrotic region of primary tumor from mice bearing 4T1 tumors. Box represents necrotic region. Green: H2B-GFP only in tumor cells; Red: CitH3; Blue: DAPI. f. Higher magnification from Extended Data Fig. 4e. Top panels: representative images of intact tumor cells; Lower panels: tumor cells undergoing nuclear expulsion. Yellow box: NETs, White boxes: Tumor cells. Green: H2B-GFP (tumor cell only), Red: CitH3, Blue: DAPI. Source data

PMC10042736

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Tumor cell NEPs enhances metastasis in a manner independent from NETosis.a. Tumor weight from parental (n = 6), empty vector (n = 5), and Padi4KO 4T1 (#135, n = 8; #137, n = 7; #341, n = 7) mouse models. n = tumors. b - c. Number of lung metastases from size-matched tumors (b) and tumor weight (c) from 4T1 empty vector versus Padi4KO cells. n = mice (b) or tumors (c). d. Padi4 Western blot of EO771-LMB cells for validating Padi4 knockout. e. Tumor weight from empty vector (n = 5) and Padi4KO EO771 (#6112, n = 5; #6116, n = 5) mouse model. n = tumors f - g. Padi4 (f) and CitH3 (g) Western of EO771-LMB cells with Padi4 knockdown and scrambled shRNA control. h - i. Western blots of Padi4 (h) and CitH3 (i) in Ly6G+ neutrophils from Padi4myeWT or Padi4myeKO mice to confirm Padi4 knockout in myeloid compartment. j. Tumor weight from Padi4myeKO and wildtype mice orthotopically injected with scrambled shRNA control or Padi4 knockdown EO771-LMB cells. n = tumors. k. Tumor weight from 4T1 tumor-bearing mice treated with vehicle (n = 6), Sivelestat (n = 4), DNaseI (n = 5) and GSK-484 (n = 3). n = tumors. All data are represented as mean ± s.e.m., and p values are based on two-tailed student’s t-test. Source data

PMC10042736

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No significant effect of Padi4 or NEPs on tumor cell proliferation, cell cycle, cell adhesion as well as immune response.a. MTT assay for relative growth of Padi4WT and Padi4KO 4T1 cells. Day 3 signals were normalized by day 0. n = 3 biologically independent experiments. b. Distribution of cell cycle phase in Padi4WT and Padi4KO 4T1 cells. n = 3 biologically independent experiments. c. Adhesion assay of Padi4KO 4T1 cells. NEPs or ApoDBs were pre-coated 4 hours before tumor cells seeded. n = 3 biologically independent experiments. d. Size (left) and number (right) of GFP+ metastatic colonies from empty vector, two caspase3 knockdown #19 or #54 in both Padi4WT and Padi4KO H2B-GFP 4T1 cells. The thin blue dotted line on the violin plot represents the upper and lower quartiles and the thick red dashed line represents the median. n = individual GFP positive colonies (4T1-Scr, n = 140; 4T1-#19, n = 156; 4T1-#54, n = 52; 4T1 Padi4KO-Scr n = 64; 4T1 Padi4KO-#19, n = 84; 4T1 Padi4KO-#54, n = 156; left) or mice (right). e. Number of metastases of Padi4WT and Padi4KO cells in immune competent mice (black) and immune deficient mice (red) (left two panels). Ratio of big mets divided by total mets (the third panel) and representative images of Indian ink staining in the right panels. n = mice. f. Flow cytometry of immune cells in the lungs after the injection of NEPs or apoDBs. Left two panels are percentage and absolute counts of CD45+ population in lung tissue, the third panel is immune cell subsets within CD45+ population. n = mice. All data are represented as mean ± s.e.m., and p values are based on two-tailed student’s t-test. Source data

PMC10042736

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3d93fbb0fb3f43359c876a7273378e4f

Chromatin bound S100a4 in NEPs mediates RAGE activation in tumor cells leading to metastatic outgrowth.a. List of proteins identified as NEPs abundant components from proteomic analysis. b. Western blots of S100a4 or histone H3 from dialyzed NEPs or NEPs re-incubated with S100a4. c. Luciferase signals of co-cultured Padi4KO 4T1 cells with NEPs upon treatment of NaCl. Same amount of salt as in Fig. 5i was added directly into culture media. n = 3 biologically independent experiments. d. Spheroid culture of Padi4KO 4T1 cells treated with dialyzed NEPs or S100a4 re-bound NEPs. n = spheres. Padi4WT, n = 7; Padi4KO+NEPs, n = 8; Padi4KO+purified chromatin, n = 7; Padi4KO+purified chromatin+rcs100a4. e. Spheroid culture of Padi4WT 4T1 cells treated with sRAGE. n = spheres. Padi4WT with vehicle (n = 6) or sRAGE (n = 8). f. Western blots of indicated signaling pathways with Padi4KO 4T1 cells co-cultured with NEPs or apoDBs upon a treatment with sRAGE. g. Mean Fluorescence Intensity (MFI) of Erk1/2 (left), Ki-67 (middle) and GFP (right) in lung metastases from mice that were treated with vehicle (n = 290), sRAGE (n = 100) or S100a4 mAb (n = 50) used in Fig. 5e. n = individual H2B:GFP labeled tumor cells. The thin dotted line on the violin plot represents the upper and lower quartiles and the thick dashed line represents the median. h. Representative IF images of Fig. 7g. Purple: phosphorylated Erk1/2, Red: Ki-67, Green: H2B-GFP (Tumor cell only), Blue: DAPI. All data are represented as mean ± s.e.m., and p values are based on two-tailed student’s t-test. Source data

PMC10042736

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Padi4 and CitH3 are associated with metastatic capacity in breast and lung cancer cell lines.a - b. Padi4 expression of mRNA (a: Q-PCR) or protein (b: Western) in metastatic derivatives of human breast cancer MDA-MB-231 cells. Asterisk indicates size ladder lane; n = 3 biologically independent experiments. c. Padi4 Western of cytosolic or nuclear protein extraction from murine breast cancer cell lines including series of 4T1, EO771 and TSA1. d - e. IF of CitH3 in various murine breast cancer cell lines including series of 4T1 (d), EO771 and TSA1 (e) with A23187 ionophore treatment. f. Padi4 mRNA expression among human cancer cell lines. g - h. Padi4 (g) and p65 (h) Western of 4T1 cells with or without p65 knockdown upon incubation with tumor or lung tissue conditioned media. All data are represented as mean ± s.e.m., Western blotting, qPCR and IF were repeated at least twice, and representative data are shown. Source data

PMC10042736

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Proteomic analysis of human NEPs and NETs, and validation of NEPs-specific markers.a. List of differential proteins from MDA-MB-231-LM3 NEPs compared to NETs. b. List of differential proteins from MDA-MB-231-LM3 NEPs compared to MDA-MB-231-Parental derived ApoDBs. c - d. Manders’ overlap coefficient values of CitH3 and HMGB3 or MPO in breast (c) and lung (d) cancer tissue arrays; n = independent samples. (c): n = 3; (d): NEPs, n = 4; NETs, n =5. e. Size of NETs and NETs found in lung, breast, and bladder cancers from human tissue arrays; n = independent samples. Lung NEPs, n = 4; Lung NETs, n = 7; Breast NEPs, n =5; Breast NETs, n = 3; Bladder NEPs, n = 3; Bladder NETs, n = 4. f. Western blots of HMGB1, 2 and 3 in icas9-MDA-MB-231-Parental and LM3. g. Luciferase signals of tumor cell growth of MDA-MB-231 cells co-cultured with HMGB1, 2 or 3 depleted NEPs or apoDBs; n = 3 biologically independent experiments. All data are represented as mean ± s.e.m., and p values are based on two-tailed student’s t-test. Western blotting was repeated at least twice, and representative data are shown. Source data

PMC10042736

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Padi4 expression correlates with poor prognosis of lung metastasis in TNBC patients.a. Paid4 expression levels in paired primary tumor and metastases from breast cancer patients. n = patients. p value was calculated by two-tailed paired t-test. b. Distant metastasis free survival (DMFS) with high or low Padi4 in Basal (left), or Luminal A (right) types of breast cancer patients within GSE7390 database. c. Distant metastasis free survival (DMFS) with high or low Padi4 in Basal, Luminal A, Luminal B, or Her2+ types of breast cancer patients (KMPLOT database). d. Metastasis free survival (MFS) of triple negative breast cancer patients with high or low Padi4 in lung, brain, bone, or liver in GSE2603+2034 dataset. e. DMFS of triple negative breast cancer patients with lung specific metastasis separated by high and low Padi1, 2 and 3 in GSE2603+2034 dataset. f. Metastasis free survival (MFS) of triple negative breast cancer patients with high or low nuclear expulsion signature in Brain, Bone or Liver in GSE2603+2034 dataset. p values for survival curves are based on log-rank (Mantel-Cox) test.

PMC10042736

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332a8e0fd75349828452c4a6b7cfbca4

Apoptosis triggers nuclear expulsion in cancer cells in a Padi4-dependent manner.a, Time-lapse microscopy of nuclear expulsion in 4T1 cells expressing H2B-GFP upon A23187 ionophore or Raptinal treatment. White arrow, nuclear expulsion; yellow arrow, apoptosis; dotted line, expelled nuclear area. b, Immunofluorescence (IF) of nuclear expulsion (left and middle) and extracellular fiber-like DNA/chromatin structures of NEPs (right) in 4T1 treated with A23187 ionophore for 30 min. Cells were stained with CitH3, β-actin and 4,6-diamidino-2-phenylindole (DAPI). White arrows, nuclear expulsion; yellow arrow, fiber-like structure; white box, higher magnified images (middle); dotted line, expelled nuclear area. c, Western blot of PARP1, caspase-3 and CitH3 of 4T1 cells treated with Raptinal (Rap) or A23187 ionophore (Iono). FL, full length; Clv, cleaved; Veh, vehicle. d, IF of MDA-MB-231-LM3, PC9, RT112 and SW780 cells treated with Raptinal. Dotted line, expelled chromatin. Green indicates CitH3; red indicates H2B:mCherry for MDA-MB-231-LM3 (left). Green indicates H2B:GFP; red indicates CitH3 for PC9, RT112 and SW780 (right). e, Representative images of apoptotic bodies and nuclear fragmentation in H2B-GFP Paidi4WT and Padi4KO 4T1 cells and nuclear expulsion in Padi4WT cells. f, Time-lapse of EO771-LMB cells going through apoptotic nuclear fragmentation followed by nuclear expulsion. The microscopic images representing baseline, condensation and decondensation are indicated in the figure. g, Histogram of chromatin expansion in EO771-LMB Padi4WT and Padi4KO cells tracked during treatment with Raptinal (1,000 cells) or A23187 ionophore (4,000 cells). Colors indicate chromatin expansion over the time course. h,i, Tracking the median chromatin expansion for thousands of EO771-LMB Padi4WT and Padi4KO cells treated with Raptinal and staurosporine (h) or with A23187 ionophore and PAF (i). j,k, Percentage of EO771-LMB cells undergoing nuclear expulsion with treatment of Raptinal (n = 8,148 of Padi4WTcells and n = 9,789 of Padi4KO cells examined over four independent experiments) and staurosporine (n = 10,731 of Padi4WTcells and n = 17,638 of Padi4KO examined over five independent experiments) (j) or A23187 ionophore (n = 11,656 of Padi4WTcells and n = 6,001 of Padi4KO cells examined over three independent experiments) and PAF (n = 13,690 of Padi4WTcells and n = 9,772 of Padi4KO cells examined over three independent experiments) (k). All data are represented as mean ± s.e.m. and P values are based on two-tailed Student’s t-test. Western blotting and IF were repeated at least twice and representative data are shown.Source data

PMC10042736

43018_2023_524_Fig1_HTML.jpg

7eaa8dd3a6ac439680727ada14154433

Caspases and calcium are necessary for apoptosis-induced nuclear expulsion.a, Percentage of Padi4WT and Padi4KO 4T1 cells treated with Z-LEHD-FMK undergoing nuclear expulsion measured by an expulsion algorithm (n = 4,852 of Padi4WTcells, n = 3,681 of Padi4WT cells with Z-LEHD-FMK and n = 6,488 of Padi4KO cells examined over three independent experiments). b, Diagram of inducible caspase-9 system. c,d, Percentage of cells undergoing nuclear expulsion (c) and median chromatin expansion (d) for Padi4WT and Padi4KO H2B-GFP E0771-LMB cells treated with iCasp9 dimerizing agent AP1903 (n = 1,368 of Padi4WTcells and n = 1,608 of Padi4KO cells examined over three independent experiments). e, Percentage of Padi4WT/Padi4KO E0771-LMB cells with or without caspase-3 knockdown undergoing nuclear expulsion with treatment of Raptinal (n = 773 of Padi4WT/caspase3Scr cells, n = 1,220 of Padi4WT/caspase3sh#19 cells, n = 1,540 of Padi4WT/caspase3sh#54 cells, n = 1,490 of Padi4KO/caspase3Scr cells, n = 1,729 of Padi4WT/caspase3sh#19 cells and n = 1,478 of Padi4KO/caspase3sh#54 cells examined over three independent experiments). f, Western blots of cell death markers, Parp1, caspase-3, Mlkl, Gsdm D/E and CitH3 in Padi4WT and Padi4KO cells upon treatment with indicated drugs, AP1903 (icas9), Raptinal (rap), A23187 ionophore (iono) and Shikonin (shi). g, Representative live IF images of X-Rhod1-AM as a calcium-signaling indicator in H2B-GFP 4T1 cells treated with Raptinal at 10 h and 16 h. h, Dynamics of calcium influx and chromatin expansion during nuclear expulsion in Padi4WT 4T1 cells treated with Raptinal. i, Dynamics of calcium influx during nuclear expulsion in Padi4KO 4T1 cells treated with Raptinal (n = 5,290 of Padi4WTcells and n = 5,862 of Padi4KO cells examined over three independent experiments) and staurosporine (n = 9,108 of Padi4WTcells and n = 7,039 of Padi4KO cells examined over three independent experiments). j,k, Median chromatin expansion (j) and percentage of cells undergoing nuclear expulsion (k) of EO771-LMB cells when treated with calcium chelators EGTA or BATPA-AM (n = 5,290 of Padi4WTcells, n = 5,862 of Padi4WT cells with EGTA, n = 9,108 of Padi4WT cells with BAPTA-AM and n = 7,039 of Padi4KO cells examined over three independent experiments). l, Median chromatin expansion for thousands of EO771-LMB iCasp9 cells when treated with calcium signaling blockers. m, Diagram comparing conventional apoptosis and apoptosis-induced nuclear expulsion. Calcium activates Padi4 to induce histone citrullination which leads to nuclear expulsion. All data are represented as mean ± s.e.m. and P values are based on a two-tailed Student’s t-test. Western blotting and IF were repeated at least twice and representative data are shown.Source data

PMC10042736

43018_2023_524_Fig2_HTML.jpg

9edea2bbd6b44df8b7750ba574c238f1

Padi4-mediated CitH3 and nuclear expulsion in mouse models of metastasis.a,b, Percentage of Padi4WT or Padi4KO H2B-GFP 4T1 with active caspase-3/7 (a) and with CitH3 positivity among cells showing active caspase-3/7 (b). Cells were collected from the lungs of the mice 24 h after TVI, n = mice. c, Imaging flow cytometry of Padi4WT 4T1 cells that have gone through apoptosis-induced nuclear expulsion in vivo. Cells were stained for CitH3 and active caspase-3/7. d,e, Representative IF images of CitH3, Ly6G and H2B-GFP in lung metastases from mice bearing EO771-LMB (d) or 4T1 tumors. Yellow arrows, NEPs; white arrows, NETs. f,g, Number of lung metastases in mice that received mammary fat pad (MFP) injection of Padi4WT or Padi4KO 4T1 cells (f) and EO771-LMB cells (g). Representative images from Indian ink staining (f right); 4T1, non-infected control; Vec, empty vector control; #135, #137, #341 (4T1) and #6112, #6116 (EO771) represent each Padi4KO clone. n = mice. h, Number of lung metastases from myeloid-specific Padi4WT or Padi4KO mice that bear EO771 tumors with Padi4 knockdown or scrambled short hairpin (sh)RNA (left), representative images from Indian ink staining (right), n = mice. i, Number of metastases from mice bearing 4T1 tumors treated with sivelestat, DNase I and GSK-484, n = mice. All data are represented as mean ± s.e.m. and P values are based on two-tailed Student’s t-test.Source data

PMC10042736

43018_2023_524_Fig3_HTML.jpg

29dc7634df47429d9184646cb9a78fc8

Nuclear expulsion promotes metastatic outgrowth.a, Schematic experimental design for non-autonomous effects of Padi4 on lung metastasis. b, Size (left, n = metastatic colonies) and number (right, n = mice) of mCherry+ metastatic colonies from Padi4WT or Padi4KO H2B-mCherry 4T1 cells (recipient) co-injected with Padi4WT or Padi4KO (donor) under indicated conditions. The thin dotted line on the violin plot represents the upper and lower quartiles and the thick dashed line represent the median. Padi4WT + Padi4WT-H2B:mCherry, n = 132; Padi4WT + Padi4KO-H2B:mCherry, n = 174; Padi4WT + Padi4KO-H2B:mCherry + DNase I, n = 145; Padi4KO-H2B:mCherry only, n = 269. c, Schematic experimental design for NEP effect on lung metastasis using intrathoracic injection. d–g, Bioluminescent imaging of metastatic burden in the lungs. Bioluminescent images (d), quantitative data (e,f, n = mice) or size of surface lung metastatic nodules (g, n = metastatic colonies). 4T1, n = 23; 4T1 Padi4KO, n = 19; 4T1 Padi4KO + NEPs, n = 15; 4T1 Padi4KO + apoDBs, n = 28; Padi4KO + sonic-NEPs, n = 23; Padi4KO + heat-NEPs, n = 18; from mice that received intrathoracic co-injection of Padi4WT or Padi4KO 4T1 cells with NEPs, apoDBs or PBS at indicated times. Sonic., sonicated NEPs; Heat inac., heat-inactivated NEPs. h,i, Size of the spheroid from Padi4WT or Padi4KO 4T1 cells co-cultured with NEPs or apoDBs (h), as well as treated with DNase or GSK-484 (i), n = spheres. 4T1, n = 13; 4T1 Padi4KO, n = 9; 4T1 Padi4KO + NEPs, n = 10; 4T1 Padi4KO + apoDBs, n = 8; i: 4T1, n = 13; 4T1 + DNase I, n = 8; 4T1 + GSK-484, n = 8; 4T1 Padi4KO, n = 8; 4T1 Padi4KO + DNase I, n = 4; 4T1 Padi4KO + GSK-484, n = 5 (h). All data are represented as mean ± s.e.m. and P values are based on a two-tailed Student’s t-test.Source data

PMC10042736

43018_2023_524_Fig4_HTML.jpg

e3410c9aac1b4f9b85c3b35fa0143fb8

Chromatin-bound S100a4 in NEPs mediates RAGE activation in tumor cells leading to metastatic outgrowth.a, Volcano plots of proteomics analysis −log10(P) versus log2 expression levels comparing NEPs to apoDBs or necrotic cell debris upon Raptinal (left) or A23187 ionophore (right) treatment. Mass spectrometry was performed using biologically independent samples (n = 3). FC, fold change. b, IF images for S100a4 and CitH3 in NEPs. cyan, CitH3; red, H2B-mCherry; green, S100a4; blue, DAPI. White arrows, S100a4 co-localized NEPs. c, Luciferase signals of tumor cell growth from co-culture of Padi4KO 4T1 cells with NEPs or apoDBs upon treatment of a sRAGE peptide (decoy RAGE receptor) or S100a4 neutralizing antibody (monoclonal antibody (mAb)), n = 3 biologically independent experiments. d, Luciferase signals of tumor cell growth from co-culture of MDA-MB-231-LM3 cells with iCasp9-generated NEPs or NETs upon treatment with sRAGE. PMA was used to induce NETs in neutrophils, n = 3 biologically independent experiments. PMA, phorbol 12-myristate 13-acetate. e–g, Bioluminescent imaging for lung metastatic burden. Representative images (e) or quantitative data (f, n = mice) and lung surface nodule size (g, n = metastatic colonies) from mice that received intrathoracic co-injection of Padi4WT or Padi4KO 4T1 cells with NEPs or apoDBs at indicated time. 4T1, n = 17; 4T1+sRAGE, n = 28; 4T1+neuAb, n = 28; 4T1 Padi4KO, n = 12; Padi4KO+NEPs, n = 40; Padi4KO+NEPs+sRAGE, n = 75; Padi4KO+NEPs+neuAb, n = 37 (g). h, Volcano plot of proteomics analysis −log10(P) versus log2 expression levels comparing washed NEPs (NEP-bound proteins) relative to unwashed NEPs (soluble and bound). Mass spectrometry was performed using biologically independent samples (n = 3). i,j, Luciferase signals of tumor cell growth from co-cultured Padi4KO 4T1 cells with salt-incubated NEPs, apoDBs or their supernatants (i), as well as with purified chromatin or recombinant S100a4 re-bound to chromatin upon treatment of sRAGE or mAb (j), n = 3 biologically independent experiments. k, Western blot of Erk1/2 with Padi4KO 4T1 cells co-cultured with NEPs or apoDBs (Apopt.) upon a treatment with sRAGE or PBS. Quantitative ratio of phosphor Erk1/2 to total Erk1/2 (right). l, Luciferase signal of tumor cell growth from co-cultured Padi4KO 4T1 cells with NEPs or apoDBs upon treatment with selumetinib or trametinib, p42/44 MAPK inhibitors, n = 3 biologically independent experiments. All data are represented as mean ± s.e.m. and P values are based on a two-tailed Student’s t-test. Western blotting and IF were repeated at least twice and representative data are shown.Source data

PMC10042736

43018_2023_524_Fig5_HTML.jpg

4df9a3d75172436bbf002e328fe3fbad

Inflammatory lung microenvironment is critical in Padi4 induction.a,b, Generation of highly metastatic derivatives of EO771. Padi4 western blot (a) or CitH3 (b) in EO771 parental and its metastatic derivatives, LM2 (second generation), LM3 (third) and LM4 (fourth). c, mRNA expression of Padi genes from in vitro cultured and in vivo sorted 4T1 cells, n = 3 biologically independent experiments. d, Fold increase of Padi4 mRNA upon treatments of TNF-α or LPS with indicated time. e,f, Padi4 western blot of 4T1 cells treated with TNF-α or LPS (e) and with or without NF-κB inhibitor BAY 11-7082 (f). g, Padi4 western blot of 4T1 cells incubated with lung supernatant from mice bearing 4T1 tumors (day 30) with or without BAY 11-7082. All data are represented as mean ± s.e.m. and P values are based on two-tailed Student’s t-test. Western blotting and qPCR were repeated at least twice and representative data are shown.Source data

PMC10042736

43018_2023_524_Fig6_HTML.jpg

387202d5c5f94521b1e8382396389434

Nuclear expulsion molecular signature correlate with poor prognosis.a, Volcano plots of proteomics analysis −log10(P) versus the log2 expression levels comparing MDA-MB-231-LM3 NEPs with NETs (top) or apoDBs (bottom) from MDA-MB-231. Mass spectrometry was performed using biologically independent samples (n = 3). b, Quantification of NEPs and NETs in patient tumor tissue arrays from multiple types of cancers. CitH3+HMGB3+ or CitH3+MPO+ were stained for NEPs and NETs, respectively. Representative images of NEPs (top) and NETs (bottom) are shown. Dashed lines represent NEP or NET boundaries. c, Schematic design for generating nuclear expulsion signature. d, List of nuclear expulsion molecular signature and fold change in two experimental conditions from RNA-seq. e, Multivariate analysis for DMFS in patients with breast cancer from GSE45255 (top) or KMPLOT (bottom) database. Nuclear expulsion signature and neutrophil signature were used as covariates for the Cox proportional hazard model. f, DMFS with high or low nuclear expulsion signature in patients with breast cancer from the GSE45255 (left) or GSE20685 (right) datasets. g, DMFS with high or low nuclear expulsion signature in basal type of patients with breast cancer from KMPLOT database. h, Lung-specific metastasis-free survival (lung-MFS) of patients with TNBC with high or low nuclear expulsion signature within GSE2603+2034 dataset. i,j, Overall survival (OS) of patients with lung cancer with high or low Padi4 (i) or with high or low nuclear expulsion signature (j) from CaArray (left) and GSE37745 (right). All P values are based on log-rank (Mantel–Cox test).

PMC10042736

43018_2023_524_Fig7_HTML.jpg

87d7f7bc121d4a9dad96ed3590c5ccb2

Apoptosis and A23187 ionophore trigger CitH3 in tumor cells in a Padi4 dependent manner.a. IF of NEPs from 4T1 cells with indicated stimuli. Green: H2B-GFP, Red: CitH3, Blue: DAPI. b. CitH3 Western blot of 4T1 cells treated with TNF-a, PMA and A23187 ionophore (Iono). c. CitH3 Western blots of various human cancer cell lines treated with A23187 ionphore. d. Padi4 Western blot for validating Padi4 deletion and CitH3 in 4T1 cells in untreated cells. e. CitH3 Western in Padi4WT and Padi4KO 4T1 cells treated with A23187 ionophore under starved condition. 10 % FBS treatment (FBS) in 4T1 cells used as normal condition. f. CitH3 Western in isolated NEPs or apoptotic debris (ApoDBs). g. IF of nuclear membrane disruption and extracellular double-strand DNA in Padi4WT and Padi4KO 4T1 cells. Cyan: Lamin A/C, Green: Double-strand DNA, Red: CitH3, Blue: DAPI. h. IF of Padi4WT and Padi4KO 4T1 upon A23187 ionophore treatment in combination with GSK-484. Green: CitH3, Red: Beta-actin, Blue: DAPI. Western blotting and IF were repeated at least twice and representative data are shown. Source data

PMC10042736

43018_2023_524_Fig8_ESM.jpg

f4eb57f055044303a49403e12457c58e

Time-lapse imaging, validation of nuclear expulsion by expulsion algorithm and CitH3.a - c. Individual traces showing chromatin expansion for 200 Padi4WT and Padi4KO H2B-GFP 4T1 cells treated with raptinal (a), A23187 ionophore (b) and PAF (c). d - f. Expulsion algorithm detects cells that went through nuclear expulsion. Expulsion values obtained for a Padi4WT (d) and a Padi4KO (e) cell. ROC curve to determine the expulsion threshold above which a cell is deemed to have gone through nuclear expulsion (f).

PMC10042736

43018_2023_524_Fig9_ESM.jpg

5459e76611164eb98dc7b964111f15f1

CONSORT (Consolidated Standards of Reporting Trials) diagram of patient enrollment, allocation, follow-up, and analysis. n = total number of subjects, ASA = American Society of Anesthesiologists, BMI = body mass index, FNB = femoral nerve block, SOC = standard of care.

PMC10043574

jbjsoa-8-e22.00144-g001.jpg

5ae28a1abf4d4452bc17e5110eee0448

BRIG (Blast Ring Image Generator) comparison of blaCTX-M-9-bearing IncHI2-ST1 plasmids found in monophasic S. Typhimurium ST34 isolates from a Spanish hospital. Each ring corresponds to a plasmid according to the color code specified on the right-hand side. pHUD 1/16 was entirely sequenced in this study and used as the reference (inner black ring). Relevant genes of this plasmid are displayed in the outer black ring, represented by arrows indicating the direction of transcription. Note that only the resistance genes are annotated for clarity in the large resistance region located between the hipA and pcoS genes (marked with an asterisk) (see Figure 2 for further details). To generate the image, the concatenated contigs of the other plasmids, as identified by PLACNETw, were used. Thus, the presence/absence of genes, but not necessarily the synteny, is accurate. Also, genes carried by these plasmids but absent in the control are excluded from the comparison but included in Figure 2.

PMC10044134

antibiotics-12-00547-g001.jpg

84b0897636f04a2ab036bb3079c88f7a

Detailed structure and comparison of the resistance clusters found in the IncHI2-ST1 plasmids of monophasic S. Typhimurium ST34 isolates. Protein coding regions (ORFs) are represented by arrows indicating the direction of transcription and colored according to their function: red, resistance; light blue, DNA mobility, with IS26 highlighted in dark blue, IS3000 and IS6000 in light purple, and ISCR elements in dark purple; orange, other roles; olive green, plasmid ORFs flanking the resistance clusters. The two copies of IS26, flanked by direct repeats that delineate a fragment that was probably inverted in pHUD 1/16, are marked with asterisks. The alignments were created with EasyFig BLASTn. The gray shading between regions reflects nucleotide sequence identities according to the scale on the figure’s right.

PMC10044134

antibiotics-12-00547-g002.jpg

f6a61e39063242cc8ca57ac610638afb

Genetic organization and comparisons of the chromosomal RR regions found in the monophasic S. Typhimurium ST34 isolates from a Spanish hospital. Coding regions are represented by arrows indicating the direction of transcription and colored according to their function: red, resistance; blue, DNA mobility, with IS26 highlighted in darker blue; orange, other roles; white, flanking chromosomal ORFs named according to accession number NC_003197, which corresponds to the chromosome of S. Typhimurium LT2 [45]. The alignments were created with EasyFig BLASTn. The gray shading between regions reflects nucleotide sequence identities according to the scale in the right bar.

PMC10044134

antibiotics-12-00547-g003.jpg

3aace13ca39f43109f7eff2e58f0aadf

Changes in vascular endothelial growth factor receptor 2 (VEGF-R2) (a), vascular endothelial growth factor receptor 3 (VEGF-R3) (b), and vascular cell adhesion molecule-1 (VCAM-1) (c) levels during somatostatin analogue (SSAs) treatment in patients with neuroendocrine tumors.

PMC10044914

biomedicines-11-00842-g001a.jpg

c115fc90fb95456a84e29b92927c69d5

Spearman’s Rank Correlations. Spearman’s coefficients of the relationships among analyzed variables in NET patients: VEGF-R2 vs. Treatment duration (the presented correlation is statistically significant, p < 0.05).

PMC10044914

biomedicines-11-00842-g002.jpg

99f045ecc90c414d8b573b3b4abe5622

Flowchart of inclusions and exclusions in the study population.

PMC10046942

cancers-15-01867-g001.jpg

64bd500e3e4d4815b696182efe5ac190

Prevalence of CAD (coronary artery disease) stages in patients hospitalized for peripheral artery disease in Germany between 2009 and 2018.

PMC10047185

diagnostics-13-01163-g001.jpg

23901d8ff689482a9035aa7b271b27e1

Median costs of hospitalizations for peripheral artery disease with and without concomitant coronary artery disease (CAD) in Germany between 2009 and 2018.

PMC10047185

diagnostics-13-01163-g002.jpg

c06998b316424ab984cb014e77e53c43

Prevalence of major and minor amputations in patients hospitalized due to peripheral artery disease with and without coronary artery disease between 2009 and 2018.

PMC10047185

diagnostics-13-01163-g003.jpg

4427287d3b624fd788e7d4e6278339ec

Prevalence of coronary interventions in patients hospitalized due to PAD between 2009 and 2018 in (%). PCI: percutaneous coronary intervention; PTCA: percutaneous transluminal coronary angioplasty; BMS: bare-metal stent; DES: drug-eluting stent.

PMC10047185

diagnostics-13-01163-g004.jpg

4afdb1d07aef42f4981f972c329f747a

Systematic review flow chart of records identification and study screening.

PMC10047331

diagnostics-13-01187-g001.jpg

046477c13b7541ffa14e8b0d1d668328

BRAF mutation and Gender correlation forest plot [7,12,14,15,16,17,18,20,24,25,26,28,30,31,33,37,38,40,42,43,45,47,48,49].

PMC10047331

diagnostics-13-01187-g002.jpg

ff2979be62314a838f575c9f21eccb08

BRAF mutation and tumor size forest plot [7,13,14,15,16,17,25,28,29,31,33,35,37,40,43,46].

PMC10047331

diagnostics-13-01187-g003.jpg

24b1bbeb292a4a8d9c9f38b0aaa195fa

BRAF mutation and Multifocality forest plot [7,14,17,18,31,38,40,41,42,45,47].

PMC10047331

diagnostics-13-01187-g004.jpg

0cd7a26ca54e4412a62804c3225e090b

BRAF mutation and Vascular invasion forest plot [15,17,29,35,40,41,42,45].

PMC10047331

diagnostics-13-01187-g005.jpg

558b2fa6b10942899d9f7f082932a694

BRAF mutation and LNM forest plot [7,12,14,16,17,18,20,24,25,28,29,31,33,35,37,38,40,41,42,45,49].

PMC10047331

diagnostics-13-01187-g006.jpg

e9638d129c704551b4269bbef0b421bf

BRAF mutation and ETE forest plot [7,12,13,15,16,17,18,20,29,31,33,37,38,40,42,45,47].

PMC10047331

diagnostics-13-01187-g007.jpg

cf050e50bf01457aaeb3eaa37ebc5d46

BRAF mutation and Distant Metastasis Forest plot [7,12,13,14,16,17,18,29,31,33,35,37,38,40,42,45].

PMC10047331

diagnostics-13-01187-g008.jpg

e88aa581a5854ab5a3c2b105e8d57551

BRAF mutation and Tumor Recurrence Forest plot [7,13,17,18,20,31,38,40,45,47].

PMC10047331

diagnostics-13-01187-g009.jpg

bbe6714437ee4f228ec90fb79c1c5428

Risk of bias summary; authors’ judgements on each risk of bias item for each included study [7,11,12,13,14,15,16,17,18,19,20,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49].

PMC10047331

diagnostics-13-01187-g010.jpg

b62c211694544e808672e72ab4b15f1a

Precision scale and sieve used to collect and weight the specimens.

PMC10047352

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1deec4b18e8c4f9eb1e0e50e62e215a5

Graph of the average of the weights obtained.

PMC10047352

dentistry-11-00068-g002.jpg

05a89bf86e1541918f934fd8ce2a2e13

Schematic representation of the causal mediation analysis. The total effect of an exposure (FTO genotype) over an outcome (concentration of circulating adiponectin) is dissected into direct and indirect effects. The indirect effect is the one being exerted via a potential mediator of interest (body adiposity). The remaining part of the effect goes directly or via mediators other than the ones under current study.

PMC10047575

children-10-00426-g001.jpg

1e92c369eff84547b4c19f4196bf84d1

Associations between circulating adiponectin concentrations in log−transformed μg/mL, with FTO genotype for SNP rs9939609, comparing carriers of the heterozygous (TA) and homozygous (AA) genotype for the mutation, with the wild-type individuals (TT, reference group), respectively. The results are expressed in beta effect estimates of and their respective 95% CIs. Model 2 was adjusted for age, sex, and Tanner’s stage. Model 3 included the adjustments in model 2 plus BMI z-score.

PMC10047575

children-10-00426-g002.jpg

85e0e37b3b3e4429a9076777babcf432

(A–C) Associations of circulating adiponectin concentrations in log−transformed μg/mL, with several measures of overall and central adiposity. (A) BMI z-score, (B) total body fat % and (C) Waist to height ratio (WHtR). The statistical model was adjusted for age, sex, Tanner’s stage and FTO rs9939609 genotype. The results are expressed in beta effect estimates of and their respective 95% CIs.

PMC10047575

children-10-00426-g003.jpg

e6e41117362a4f5b8fce559e1d3dd264

The experimental graphomotor setup with a pen tablet, school desk, and chairs. The experimenter was seated on the right side and could monitor the pen movements in real time on a laptop.

PMC10047900

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71247c149e5143ae8fcac15e3420ee59

BHK text (Portuguese version obtained from the French version [15]—Figure A1 in Appendix A). Participants copied the BHK text, composed of progressively more complex words, for 5 min.

PMC10047900

children-10-00445-g002.jpg

c1af12454b8e45fc81dc9aaf698d1ecc

Handwriting example of the BHK text (Portuguese version is shown in Figure 2 and the English translation is shown in Appendix A) copied for 5 min by a participant on a blank A4 sheet of paper in landscape orientation. (Image was reduced and cropped. The border measured in reality 18.4 cm × 13.5 cm.)

PMC10047900

children-10-00445-g003.jpg

6c0fee21510942be875b4ec3db87cbaa

The only significant group differences are between boys and girls for the number of strokes (left panel) and the slant in radians (right panel) (1.27 rad = 73 deg, 1.36 rad = 78 deg).

PMC10047900

children-10-00445-g004.jpg

6fdda1bdcd6640c998e05a20b02db390

The French BHK text [15] (left panel) used to generate the cross-culturally adapted Portuguese version (shown in Figure 2) and the literal translation of the Portuguese version into English (right panel).

PMC10047900

children-10-00445-g0A1.jpg

1343a33983e64950a2b67cf7f036fc2b

Comparative evaluation of the 1H NMR spectral profiles of bulk major lipids in an unheated (control) sample of SBO acquired at (a) 60 and (b) 400 MHz operating frequencies. Assignment labels for resonances are available in Table 1. (c) Simulated partial 1H NMR spectrum of the glycerol backbone resonances of a typical triacylglycerol species (expanded Resonance 9A and 9B regions (3.90–5.50 ppm)). Resonances A and B had centralised chemical shift values of 4.22 and 5.30 ppm for this simulation. The 2JAB coupling constant utilised was 10.8 Hz, with 3JAX = 6.0 Hz, and 3JBX = 5.2 Hz.

PMC10048026

foods-12-01254-g001a.jpg

b1ceda4b4b9a42f7b05650e002f5dcd5

High-frequency (8.10–10.20 ppm) aldehydic proton region of the LF (60 MHz) 1H NMR spectrum of SBO heated at 180 °C for a 90 min duration. Numbered label assignments correspond to those in Table 2. Aldehyde signals labelled 3 and 5 represent only one line from the doublet resonance usually observed for these α,β-unsaturated aldehydes at MF strength (400 MHz), as shown in Figure 3.

PMC10048026

foods-12-01254-g002.jpg

8feed96d4ab648028e4509e229feb6d5

High frequency (8.10–10.20 ppm) aldehydic proton (-CHO) regions of the LF (60 MHz) and MF (400 MHz) 1H NMR spectra of an olive oil (MOO) sample before and after heating for a period of 90 min at 180 °C. Numbered label assignments correspond to those available in Table 2.

PMC10048026

foods-12-01254-g003.jpg

6d787cbf0db849a3a2a4821e9f4560e8

Partial aldehydic proton (-CHO) regions of the 1H NMR spectra of n-hexanal (δ = 9.75 ppm, t) and trans-2-octenal (δ = 9.48 ppm, d) in a combined CDCl3/unheated MOO medium at analyte solution concentrations of 5.40–54.98 and 2.42–40.40 mmol/L, respectively. Spectra acquired at (a) 60 and (b) 400 MHz operating frequencies are shown. The analyte media for these experiments were prepared according to one of the methods described in Section 2.3, and contained 0.20 mL of control (unheated) olive oil in a total volume of 0.76 mL. Numbered label assignments correspond to those available in Table 2.

PMC10048026

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(a,b), Plots of the ratio of aldehydic proton (-CHO) intensity to that of the terminal FA-CH3 group of a fixed volume of an unheated (control) olive oil solution co-calibrant additive versus n-hexanal calibrant concentration for spectra acquired at both 60 (blue datapoints) and 400 MHz (red datapoints) operating frequencies, respectively. (c,d), As (a,b), respectively, but for the trans-2-octenal calibration standard. Analytical calibrant solutions contained 0.20 mL of each aldehyde stock solution (0.00–54.98 mmol/L for n-hexanal, and 0.00–40.40 mmol/L for trans-2-octenal) in CDCl3, 0.20 mL of the olive oil co-calibrant, a further 0.20 mL of the CDCl3 solvent, and 0.10 and 0.06 mL aliquots of the 2,5-DTBHQ antioxidant (67.0 mmol/L) and the additional TCB 1H NMR reference standard (19.8 mmol/L) solutions, also in CDCl3. Abbreviations for the correlation plots shown in (a–d): - - - - -, grey, 95% confidence intervals (CIs) for means; --------, black, 95% CIs for observations.

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Plots of signal-to-noise (STN) ratio versus concentration of aldehyde calibrants. (a,b), n-Hexanal in ‘neat’ CDCl3 and olive oil/CDCl3 analytical solutions, respectively; (c,d), as (a,b), respectively, but for trans-2-octenal calibrant solutions. Abbreviations for the correlation plots shown in (a–d): as described in Figure 5.

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Plots of the concentrations of (a) n-hexanal (t, δ = 9.74 ppm) and (b) trans-2-octenal (d, δ = 9.48 ppm) determined at a 60 MHz operating frequency on a LF benchtop NMR spectrometer versus those obtained on a conventional MF 400 MHz NMR facility. Abbreviations for the correlation plots shown in (a,b): - - - - -, grey, 95% confidence intervals (CIs) for means; --------, black, 95% CIs for observations. (c,d), Corresponding Bland–Altman style dominance plots (BAPs) for comparisons of analytical results generated by the 60 and 400 MHz NMR facilities, respectively. For the BAP plots, the dark blue and red lines represent the null hypothesis zero difference and determined mean difference values, respectively. The dotted black lines represent 95% CIs for the mean difference (depicted as the red line), and the dotted green lines display the ±1.96sd limits for the latter; sd represents the standard deviation of the difference values for each plot.

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Aldehydic proton (-CHO) region (9.40–9.90 ppm) of the MF (400 MHz) 1H NMR spectrum of a soybean oil (SBO) sample heated for periods of 0, 30, 60 and 90 min at 180 °C according to our LSSFEs. Numbered label assignments correspond to those available in Table 2. The triplet resonance located at δ = 9.817 ppm is this spectrum is attributable to low-molecular-mass n-alkanal species, which arise from the thermo-oxidative deterioration of ω-3 FAs, and higher levels of these are generally characteristic of oils containing significant contents of these highly unsaturated FAs (≥5% (w/w)) when exposed to high-temperature thermal stressing episodes [28]. Colour key code: blue, 0 min; red, 30 min; green, 90 min; violet, 90 min.

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ANOVA of saturated aldehydic LOP concentration dataset from experiments involving the exposure of four different culinary oil products to LSSFEs. (a–c) Bar diagrams showing mean ± 95% CI values for saturated aldehydic LOP concentrations generated in culinary oils heated according to our LSSFEs at a temperature of 180 °C for periods of 60 and 90 min, and showing differences between heating time-points, NMR spectrometer field strength and oil products evaluated, respectively. (d) Plot of observed saturated aldehyde concentration versus those predicted from this ANOVA model (Equation (2)), which included a statistically significant first-order oil product x spectrometer operating frequency interaction effect (R2 = 0.828). Abbreviations: OF, NMR spectrometer operating frequency; CSO, chia seed oil; MOO, refined olive oil; RSO, rapeseed oil; SBO, soybean oil.

PMC10048026

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ANOVA of α,β-unsaturated aldehydic LOP concentration dataset from experiments involving the exposure of four different culinary oil products to LSSFEs. (a–c) Bar diagrams showing mean ±95% CI values for α,β-unsaturated aldehydic LOP concentrations generated in culinary oils heated according to our LSSFEs at a temperature of 180 °C for periods of 60 and 90 min, and showing differences between heating time-points, NMR spectrometer operating frequencies and oil products evaluated, respectively. (d) Plot of observed concentration versus those predicted from this ANOVA model (Equation (3)), which included no interaction effects (R2 = 0.876). Abbreviations: OF, NMR spectrometer operating frequency; CSO, chia seed oil; MOO, refined olive oil; RSO, rapeseed oil; SBO, soybean oil.

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Expanded 7.80–12.00 region of the 1H NMR spectrum of a sample of sunflower oil thermally stressed according to our LSSFEs (180 °C for a period of 90 min). Abbreviations: LHP-OOH, broad lipid hydroperoxide signals attributable to CHPDs and/or HPMs. Numbered label assignments correspond to those in Table 2.

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(A) Chemical structure of crosslinked hydrogels prepared by thermal initiation of HEMA (blue), EGDMA (green) and RAFT (pink) (PCG 2). (B) Grafting of PAA chains (red) onto HEMA via RAFT chain extension (PCG 2B). (C) Chemical reaction route to prepare hydrogels copolymerized with statistical AA comonomer in initial reaction feed (PCG 3). (D) Overall structure of copolymer AA throughout HEMA gel (PCG 3).

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(A) IR analysis of HEMA + RAFT–HEMA films. (B) Raw titration data of RAFT–HEMA films. (C) Sw of HEMA and RAFT–HEMA films at different pH levels.

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(A) IR analysis of AA-grafted RAFT–HEMA films. (B) Raw titration data of AA-grafted RAFT–HEMA films. (C) Sw of AA-grafted RAFT–HEMA films at different pH levels.

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(A): Raw titration data of AA-copolymerized RAFT–HEMA films. (B) Sw of AA-copolymerized RAFT–HEMA films at different pH levels.

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Representative engineering stress/strain curves of PCN 1, 2, 2B and 3A.

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