Background: Fluorescence-guided brain tumor surgery using fluorescein sodium (FNa) for contrast is effective in high-grade gliomas. However, the effectiveness of this technique for visualizing noncontrast-enhancing and low-grade gliomas is unknown. This report is the first documented case of the concurrent use of wide-field fluorescence-guided surgery and confocal laser endomicroscopy (CLE) with high-dose FNa (40 mg/kg) for intraoperative visualization of tumor tissue cellularity in a nonenhancing glioma.Case Description: A patient underwent fluorescence-guided surgery for a left frontal lobe mass without contrast enhancement on magnetic resonance imaging. The patient received 40 mg/kg FNa intravenously at the induction of anesthesia. Surgery was performed under visualization with a Yellow 560 filter and white-light wide-field imaging. Intraoperative CLE produced high-quality images of the lesion 1.5 h after FNa injection. Frozen-section analysis demonstrated findings comparable to those of intraoperative CLE visualization and consistent with World Health Organization (WHO) glioma grades II–III. The patient recovered without complications. Analysis of the permanent histologic sections identified the tumor as an anaplastic oligodendroglioma, IDH-mutant, 1p/19q co-deleted, consistent with WHO grade III because of discrete foci of hypercellularity and increased mitotic figures, but large regions of the lesion were low grade.Conclusions: The use of high-dose FNa in this patient with a nonenhancing borderline low-grade/high-grade glioma produced actionable wide-field fluorescence imaging using the operating microscope and improved CLE visualization of tumor cellularity. Higher doses of FNa for intraoperative CLE imaging and possible simultaneous wide-field fluorescence surgical guidance in nonenhancing gliomas merit further investigation. Fluorescein sodium (FNa) is a fluorescent biomarker available as a water-soluble dye that has been widely used in ophthalmology since the early 1960s. Moore et al. –5. In a FNa is commonly used for retinal angiographic procedures, and it has been shown to have few side effects with oral ingestion; the most common adverse effects are nausea and vomiting , 8. AddiFNa is useful in identifying intracranial malignancies because it extravasates from cerebral vessels in places where the blood-brain barrier has been damaged. This mechanism allows the dye to concentrate, localizing the area where tumor invasion has disrupted vascular integrity. The pattern of FNa extravasation is relatively unique in HGG compared to that in simple surgical trauma to brain tissue, and the pattern indicates a disruption in the blood-brain barrier . FNa is In conjunction with dedicated fluorescein filters on surgical microscopes, the increasing use of intraoperative histopathologic examination with confocal laser endomicroscopy (CLE) using FNa has produced a tool for optically interrogating gliomas with a sensitivity and specificity comparable to the examination of a frozen section –18. Our A patient with a 4-year history of headaches was admitted for new-onset generalized seizure. Magnetic resonance imaging (MRI) showed a nonenhancing 1.6 × 1.3-cm heterogeneous posterior left frontal lobe mass just anterior to the precentral gyrus, with a surrounding abnormal high signal on T2-weighted and fluid-attenuated inversion recovery (FLAIR) imaging, interpreted as probably representing edema . The mosex vivo CLE imaging . CLE imaging demonstrated hypercellular brain architecture, with abnormal cells suggestive of a cellular tumor in all biopsy specimens (Four biopsies from within the tumor region (the last obtained about 1.5 h after FNa administration) were subjected to immediate intraoperative pecimens . Histolopecimens . FurtherCLE concurrent with high-dose FNa provided extremely clear images of cellular architecture, mitotic figures, endothelium of vessels, and swollen axons . The briGliomas are classified according to WHO grades I–IV, with LGG encompassing grades I and II. Still, they are representative of a heterogeneous group of tumors with wide variability in terms of prognosis and treatment. In the case description, we present the immunohistochemistry and pathologic characteristics identifying the tumor as an anaplastic oligodendroglioma, IDH-mutant, 1p/19q codeleted, with ATRX expression isolated within a vast majority of areas that were lower-grade glioma. Although the tumor was classified as a grade III glioma because of regions of increased aggressive characteristics, the prognostic significance of grade II vs. III in this tumor class compared with that of a nonanaplastic oligodendroglioma with an otherwise similar profile is not clearly understood, with the diagnostic criteria for histopathologic grading introducing subjectivity in interobserver variability in grading these lesions , which they referred to as nonenhancing gliomas. All five patients received 5 mg/kg FNa, and the Yellow 560 filter was used as needed. These investigators found that, despite the lack of contrast enhancement on MRI in all patients, all five tumors demonstrated some degree of fluorescence, which correlated with PET metabolic activity. One grade II tumor was visible only under the fluorescence-detecting filter. Final grading identified two LGG and three HGG as grade III. Nonetheless, Schebesch et al.  with a high dose of FNa in a patient with a nonenhancing glioma up to 1.5 h after FNa administration. This report supports the investigation of higher doses of FNa in nonenhancing gliomas and gliomas suspected to be a lower grade that usually do not exhibit 5-ALA-mediated fluorescence. Given the highly informative images, the minimal adverse effects of FNa, and the good postoperative outcome of this case, higher doses of FNa (20–40 mg/kg) should be considered or trialed with intraoperative CLE.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by St. Joseph's Hospital and Medical Center Institutional Review Board for Human Research. The patients and participants provided written informed consent to participate in this study.EB, PN, and MP: study planning and coordination. EB and IA: acquisition of confocal images. EB, NO, XZ, and IA: processing and organizing of the data and confocal images. EB, XZ, IA, JE, PN, and MP: assessment of confocal images. EB, NO, and MP: writing the draft. MP: final approval. All authors: review of the draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In comparing the three risk groups of risk for in-hospital bleeding, the high/very high-risk group had significantly higher dp-ucMGP levels from both very low/low group  and the moderate group . Overall, since higher dp-ucMGP levels were associated with elevated CRUSADE score and prolonged hemostasis parameters, this may suggest that there is a biological link between dp-ucMGP plasma levels and the risk of bleeding in patients who present with AMI.The “Can Rapid risk stratification of Unstable angina patients Suppress ADverse outcomes with Early implementation of the ACC/AHA guidelines” (CRUSADE) score emerged as a predictor of major bleeding in patients presenting with the acute coronary syndrome. On the other hand, previous studies established the association of dephosphorylated-uncarboxylated Matrix Gla protein (dp-ucMGP) and vitamin K, as well as their subsequent impact on coagulation cascade and bleeding tendency. Therefore, in the present study, we explored if dp-ucMGP plasma levels were associated with CRUSADE bleeding score. In this cross-sectional study, physical examination and clinical data, including plasma dp-ucMGP levels, were obtained from 80 consecutive patients with acute myocardial infarction (AMI). A significant positive correlation was found between CRUSADE bleeding score and both dp-ucMGP plasma levels (r = 0.442,  Appearing in various clinical presentations and severity, coronary artery disease (CAD) represents one of the major challenges of 21st century medicine, with acute coronary syndrome (ACS) being one of its most dramatic manifestations ,4. AccorAmong several bleeding risk scores that developed over the years, the “Can Rapid risk stratification of Unstable angina patients Suppress ADverse outcomes with Early implementation of the ACC/AHA guidelines” (CRUSADE) score emerged as one of the robustly validated tools for estimation of major bleeding probability in ACS ,7. SinceMatrix γ-carboxyglutamate (Gla) protein (MGP) is a protein dependent on vitamin K and is mainly produced by chondrocytes, vascular smooth muscle cells, and endothelial cells ,14,15,16Due to the established association between dp-ucMGP and vitamin K, as well as between vitamin K and the risk of bleeding, in this study we hypothesized that dp-ucMGP plasma levels will be associated with CRUSADE bleeding score in patients with acute myocardial infarction (AMI).The present study was cross-sectional and it was performed at the University Hospital of Split, Split, Croatia, during the period from June 2018 to December 2019. The study has been approved by the Ethics Committee of the University Hospital of Split  and was conducted per all ethical principles of the Helsinki Declaration from 2013. Before enrolment in the study, every participant was informed about the procedures, goals, and grounds of this research, and each participant individually signed informed written consent.The present study included 80 consecutive patients ≥18 years of age with the chief complaint of chest pain that were subsequently diagnosed with AMI and transferred to Cardiology Department, of which 40 were patients with ST-elevation myocardial infarction (STEMI) and 40 had a non-ST-elevation myocardial infarction (NSTEMI), while patients with unstable angina (UA) were not included in the study. All included patients had a positive high-sensitivity cardiac troponin I (hs-cTnI) test with values measured beyond the upper 99th percentile at the time of hospital admission (32.4 ng/L for men and 15.6 ng/L for women) accompanied with relevant electrocardiographic (ECG) changes. Finally, patients were diagnosed and treated in accordance with the European Society of Cardiology (ESC) guidelines for the treatment of ACS ,22.For this study, additional exclusion criteria were applied as follows: patients with atrial fibrillation or atrial flutter, patients treated with anticoagulants , vitamin K supplementation in the last year, patients previously treated with antiplatelet agents within the last year (except used for the current ACS management), state of hemodynamic shock at admission (defined by the current ESC guidelines), active malignancy, medications/supplements that significantly alter bone metabolism , and disorders of bone metabolism .For the body weight (kg) and height (cm) measurements, a calibrated scale  was used, and the BMI was calculated by the body weight (kg) being divided by height squared. Physical examination, clinical history, and pharmacotherapy use were directly collected from all patients included in the study on the first day of their hospital admission. Standard 12-lead electrocardiography recording was obtained multiple times during in-hospital stay using AliveCor Kardia mobile device system . Furthermore, a standard transthoracic echocardiography (TTE) examination was performed within the first 24 h of admission in all patients. All echocardiographic measurements were taken while patients were at rest and in the left lateral decubitus position. Left ventricular ejection fraction  was measured several times by the 2D biplane method, according to the modified Simpson’s rule, and the average value was recorded. All images were acquired using the Vivid 9 ultrasound system  and analyzed on the Echo PAC workstation .The in-hospital mortality risk was assessed by the Global Registry of Acute Coronary Events (GRACE) score, a scoring system intended to stratify ACS patients according to the risk of in-hospital and 6-month to 3-year mortality . AccordiThe risk of major bleeding in patients presenting with NSTEMI or STEMI was estimated by the use of the CRUSADE score . This scPeripheral blood samples were collected in test tubes with anticoagulant on admission and were afterward centrifuged and stored at −80 °C for further analyses. Blood samples were analyzed by the same medical biochemist, blinded to the subject group in the study. Plasma dp-ucMGP levels were analyzed via standard chemiluminescent immunoassay (CLIA) method by using IDS-iSYS InaKtif MGP  according to the manufacturer’s instructions. We performed a paired measurement based on which the mean value was determined. The minimal limit of detection was set at 200 pmol/L. Inter-assay and intra-assay coefficients of variability (CV) were 7.9% and 4.5%, respectively. High sensitivity cardiac troponin I (hs-cTnI) was determined by chemiluminescent microparticle immunoassay (CMIA) using ARCHITECT STAT High Sensitive Troponin-I assay  according to the manufacturer’s instructions. The remaining laboratory parameters were measured according to standards of good laboratory practice.t-test and Mann–Whitney U test depending on variable distribution normality while differences in categorical values between two groups were estimated by using a chi-squared test (χ2) or Fisher’s exact test, depending on sample size.The data for this study were analyzed using SPSS Statistics for Windows  and Prism 6 for Windows . Categorical data were shown as absolute numbers (N) and percentages (%), whereas continuous data were shown as mean ± standard deviation (SD) or median (interquartile range). The normality of distribution was assessed using the Kolmogorov–Smirnov test. Differences in continuous variables between the two main groups of interest (NSTEMI and STEMI) were assessed by using independent samples p-values were reported.To determine if plasma dp-ucMGP levels were different across three categories of patients stratified according to their major bleeding risk assessed by the CRUSADE score (low or very low vs. moderate vs. high or very high risk) we used one-way ANOVA analysis with post hoc Bonferroni correction. Furthermore, we underwent to evaluate if dp-uc MGP levels were independently associated with a high or very high risk of in-hospital bleeding (defined as the CRUSADE score > 40 points), and in this analysis, we employed a multivariable logistic regression that was adjusted for prespecified covariates of age, thrombocyte count, prothrombin time (PT) expressed as an international normalized ratio (INR), activated partial thromboplastin time (aPTT), presence of multivessel disease detected on coronary angiography, and type of the ACS event (NSTEMI or STEMI). Other important variables that are established to modify bleeding risk such as sex, hematocrit, eGFR and history of vascular disease and diabetes mellitus were not included as covariates in the model to avoid statistical collinearity since these variables were already incorporated into the CRUSADE score that was designated as the binary outcome variable in the model. Similarly, to avoid model overfitting respective to sample size, we used 6 prespecified covariates along with dp-ucMGP levels, as elaborated previously. For this analysis, respective odds ratios (ORs), 95% confidence intervals (CIs), and two-tailed t-test was used for the analysis.Patients were further analyzed according to plasma dp-ucMGP tertiles. In this analysis, patients in lowest dp-ucMGP terile were compared to patients in highest dp-ucMGP tertile with respect to age, GRACE and CRUSADE score values and variables interacting with hemostasis and bleeding tendency including eGFR, hematocrit, thrombocyte count, PT-INR time, and aPTT. Due to normal distribution of these variables, p) values and respective correlation graphs were generated. The p-value < 0.05 was considered statistically significant in all analyses.Finally, to inspect linear association of plasma dp-ucMGP levels with CRUSADE bleeding score, as well as CRUSADE bleeding score with a risk of in-hospital mortality (GRACE score), a Pearson’s bivariate correlation analysis was used. In this analysis, the r correlation coefficient (rho), two-tailed significance (p = 0.034), CRUSADE score (p = 0.011), dp-ucMGP (p = 0.001) levels, and prevalence of patients with multivessel disease (p = 0.005) were significantly higher among patients with NSTEMI, whereas the estimated glomerular filtration rate (p = 0.029) was higher in the STEMI subgroup. In respect of other baseline characteristics, there were no significant differences observed between the two groups.The baseline characteristics of patients with the respect to AMI type are presented in p = 0.430). However, patients at high/very high risk of major bleeding had significantly higher dp-ucMGP levels compared to patients at very low/low and moderate risk of major bleeding   .p = 0.023). The other incorporated covariates showed no predictive value in the regression model , PT-INR (p = 0.038) and CRUSADE score (p < 0.001) were significantly higher in the highest dp-uc MGP tertile in comparison to the lowest tertile. Similarly, patients in the highest dp-uc MGP tertile had more impaired renal function (p < 0.001) and lower hematocrit (p = 0.004) compared to patients in the lowest dp-ucMGP tertile, as shown in Among laboratory parameters reflecting hemostasis and risk scoring systems used it was obtained that aPTT (p < 0.001) (p < 0.001)   and GRAC< 0.001) , respectThe results from the present study show that laboratory parameters reflecting hemostasis and bleeding tendency (aPTT and PT-INR), as well as validated scoring system estimating the risk of in-hospital major bleeding (CRUSADE score) were significantly higher among patients that had the highest dp-uc MGP plasma levels when compared to patients within the lowest side of the dp-uc MGP level spectrum. Similarly, apart from positively correlating with CRUSADE score, dp-ucMGP levels emerged as an independent predictor of high or very high in-hospital major bleeding risk, beyond age and other hemostatic and clinical parameters. In addition, dp-ucMGP plasma levels were significantly higher in patients with a high/very high risk of bleeding in comparison to patients with either moderate or low/very low risk of bleeding. Finally, the CRUSADE bleeding score positively correlated with the risk of in-hospital mortality, as estimated by the GRACE score.The multiple correlations with respect to CRUSADE and dp-ucMGP that we observed in the present study indicate a plausible link between dp-ucMGP plasma levels and the risk of major bleeding in patients that present with ACS. Even though this relationship has not been explored in other works and in population such as in this study, there are several reports that support the biological plausibility of our findings. Firstly, Cranenburg et al. were the first to establish a causal association between dp-ucMGP levels and vitamin K status . The autIn line with this, circulating dp-ucMGP levels increase with age and with worsening of renal function in the general population, an observation that might be explained in part by vitamin K deficiency . Since iIn the present study, we intentionally included patients with STEMI and NSTEMI equally, since, in our previously published study, we demonstrated that dp-ucMGP levels were significantly higher in patients with NSTEMI in comparison to patients with STEMI, likely reflecting the more calcified and complex coronary lesions, and higher vascular calcification burden ,31,32,33Our study has several limitations. The cross-sectional nature of the study allows us only to ascertain potential associations, and not causal relationships. Furthermore, our study only measured risk of in-hospital mortality and major bleeding, while we did not prospectively register potential bleeding and mortality events, both in-hospital and during the eventual follow-up. Moreover, as sample size is fairly small, several other potentially significant predictors of high or very high bleeding risk may be missed in this study. Finally, a single measurement of dp-ucMGP levels was performed; hence, data on the possible dynamic of the measured MGP fraction in this setting remains elusive.In conclusion, our findings suggest that elevated dp-ucMGP fractions measured in the ACS patients could indicate bleeding tendency, as reflected in prolonged parameters of hemostasis, and might also serve as a proxy for the vitamin K deficiency, as was shown in the previous studies. Furthermore, such notion is reinforced by the positive correlation between dp-ucMGP levels and robustly validated score that estimates risk of major bleeding in the ACS such as CRUSADE. Finally, it is evident from this example that precision medicine and individual estimation of ischemic and bleeding risk is a complex issue that needs to be employed in contemporary practice in order to fine-tune therapeutic modalities and guide clinical decision making. Hence, further prospective studies that will be designed to prospectively evaluate the relationship of dp-ucMGP with clinical bleeding and ischemic outcomes are warranted.


Algorithmic cellular segmentation is an essential step for the quantitative analysis of highly multiplexed tissue images. Current segmentation pipelines often require manual dataset annotation and additional training, significant parameter tuning, or a sophisticated understanding of programming to adapt the software to the researcher’s need. Here, we present CellSeg, an open-source, pre-trained nucleus segmentation and signal quantification software based on the Mask region-convolutional neural network (R-CNN) architecture. CellSeg is accessible to users with a wide range of programming skills.CellSeg performs at the level of top segmentation algorithms in the 2018 Kaggle Data Challenge both qualitatively and quantitatively and generalizes well to a diverse set of multiplexed imaged cancer tissues compared to established state-of-the-art segmentation algorithms. Automated segmentation post-processing steps in the CellSeg pipeline improve the resolution of immune cell populations for downstream single-cell analysis. Finally, an application of CellSeg to a highly multiplexed colorectal cancer dataset acquired on the CO-Detection by indEXing (CODEX) platform demonstrates that CellSeg can be integrated into a multiplexed tissue imaging pipeline and lead to accurate identification of validated cell populations.CellSeg is a robust cell segmentation software for analyzing highly multiplexed tissue images, accessible to biology researchers of any programming skill level. Tissue imaging and single-cell analysis can reveal previously undetected biological structure and uncover subtle spatial relationships between cells. Recently, the development of antibody-based multiplexed imaging methods has enabled deep single-cell phenotyping of tissue microenvironments –9. This Commonly used segmentation algorithms include watershed (WTS) combined with thresholding , 19 and While pre-trained deep learning architectures exist, such as StarDist  and Cellhttps://michaellee1.github.io/CellSegSite/index.html), an easy-to-use, pre-trained, Mask R-CNN-based cell segmentation and pixel quantification software. Users supply a set of tissue images to CellSeg, and the software returns the segmented images and a table of single-cell statistics including each cell’s location, nucleus size, and mean pixel values in each imaging channel. CellSeg is open-source and available for Windows, Mac, or Linux. It is capable of segmenting JPG, PNG, and TIFF images of any image size, subject to hardware requirements reported below. CellSeg is accessible to individuals of all programming levels and requires minimal user input. For most uses, the pre-trained CellSeg model requires no additional manual annotation of training data, no additional training, and limited parameter tuning to produce a high-quality segmentation. The software has been designed to work as a library, so more advanced Python users can customize the pipeline to fit their needs. Applications detailed below demonstrate that CellSeg robustly segments highly multiplexed fluorescence images from a variety of healthy and cancerous tissues. CellSeg exceeds an established WTS segmentation pipeline both in terms of user friendliness as well as segmentation accuracy and performs at the level of two state-of-the-art deep learning segmentation algorithms.Here, we present CellSeg (The CellSeg software is implemented in Python and run using Jupyter Notebook . CellSeghttps://cellengine.com), Cytobank (https://www.cytobank.org), or FlowJo (https://www.flowjo.com), as well as cell clustering programs like VorteX  with all other parameters set to default. Brightfield images were segmented using parameters channels = , invert = True, and flow_threshold = 0.8 with all other parameters set to default. Predicted masks for each segmentation algorithm were saved and uploaded to Kaggle to obtain the mAP score.CellSeg and an optimized WTS segmentation algorithm  were botWe implemented two mask expansion algorithms. The first algorithm expands the boundary of each mask by a user-defined number of pixels. If this expansion leads to two overlapping masks, the algorithm assigns each pixel in the overlapping region to the mask whose center is closest to the pixel. The first mask expansion algorithm is computationally efficient and works well for tissue images with cells of similar size. However, the algorithm biases pixel assignment towards smaller masks, since the center of these masks are generally closer to the overlap region than the centers of larger masks. To correct for this, the second expansion algorithm iterates over the masks, expanding each mask by 1 pixel until it collides with another pre-existing mask boundary, at which point growth in that direction stops. The algorithm proceeds until each mask has been expanded by the user-defined number of pixels. This algorithm mitigates the need for assigning pixels based on distance to cell center at the cost of more computation time. Through iterative visual inspection of masks with and without mask expansion, we found that growth by 1 or 2 pixels is usually sufficient to capture most membrane protein signal. The lateral bleed compensation algorithm implemented in the CellSeg pipeline is the same as in previously published work from our group, readers are directed to the original paper for the details of the algorithm .https://cellengine.com). When evaluating fluorescent bleed compensation on the CRC dataset, samples were aggregated by TMA, resulting in two gates for each cell population, one corresponding to each TMA. When performing population correlation analysis, cell types were gated by sample, resulting in tailored gates for the 140 samples. Gating was performed independently for the WTS dataset and the CellSeg dataset in a blinded fashion (J.S.B.). Resulting identified populations were validated by an expert in flow cytometry (C.M.S.). Population correlation analysis was performed in R Studio. The script is available upon request. Plots in Fig. Segmentation of the CRC dataset and multi-tissue TMA was performed on a Dual Intel® Xeon® Silver 4114 10-core CPU. Resulting segmented data was gated in CellEngine (https://michaellee1.github.io/CellSegSite/index.html. Operating system(s): Windows, MacOS, or Linux. Programming language: Python. Other requirements: Web browser, internet connection, Conda, Jupyter, minimum 16 GB RAM, other Python package dependencies listed on project home page. License: MIT. Restrictions for Non-academics: None.Project Name: CellSeg. Project home page:


N = 30,275) from the mobile health App users in Japan, we show age-dependent lifestyle changes during a nonpunitive “mild lockdown” (from April to May 2020).The stay-at-home order during the COVID-19 pandemic has restricted individuals’ social behaviors, and therefore, effected their lifestyle including sleep, diet, and physical activity. Using the cross-sectional study design with a large sample size , we investigated an association between lifestyle change and body-weight change. Participants who reported advanced sleep phase during mild lockdown described a weight decrease. In contrast, the delayed sleep phase reported a weight gain. The results were significant after adjustment of confounding factors including physical activity and meal changes.Although there is cumulative evidence showing a relationship between late chronotype and obesity, it is still unclear about the potential benefit of the chronotype management to control body weight. Thus, to the best of our knowledge, this is the first study investigating the association between chronotype and weight changes by leveraging a large cohort. The potential impact of lifestyle change on health outcomes during the COVID-19 pandemic has received substantial attention. The stay-at-home order has restricted individuals’ social behaviors, and therefore, effected their lifestyle including sleep, diet, and physical activity. Since these lifestyles are strongly associated with obesity, which is one of the major public health issues worldwide –4. In JaN = 30,275) from an online survey in this App, we show age-dependent sleep changes by this mild lockdown. Moreover, we are the first to describe an association between sleep phase shift and body-weight change during the pandemic.The aim of this study is to find the effect of COVID-19-related social restrictions on the mobile health App “Asken” users, who generally intend to lose weight. Asken is a food-log and food-coaching App and was downloaded about 5500,000 times in Japan. The App gives feedback of food contents based on the Dietary Reference Intakes for Japanese decided by the Ministry of Health, Labor, and Welfare. By cross-sectional large data  excluding shift workers and subjects who answered outliers were retrieved . Consistent with these findings, the social jetlag (weekly jetlag appeared by the sleep phase difference between work-days and free-days) was decreased especially in younger ages . Taken together, mild lockdown is associated with phase-delay and increased sleep duration in work-days, and decreased social jetlag.From 40,513 answers, a total of 30,275 subjects  recorded height and body weight using this App. We confirmed BMI change correlated with MSW and MSF after adjusting the age, sex, and those lifestyle changes (p < 0.01). In summary, chronotype shift during the mild lockdown was associated with the body-weight change.Then, we analyzed body-weight change during the mild lockdown to understand the relation with a lifestyle change. Averaged weight change was 0.08 kg in this cohort Table ; it mighHere we found the improvement of social jetlag and sleep duration by the mild lockdown in Japan. Previous reports of delayed sleep phase on work-days and decreased social jetlag during the COVID-19 pandemic in Argentina  and a woTo the best of our knowledge, this is the first study investigating the association between chronotype management and body-weight change during the COVID-19 pandemic using a large cohort from an online survey database. In addition, we adjusted for several lifestyle-related factors in our model. Social jetlag often seen in late chronotype is related to obesity . Late chThe limitations of our study include misclassification due to the self-reports, unmeasured and uncontrolled confounding, unclear temporal ordering between variables, and lack of generalizability to other populations given the various pandemic situations and political decisions. In summary, our findings suggest the possible modifications in sleep pattern and its association with body-weight change among the Japanese population during the mild lockdown. As obesity or disrupted circadian clock by social jetlag or irregular lifestyle exacerbate immune function , furtherSupplemental information


Although immune checkpoint inhibitors (ICIs) have been shown to improve overall survival (OS) in advanced non-small-cell lung cancer (NSCLC) patients, ICIs sometimes cause various types of immune-related adverse events (irAEs), which lead to the interruption of ICI treatment. This study aims to evaluate the clinical significance of the continuation of ICIs in NSCLC patients with irAEs and to assess the safety and efficacy of the readministration of ICIs after their discontinuation due to irAEs.We retrospectively identified patients with advanced NSCLC who were treated with first- to third-line anti-programmed cell death-1 (PD-1) therapy from January 2016 through October 2017 at multiple institutions belonging to the Niigata Lung Cancer Treatment Group. Progression-free survival (PFS) and OS from the initiation of ICI treatment were analyzed in patients with and without irAEs, with and without ICI interruption, and with and without ICI readministration. A 6-week landmark analysis of PFS and OS was performed to minimize the lead-time bias associated with time-dependent factors.vs. not reached (95% CI: 22.4–NE); p = 0.025]. Of 52 patients who discontinued ICIs, 14 patients (6.1%) readministered ICIs, and OS in patients with ICI readministration was significantly longer than that in patients without ICI readministration .Of 231 patients who received anti-PD-1 antibodies, 93 patients (40%) developed irAEs. Of 84 eligible patients with irAEs, 32 patients (14%) continued ICIs, and OS was significantly longer in patients who continued ICIs than that in patients who discontinued ICIs [not reached (95% CI: NE-NE) The current study demonstrated that both the continuation and readministration of ICIs after irAE occurrence improved OS compared to the permanent interruption of ICIs in NSCLC patients with ICI-related irAEs. Immune checkpoint inhibitors (ICIs), such as anti-programmed cell death-1 (PD-1) and anti-programmed cell death ligand-1 (PD-L1) antibodies, have achieved durable responses in some patients with non-small-cell lung cancer (NSCLC) . Anti-PDTreatment with ICIs is often accompanied by immune-related adverse events (irAEs), and irAEs can be lethal or the main reason for the discontinuation of ICIs. The decision of whether to continue or discontinue ICIs after the occurrence of irAEs is generally based on the type of irAE and its severity . We prevThis study aims to assess the significance of the continuation of ICIs in NSCLC patients who developed irAEs and to evaluate the safety and efficacy of the readministration of ICIs in patients who discontinued ICI treatment due to ICI-related irAEs.We retrospectively analyzed the medical records of patients with advanced NSCLC who were treated with single-agent anti-PD-1 as first- to third-line therapy at multiple institutions belonging to the Niigata Lung Cancer Treatment Group from January 2016 to October 2017. To prevent selection bias, all consecutive patients who met eligibility were enrolled. This study was approved by the institutional review board of each participating institution.The following data were collected retrospectively for all patients: demographics, phenotypes of cancers, types of anti-PD-1 therapies, and irAEs. Treatment responses were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria version 1.1. Each irAE was graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Progression-free survival (PFS) was measured as the time from the start of anti-PD-1 therapy to progressive disease (PD) or death due to any cause. OS was measured as the time from the first administration of immunotherapy to death due to any cause. The objective response rate (ORR) was defined as the percentage of patients assessed as having complete response (CR) or partial response (PR) of all patients treated with anti-PD-1 therapy. The disease control rate (DCR) was defined as the percentage of patients assessed as having CR, PR, or stable disease (SD) of all patients treated with anti-PD-1 therapy. Treatment interruption was defined as either the delay or cessation of ICI treatment due to irAEs. Patients with ICI readministration were defined as those who were readministered a PD-1 inhibitor at least one time after the interruption of ICI treatment due to irAEs. Patients without ICI readministration were defined as those whose ICI treatment was permanently stopped due to irAEs.t-tests. Comparisons between groups were performed by Fisher’s exact test or the chi-square test. To minimize the lead-time bias associated with time-dependent factors, we performed a 6-week landmark analysis including only patients who were alive or whose disease was under control at 43 days after the initiation of anti-PD-1 therapy, which is the median time of onset of irAEs, for PFS and OS. For this 6-week landmark analysis, we excluded 66 patients for PFS and 17 patients for OS in the analysis of Kaplan–Meier survival curves were constructed for PFS and OS, and differences between groups were identified using the log-rank test. The univariate Cox proportional hazards model was used to assess the effects of the presence of irAEs, the continuation of ICIs, and the readministration of ICIs on PFS and OS. Continuous variables are presented as the median (range) and were compared by two-sided EGFR) mutations was lower in patients with irAEs than that in patients without irAEs. Other clinical features, including age, Eastern Cooperative Oncology Group performance status, treatment line, and PD-L1 expression, were not significantly different.In total, 231 patients were enrolled in this study. Among these patients, 93 patients (40%) developed irAEs Figure 1vs. 4.8 (95% CI: 3.2–7.6); p < 0.001]. The median OS was also significantly longer in patients with irAEs than that in patients without irAEs . The hazard ratios estimated by the Cox proportional hazards model were as follows: the PFS hazard ratio was 0.51 , and the OS hazard ratio was 0.49 . Furthermore, the ORR and DCR were significantly higher in patients with irAEs than those in patients without irAEs (The distribution of irAEs is shown in ut irAEs Table 3.vs. 15.3 (95% CI: 8.3–NE); p = 0.76]. However, the median OS was significantly longer in patients who continued ICIs than that in patients who discontinued ICIs . The hazard ratios estimated by the Cox proportional hazards model were as follows: the PFS hazard ratio was 0.9 , and the OS hazard ratio was 0.27 . In terms of irAE phenotypes, the percentage of patients who experienced immune-related pneumonitis was higher in the anti-PD-1 treatment interruption group than that in the anti-PD-1 continuation group ; p = 0.17]. On the other hand, the median OS was significantly longer in patients with ICI readministration than that in patients without ICI readministration . The hazard ratios estimated by the Cox proportional hazards model were as follows: the PFS hazard ratio was 0.46 , and the OS hazard ratio was 0.15 . The characteristics of the initial irAEs stratified by readministration are shown in In total, 52 patients discontinued anti-PD-1 treatment, and 14 patients were readministered ICIs (This study demonstrated the prognostic significance of the occurrence of irAEs, ICI continuation after the development of irAEs, and ICI rechallenge after the interruption of ICIs due to irAEs. Patients with irAEs had a better prognosis than those without irAEs Figure 2+ T cells is transient (via continuation or readministration.There have been no reports that simultaneously evaluated the significance of ICI continuation and readministration after the occurrence of irAEs in NSCLC patients. In a retrospective study verifying the impact of ICI interruption due to irAEs, the median OS was worse in patients with ICI interruption than that in others with continuous ICI administration . Severalransient . Althougransient . CollectIn the current study, there was no PFS advantage from the continuation or readministration of ICIs in patients with ICI-related irAEs 4. As a Unexpectedly, our study demonstrated no difference in the frequency of irAEs whose CTCAE grade was over 3 between patients with ICI readministration and those with permanent ICI interruption 5. This vs. 37%, p = 0.057; The limitations of the current study include the relatively small number of patients with ICI readministration and the retrospective nature of the study. Clinicians might have tended to avoid continuing and readministering ICIs to patients with irAEs such as pneumonitis, which could be fatal if exacerbated. There is no detailed analysis for each irAE in this study. In addition, response rate to initial anti-PD-1 therapies in patients with readministration tended to be higher than that in patients without readministration (ORR 71% In summary, we retrospectively investigated the clinical significance of the continuation and readministration of single-agent anti-PD-1 therapy in NSCLC patients with ICI-related irAEs. The continuation and readministration of ICIs significantly prolonged OS, and their safety was acceptable. A future prospective study is needed to establish optimal treatment strategies for patients with irAEs.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Niigata University Graduate School of Medical and Dental Sciences, Niigata Prefectural Shibata Hospital, Niigata City General Hospital, Niigata Cancer Center Hospital, Saiseikai Niigata Hospital, Nishi-Niigata Chuo National Hospital, Niigata Prefectural Central Hospital, Nagaoka Chuo General Hospital, Nagaoka Red Cross Hospital, Niigata Medical Center. The patients/participants provided their written informed consent to participate in this study.TO devised this study. TO and SW designed the protocol. SW, TO, SS, KN, KI, RK, TM, TA, SM, HT, MO, MTe, NM, TI, AI, KS, HY, MH enrolled patients into the study. TF and SW co-wrote this manuscript, conducted data analysis, and prepared figures and tables. All authors contributed to the article and approved the submitted version.The primary author was supported by a grant from the Niigata Medical Association, Japan.SW reports grants and personal fees from AstraZeneca, personal fees from Chugai Pharma, personal fees from Ono Pharmaceutical, personal fees from Bristol-Myers, grants and personal fees from Boehringer Ingelheim, personal fees from Eli Lilly, personal fees from MSD, personal fees from Taiho Pharmaceutical, personal fees from Pfizer, personal fees from Novartis, and personal fees from Daiichi Sankyo outside the submitted work. TO reports personal fees from Boehringer Ingelheim, personal fees from MSD, personal fees from Eli Lilly, personal fees from AstraZeneca, personal fees from Chugai-pharm, and personal fees from Bristol-Myers Squibb outside the submitted work. SS reports personal fees from AstraZeneca, personal fees from Chugai Pharma, personal fees from Taiho Pharmaceutical, and personal fees from MSD outside the submitted work. KN reports personal fees from AstraZeneca, personal fees from Boehringer Ingelheim, personal fees from Taiho Pharmaceutical, and personal fees from MSD outside the submitted work. AI reports personal fees from AstraZeneca, personal fees from Chugai Pharma, personal fees from Bristol-Myers, personal fees from Boehringer Ingelheim, personal fees from Ono Pharmaceutical, personal fees from Taiho Pharmaceutical, personal fees from Novartis International AG, and personal fees from Daiichi Sankyo Company outside the submitted work. SH reports personal fees from GlaxoSmithKline Inc. outside the submitted work. TA reports personal fees from Eli Lilly Japan, personal fees from Chugai Pharmaceutical, personal fees from Taiho Pharmaceutical, personal fees from Ono Pharmaceutical, personal fees from Bristol-Myers Squibb, personal fees from AstraZeneca, and personal fees from Mylan outside the submitted work. SM reports personal fees from Chugai Pharmaceutical, personal fees from ONO Pharmaceutical, personal fees from AstraZeneca, personal fees from Eli Lilly, personal fees from MSD, personal fees from Boehringer Ingelheim, personal fees from Taiho Pharmaceutical, personal fees from Novartis, personal fees from Bristol-Myers Squibb, and personal fees from Kyowa Hakko Kirin outside the submitted work. HT reports grants and personal fees from Bristol-Myers Squibb, grants and personal fees from Eli Lilly, grants and personal fees from MSD, grants and personal fees from Taiho pharmaceutical, grants and personal fees from Pfizer, grants and personal fees from Novartis, grants and personal fees from Chugai pharmaceutical, grants and personal fees from Astra Zeneca, grants and personal fees from Boehringer Ingelheim, grants and personal fees from Ono pharmaceutical, and grants and personal fees from Merck outside the submitted work. MO reports personal fees from AstraZeneca, personal fees from Ono Pharmaceutical, personal fees from Bristol-Myers, personal fees from Boehringer Ingelheim, personal fees from MSD, and personal fees from Taiho Pharmaceutical outside the submitted work. NA reports personal fees from Meiji Seika Pharma, grants from MSD, and personal fees from MSD outside the submitted work. MH reports personal fees from Boehringer-Ingelheim Japan, personal fees from AstraZeneca, personal fees from Taiho Pharmaceutical, and personal fees from Daiichi Sankyo outside the submitted work. YO reports personal fees from Boehringer-Ingelheim Japan and personal fees from Meiji Seika Pharma outside the submitted work. TKo reports personal fees from AstraZeneca, Boehringer-Ingelheim, Sanofi Genzyme, Novartis, Daiichi Sankyo, Kyorin Pharmaceutical, and GlaxoSmithKline outside the submitted work. TKi reports grants and personal fees from Chugai Pharma, grants and personal fees from Boehringer Ingelheim, grants and personal fees from Eli Lilly, grants and personal fees from MSD K.K., personal fees from Astellas Pharma Inc., grants and personal fees from Taiho Pharmaceutical Co., Ltd., personal fees from Bristol-Myers Squibb Company, personal fees from Pfizer Japan Inc., grants and personal fees from Daiichi Sankyo Co., Ltd., personal fees from Taisho Toyama Pharmaceutical Co., Ltd., personal fees from Janssen Pharmaceutical K.K., personal fees from Japan BCG Laboratory, grants and personal fees from Ono Pharmaceutical Co., Ltd., personal fees from Novartis Pharma K.K., personal fees from Mylan N.V., grants and personal fees from AstraZeneca, personal fees from Roche Diagnostics K.K., grants and personal fees from Shionogi & Co., Ltd., grants from TEIJIN PHARMA Ltd., and grants from KYORIN Pharmaceutical Co., Ltd., outside the submitted work.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Drosophila melanogaster sense RNA and control 3′-2′-cGAMP signaling. In this review, we summarize and discuss in depth recent studies that identified or implied cGAS as an RBP. We also comprehensively summarized current experimental methods and computational tools that can identify or predict RNAs that bind to cGAS. Based on these discussions, we appeal that the RNA-binding activity of cGAS cannot be ignored in the cGAS-mediated innate antiviral response. It will be important to identify RNAs that can bind and regulate the activity of cGAS in cells with or without virus infection. Our review provides novel insight into the regulation of cGAS by its RNA-binding activity and extends beyond its DNA-binding activity. Our review would be significant for understanding the precise modulation of cGAS activity, providing the foundation for the future development of drugs against cGAS-triggering autoimmune diseases such as Aicardi-Gourtières syndrome.cGAS, a DNA sensor in mammalian cells, catalyzes the generation of 2’-3’-cyclic AMP-GMP (cGAMP) once activated by the binding of free DNA. cGAMP can bind to STING, activating downstream TBK1-IRF-3 signaling to initiate the expression of type I interferons. Although cGAS has been considered a traditional DNA-binding protein, several lines of evidence suggest that cGAS is a potential RNA-binding protein (RBP), which is mainly supported by its interactions with RNAs, RBP partners, RNA/cGAS-phase-separations as well as its structural similarity with the dsRNA recognition receptor 2’-5’ oligoadenylate synthase. Moreover, two influential studies reported that the cGAS-like receptors (cGLRs) of fly  As highlighted by the current COVID-19 pandemic, viral infection poses a great threat to human health and economics . The innAlthough cGAS is traditionally recognized as a DNA-binding protein , numerouHerein, we highlighted recent studies supporting or implying the RNA-binding activity of cGAS. Simultaneously, we discussed the subcellular location of cGAS based on the reported membrane, cytosol, and nucleus localization. Moreover, we summarized current experimental and computational methods that can identify or predict the RNA interactors of cGAS. We used bioinformatics tools to analyze the interaction propensity between mouse- or human-derived cGAS and RNA. This review provides novel insight into the noncanonical regulatory manner of cGAS activity in the innate antiviral response.via the carboxyl terminus to phosphorylate IRF-3  gapmer antisense oligonucleotides (ASOs) exhibit sequence-dependent inhibition of sensing by the RNA sensor TLR7  and inhibit cGAS activity when the dsDNA concentration is high , a class of noncoding RNAs, are characterized by a covalent bond linking the 3’ and 5’ ends during the process of RNA splicing , 78. ciaIn vitro studies also showed that recombinant cGAS could produce cGAMP in the presence of RNA : DNA hybrids , which exerts antiviral immunity in conjunction with the NF-κB pathway , such as hnRNP K homology domains, RNA recognition motifs, dsRNA-binding domains, and ZnF domains –106. Howith cGAS . cGLRs r pathway . It can http://service.tartaglialab.com/page/catrapid_group) is an online algorithm predicting the propensity of protein-RNA interactions based on the respective contributions of secondary structure, hydrogen bonding, and Van Der Waals interactions , RNA pull-down, systematic evolution of ligands by exponential enrichment (SELEX), RNAcompete, and RBNS (in cells assays mainly include RNA binding protein immunoprecipitation (RIP) and crosslinking immunoprecipitation (CLIP) assays  assays . Furthernfluence . TherefoAlthough experimental methods could provide convincing evidence supporting the RNA-protein interaction, most of the experimental methods are technically challenging and time-consuming Table 3.In this review, we highlighted the RNA-binding activity of cGAS based on the RNA interactors of cGAS, the numerous RBP partners of cGAS, and its structural similarity with 2′-5′-OAS. We also elucidated and discussed current experimental methods and computational tools that can be used for exploring and calculating cGAS-RNA interactions. These clues indicated that the RNA-binding activity of cGAS may play an important role in the innate antiviral response, which needs to be further confirmed in the future. Although cGAS showed potential RNA-binding activity, it should be noted that the RNA binding activity of cGAS is different from that of known RNA sensors, such as RIG-1 and TLR3, which mainly recognize viral RNA . In contYM, XW, HS, YilW, WS, JX, and WL: conception and design, collection and/or assembly of references, data analysis and interpretation, and manuscript writing; HS, ZR, YifW, and YilW: final interpretation and helpful discussion of the manuscript. All authors read and approved the final manuscript and agreed to this publication. YM and XW contributed equally to this article.This work was supported by grants from the National Natural Science Foundation of China (Nos. 81872908 and 82072274), Key Laboratory of Virology of Guangdong Province, Guangdong Provincial Natural Science Foundation Project (No. 2019A1515010046), Study on the mechanism of estrogen promoting the repair of urethral function in the treatment of female stress urinary incontinence with hUCMSCs (No. 2021A1515011221), and Guangdong Modern Agricultural Industrial Technology System Innovation Team (No. 2017YFC1701100).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The ‘gold standard’ treatment of severe neonatal jaundice is phototherapy with blue–green light, which produces more polar photo-oxidation products that are easily excreted via the bile or urine. The aim of this study was to compare the effects of bilirubin (BR) and its major photo-oxidation product lumirubin (LR) on the proliferation, differentiation, morphology, and specific gene and protein expressions of self-renewing human pluripotent stem cell-derived neural stem cells (NSC). Neither BR nor LR in biologically relevant concentrations (12.5 and 25 µmol/L) affected cell proliferation or the cell cycle phases of NSC. Although none of these pigments affected terminal differentiation to neurons and astrocytes, when compared to LR, BR exerted a dose-dependent cytotoxicity on self-renewing NSC. In contrast, LR had a substantial effect on the morphology of the NSC, inducing them to form highly polar rosette-like structures associated with the redistribution of specific cellular proteins (β-catenin/N-cadherin) responsible for membrane polarity. This observation was accompanied by lower expressions of NSC-specific proteins  together with the upregulation of phospho-ERK. Collectively, the data indicated that both BR and LR affect early human neurodevelopment in vitro, which may have clinical relevance in phototherapy-treated hyperbilirubinemic neonates. Bilirubin (BR) is the end product of the heme degradation pathway. While in the past BR was considered a potentially neurotoxic waste product of heme catabolism, numerous in vitro, in vivo, and clinical studies over recent decades have demonstrated that when it is only mildly elevated it is also an endogenous antioxidant with potent anti-inflammatory and cytoprotective effects, mediated via true endocrine mechanisms .Elevated serum BR concentrations are present in most newborn infants . The phyZ-lumirubin (LR), E,Z-BR, and Z,E-BR being the principal compounds. Additional BR photoproducts include monopyrrolic (Z-BOX A-D), dipyrrolic (propentdyopents), and tripyrrolic (biopyrrin A/B) compounds ,2524,25]p < 0.05)  B,C. In aThe data strongly suggested that LR-treated self-renewing NSC acquire a significantly different morphology reminiscent of immature rosettes, with apically localized cell polarity proteins; a phenomenon accompanied by changes in various transcription factors and activation of an ERK signaling pathway.Lastly, in order to determine if BR or LR affected the ability of the NSC to terminally differentiate into neurons, the NSC were exposed to both pigments and the extent of their terminal differentiation was subsequently assessed. NSC were seeded in a standard conditioned growth medium on Day 0, and the treatment was started three days after plating , glioblastoma (U-87), and microglial cells (HMC3) , did not affect cell viability at all, even at the biologically relevant concentrations 25 µM) . HoweverY, gliobl5 µM . HoBased on these findings, and since NSC have never been studied for their susceptibility to the potentially harmful effects of BR, we focused on the possible cytotoxic effects of BR and LR on these cells. We observed a significantly decreased viability and metabolic activity in those cells treated with gradually increasing concentrations of BR, while LR only had a significant effect only at the highest concentration. As discussed above, BR in high concentrations clearly exerts cytotoxicity, in particular on cells of the central nervous system (CNS). However, the pathogenesis of the whole process is not fully understood, with significant differences in susceptibility to BR toxicity across various brain regions and cell populations ,33. PrevInterestingly, while performing these studies on the cytotoxicity of BR and LR, we noticed that LR induced major morphological changes in our self-renewing NSC, while no such changes were noticed after exposure to BR. This observation may be of clinical importance, since cellular polarity plays a significant role during the development of the CNS . During Finally, we also assessed the capacity of BR or LR to affect the terminal differentiation of NSC. Previously, it has been shown that moderate to severe hyperbilirubinemia could induce neurological dysfunction and potentially impair brain myelination with long-term sequelae, particularly in preterm infants . HoweverOur study has assessed the possible impact of BR as well as its major photoisomer LR on human neurodevelopment using an in vitro model of hPSC-derived NSC. Our data demonstrate that neither of these compounds significantly affected the extent of terminal differentiation of neurons and astrocytes. However, compared to LR, BR exerted a higher cytotoxicity on self-renewing NSC, and this effect was dose-dependent and accompanied by mildly elevated pro-apoptotic markers. On the other hand, LR had a dose-dependent effect on the morphology of self-renewing NSC, inducing them to form highly polarized structures with lower expressions of some NSC-specific markers, but with activation of ERK signaling. Considering the tightly orchestrated processes during the formation of the CNS, this effect of changed cell polarity and identity could potentially have a detrimental effect on the developing brain. Although complex mechanisms behind these phenomena remain to be addressed, both BR and LR clearly affect early human neurodevelopment in vitro.


Simulation-based prediction of mechanical properties is highly desirable for optimal choice and treatment of leather. Nowadays, this is state-of-the-art for many man-made materials. For the natural material leather, this task is however much more demanding due to the leather’s high variability and its extremely intricate structure. Here, essential geometric features of the leather’s meso-scale are derived from 3D images obtained by micro-computed tomography and subsumed in a parameterizable structural model. That is, the fiber-bundle structure is modeled. The structure model is combined with bundle properties derived from tensile tests. Then the effective leather visco-elastic properties are simulated numerically in the finite element representation of the bundle structure model with sliding contacts between bundles. The simulation results are validated experimentally for two animal types, several tanning procedures, and varying sample positions within the hide. Finally, a complete workflow for assessing leather quality by multi-scale simulation of elastic and visco-elastic properties is established and validated. Leather is a renewable material that is primarily used in manufacturing of high-priced consumer goods like shoes, clothing, upholstered furniture, or automotive interiors. Due to the naturally grown raw material from animals’ skins, each up-cycled tanned hide for leather production is unique. Being proclaimed as a marketing advantage on the one hand, this high variability represents, on the other hand, a considerable challenge for production, processing, and final application of leather.It is well-known that all biological tissues and materials are visco-elastic, see for example, ,2. Thus,The collagen-based hierarchical 3D structure of the animal hide is essentially retained in manufacturing. Affecting the properties of the finished leather, the native structure causes a material-inherent large variance and pronounced anisotropy of the physical properties  due to  have been applied to capture structural features of the collagen fiber-bundles ,9,10. TrLeather properties are determined significantly by the fiber-bundle structure and their elastic and relaxation properties ,13. The The approach sketched above relies on a geometric model for the collagen fiber-bundle network reproducing contacts or branching, in particular the winding-contact-branching. This essential geometric information is deduced from 3D image data obtained by micro-computed tomography of leather snippets of the size of a few millimeters. 3D quantitative analysis of the network structure based on these images emphasizes the high variability with respect to animal, position within the hide, and tanning. Segmentation and successive analysis of representative numbers of fiber-bundle segments and contact elements failed, as all attempts reported on previously ,11,27,28Together with experimentally determined (visco-)elastic and relaxation properties of fiber bundles, the network model yields a meso-structural simulation tool for comparing several species, races, as well as individual body parts and tanning processes.Leather samples and fiber bundles from back, flank, and axilla of metal-free vegetable-tanned, non-dyed bovine hides from the Simmental breed (leathers L1 and L2), zebu breed (leather L3), and synthetically tanned bovine hide (leather L4) were investigated . All leather samples were reduced to their reticular layers by splitting off the grain and papillary layers. The sample conditioning and preparation followed ISO 2418, ISO 2419, and ISO 2420.Light and electron microscopic imaging of complete leather structure areas, as well as selected substructures and structure elements like collagen fibers and collagen fiber-bundles confirmed the hierarchical leather structure, as well as gradients in density . Weave aE [MPa] are recalculated as a slope of the linear part of the stress-strain curves.Macroscopic bulk densities and thicknesses are determined according to ISO 2420 and ISO 2589, respectively. For leathers L1–L3, identical technological processing resulted in similar raw densities (0.51 to 0.71 g/ mm) see . ResultsThe micro-mechanical properties of collagenous fibers and fiber-bundles have to be measured as they determine the macroscopic leather material behavior to a high extent. To this end, collagen fiber-bundles are gently manually separated from the back, flank, and axilla of leathers L1–L4. They are imaged by light microscopy to determine their cross-sectional shapes and diameters see . The thiFiber-bundles are subject to two types of load scenarios: (1) Classical uniaxial tensile test up to fiber-bundle breakage, and (2) incremental loading up to a pre-defined elongation followed by recording the force drop. Relaxation results for fiber-bundles and leathers are discussed in Individual micromechanical stress-strain experiments using a test stage of Kammrath & Weiss GmbH yield force and elongation parameters on fibre-bundle scale. All the experiments were conducted on samples that were 2 mm in length, clamped in the testing machine from both sides. Tension was applied by increasing displacements. Videos help to observe the tensile load-induced deformation process on a microstructure level, including complete fiber-bundle reorientation a,b, fibeTypical force-elongation diagrams for loaded fiber-bundles are presented in Dumbbell-shaped leather specimen from the back, flank, and axilla are tested according to ISO 3376. Six specimens are chosen in each of the three sample positions within the hide: three specimens in longitudinal direction, parallel to the back line of the animal, and three specimens perpendicular to the back line (transverse direction). See Overall, 66 specimens were tested. Typical stress-strain diagrams  for the The zebu leather samples from L3 yield lower maximal stresses than Simmental breed samples , while L4 features the largest magnitudes. The strain level of L4 is higher (40–50%) than those of L1–L3  image without tunnels or wholes within the component to be skeletonized. Thus, all bundle substructures captured in the rithm of .Cheng’s algorithm contracts skeleton nodes. Consequently, the valence of these new nodes increases. Nevertheless, a clear predominance of Y nodes is observed.Structural modeling is needed to generate geometrically simple elements including proper connectivity. The network structure of the fiber-bundles features predominantly Y-shaped nodes, as described above. Moreover, the fiber-bundle network is forming essentially one connected component. These two observations motivate the choice of a structural model based on the edge system of a Voronoi tessellation. More precisely, the edge systems of planar Voronoi tessellations generated by macroscopically homogeneous, random Poisson point processes are rotated spatially, and interwoven. This allows for control by a very small set of parameters, captures the microscopic spatial variation, and naturally reflects the strong prevalence of Y nodes. The latter is due to the fact that vertices in planar macroscopically homogeneous random Poisson-Voronoi tessellations are almost surely of valence three .The planar edge systems as shown in The model is controlled by the mean number The mechanical behavior of the leathers is modeled using our in-house software-tool FISFT which includes a 1D finite element method for large deformations and friction evolution, see . TheoretAt low friction, bending or compression of the material does not influence the torsion at the contacts  of leather very well. Realizations of this model enable mechanical simulation, accounting for frictional sliding and visco-elastic behavior of the leather on the basis of known (measured) behavior of its fiber-bundles. Simulations were based on advanced mathematical modelling, reducing the dimension of contact problems and numerical techniques, including complicated mechanical issues as frictional sliding at the complex contact junctions. All simulations were validated by mechanical tests and show very good agreement on the investigated scale.The anisotropy of leather on several scales was discussed in detail in


NG) group of arginine in proteins can be methylated by protein-arginine methyl-transferase (PRMT) and citrullinated by protein-arginine deiminase (PAD) , symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA), with ADMA being hydrolyzed to its major metabolites dimethylamine (DMA) and l-citrulline  do not only change the physicochemical properties including charge and solubility of proteins, but they have far-reaching consequences for health, disease, and even death in living organisms. Reportedly, the first scientific publications appeared as early as the 1940s and evolved drastically over the last two decades, indicating the strong interest of researchers from many different disciplines. With 6604 and 6139 articles, acetylation and methylation belong to the most common investigated PTMs, respectively. Amino acid residues such as arginine and lysine undergo many different PTMs. For example, the terminal guanidine (AD) Fig. . Althougine Fig. . These erol Bunn .Fig. 1SiAmino Acids, a journal dedicated to amino acid, peptide and protein research, on PTMs including two review articles and five original research papers.This volume presents the first part of a special issue of Samuel and colleagues provide an up-to-date review of the literature on mechanisms for PRMT regulation by endogenous modulators and synthetic inhibitors , dimethyllysine (DML) and trimethyllysine (TML) derivatives. The significance of TML as a vascular risk factor was investigated in a clinical study on acute ischemic stroke using LC–MS/MS (Schwedhelm et al. S-transnitrosylation of cysteine residues in proteins is a less abundant but not less important PTM. S-Nitrosoalbumin is one of the most abundant endogenous S-nitrosylated proteins in human plasma and may serve as a carrier and source of NO-related bioactivity in human circulation including platelets and red blood cells. In an in vitro study, extracellular low-molecular-mass thiols including l-cysteine were found to mediate the inhibitory action of synthetic S-nitrosoalbumin on human platelet aggregation via S-transnitrosylation of the platelet surface proteins (Tsikas Like tyrosine nitration in peptides and proteins, s Tsikas .We thank the authors for their contributions and the reviewers for their honorary engagement.


Advances in multi-omics have led to an explosion of multimodal datasets to address questions from basic biology to translation. While these data provide novel opportunities for discovery, they also pose management and analysis challenges, thus motivating the development of tailored computational solutions. Here, we present a data standard and an analysis framework for multi-omics, MUON, designed to organise, analyse, visualise, and exchange multimodal data. MUON stores multimodal data in an efficient yet flexible and interoperable data structure. MUON enables a versatile range of analyses, from data preprocessing to flexible multi-omics alignment.The online version contains supplementary material available at 10.1186/s13059-021-02577-8. Multi-omics designs, that is the simultaneous profiling of multiple omics or other modalities for the same sample or cells, have recently gained traction across different biological domains. Multi-omics approaches have been applied to enable new insights in basic biology and translational research , 2.On the one hand, the emerging multi-omics datasets result in novel opportunities for advanced analysis and biological discovery . CriticaWhile specialised frameworks for the analysis of different omics data types have been proposed, including for bulk and single-cell RNA-seq –9 or epimultimodal omics analysis), an analysis framework that is designed from the ground-up to organise, analyse, visualise, and exchange multimodal data. MUON is implemented in Python and comes with an extensive toolbox to flexibly construct, manipulate and analyse multi-omics datasets. At the core of the framework is MuData, an open data structure standard, which is compatible with and extends previous data formats for single omics . In fact, the software incorporates ideas and extends it in a modular fashion, similar to the existing practice in the Bioconductor community [MuData and MUON take inspiration and build on concepts from AnnData  and Scanommunity .MuDataAhttps://support.10xgenomics.com/single-cell-multiome-atac-gex/datasets). Log-normalisation was used for both gene and peak counts, and respective values for highly variable features scaled and centred to zero mean and unit variance were then used as input to discussed algorithms such as PCA, as implemented in scikit-learn [Single-cell multiome ATAC + gene expression demonstration data for peripheral blood mononuclear cells (PBMCs) from a healthy donor with granulocytes removed through cell sorting processed with ARC 1.0.0 pipeline were provided by 10X Genomics . Log-normalisation was used for gene counts, and dsb [https://muon-tutorials.readthedocs.io/en/latest/cite-seq.CITE-seq data for PBMCs from a healthy donor were provided by 10X Genomics ( and dsb  was usedAdditional file 1. Figure S1Additional file 2. Review history


Training pre-service EArly CHildhood educators in physical activity (TEACH) study is to develop and implement an e-Learning course in physical activity and sedentary behaviour to facilitate improvements in: pre-service ECEs’ self-efficacy and knowledge to lead physical activity and outdoor play opportunities and minimize sedentary behaviours in childcare. This study will also explore pre-service ECEs’ behavioural intention and perceived control to promote physical activity and outdoor play, and minimize sedentary behaviour in childcare, and the implementation of the e-Learning course. Methods/Design: A mixed-methods quasi-experimental design with three data collection time points  will be employed to test the e-Learning course in early childhood education programs  across Canada. Pre-service ECEs enrolled in colleges/universities assigned to the experimental group will be required to complete a 4-module e-Learning course, while programs in the comparison group will maintain their typical curriculum. Pre-service ECEs’ self-efficacy, knowledge, as well as behavioural intention and perceived behavioural control will be assessed via online surveys and module completion rates will be documented using website metrics. Group differences across timepoints will be assessed using linear mixed effects modelling and common themes will be identified through thematic analysis. Discussion: The TEACH study represents a novel, evidence-informed approach to address the existing gap in physical activity and sedentary behaviour-related education in Canadian post-secondary early childhood education programs. Moreover, e-Learning platforms, can be employed as an innovative, standardized, and scalable way to provide ECEs with consistent training across jurisdictions.Background: Early childhood educators (ECEs) influence young children’s early uptake of positive health behaviours in childcare settings and serve as important daytime role models. As such, it is imperative that post-secondary early childhood education programs provide students with the foundational knowledge and professional training required to confidently facilitate quality active play opportunities for young children. The primary objective of the  Physical activity is a critical behaviour which supports young children’s <5 years) physical, psychological, and academic development , while a years phIn Canada, two-thirds of young children attend childcare , equatinFor young children attending childcare, early childhood educators (ECEs) serve as their primary daytime role models; they are responsible for scheduling physical activity within their daily programming, while also facilitating active play. Therefore, these individuals have the potential to influence young children’s developmental experiences and day-to-day well-being . With ren = 1292) enrolled at 61 Canadian colleges/universities revealed that only ~30% reported receiving physical activity and screen-viewing-specific training in their program and a mere 15% were familiar with national movement guidelines for young children [Of particular concern, ECEs have communicated that they lack the self-efficacy needed to confidently facilitate physical activity for young children in childcare ,18 and hchildren . More pochildren . Accordichildren , self-efchildren . As suchRequired learning outcomes for early childhood education programs delineate that graduates must be able to “promote regular, healthy physical activity in all children” ; howeverPhysical activity interventions which include professional development/training show promise for supporting improved physical activity levels among young children. For example, Pate et al.’s intervention, which trained childcare teachers in structured and unstructured physical activity opportunities and integrating physical activity into lesson plans, resulted in improved physical activity levels among preschoolers . A diffeThe need to support pre-service ECEs’ self-efficacy and knowledge related to promoting physically active behaviours among young children in childcare is clear. Supplementary professional learning opportunities related to children’s physical activity, movement skill development, and appropriate sedentary behaviours, is likely to increase their confidence and ability to facilitate supportive physical activity environments, policies, programming, and practices in the childcare setting . The traThe primary objectives of the Training pre-service EArly CHildhood educators in physical activity (TEACH) study are to examine if the e-Learning course increases pre-service ECEs’: 1. self-efficacy to engage children in physical activity and outdoor play, and minimize sedentary behaviour in childcare; and, 2. physical activity and sedentary behaviour-related knowledge. The secondary objectives of the study are to explore: 1. pre-service ECEs’ behavioural intention and perceived behavioural control to promote physical activity and outdoor play and minimize sedentary behaviour in childcare; and, 2. implementation  and potential scalability (via the Consolidated Framework for Implementation Research (CFIR) checklist) of the e-Learning course. n = 9) or comparison (n = 9) condition. A 4-module e-Learning course (hosted on a secure learning management system platform) will be implemented by schools assigned to the experimental condition, while comparison schools will continue with their typical curriculum. Data will be collected at baseline, post-course completion, and 3-month follow-up. The study protocol and tools for the study have been approved by the Non-Medical Research Ethics Board  at Western University and ethics approval for the full-scale implementation will be sought prior to recruitment and data collection. Additional approval from participating college/university ethics boards will be completed, if requestedA mixed-methods quasi-experiential design will be employed to test the effectiveness of the TEACH e-Learning course at improving pre-service ECEs’ physical activity and sedentary behaviour-related self-efficacy and knowledge. Purposefully selected English-speaking colleges/universities offering an early childhood education program in Canada  will be randomly assigned to the experimental  can combine to produce logical mental constructs that facilitate knowledge acquisition . Each inn = 39) within this framework to ensure the research project is appropriately designed and scalable if proven effective. The CFIR comprises five domains: 1. intervention characteristics ; 2. outer setting ; 3. inner setting ; 4. individual characteristics ; and, 5. implementation process . Following these implementation constructs throughout the research project will act as a marker for intervention scalability.The CFIR is commonly used to promote the translation of research to practice; as such, the TEACH study will follow constructs  and provide acceleration of expertise through the use of video-based scenarios . Givn = 26) proposed their top content areas to include in the course [Our team has developed specific e-Learning course content by way of a Delphi study, wherein international physical activity and sedentary behaviour experts ; the software will generate a number of 1 or 0) to either receive the training  or not .To help ameliorate potential recruitment challenges and secure a diverse sample with representation from a number of Canadian provinces, we will draw on our established rapport with early childhood education post-secondary programs and will purposefully select colleges/universities (see sample size explanation below) and recruit their ECE programs to participate by emailing ECE program coordinators. Once 16 colleges agree to participate, allocation to the experimental and comparison conditions will occur. To safeguard the internal validity of the intervention, it is important to avoid randomly allocating individuals within each college to receive the training (or not), given the e-Learning course may be completed during pre-service ECEs’ class time and contamination may occur. As such, participating colleges/universities will act as the unit of randomization . Once consent has been obtained from the college/university representative , the clusters will be stratified by province, college/university size, and college/university ECE program delivery mode . Blocked randomization will be performed to allocate clusters (1:1) using a computer-generated randomization tool . Prior to completing the baseline survey, pre-service ECEs must read the letter of information and consent. Voluntarily beginning the baseline survey will signify their consent to participate in the study. Programs and instructors will be invited to integrate the e-Learning course itself into their course . Pre-service ECEs’ participation in the research study itself  will be voluntary . Instructors at colleges/universities will be emailed a recruitment letter to ask if they would like to review the e-Learning course and provide their feedback about its content, functionality, and feasibility for the post-secondary early childhood education setting via a process evaluation survey and optional interview.Canadian universities and colleges that offer an early childhood education program, where the pre-service ECEs are English-speaking, the program coordinator/chair agrees to the participation of their institution, and instructors are willing to participate, will be eligible to participate in this study.English-speaking individuals who are enrolled in any early childhood education program (regardless of year of study or program type) within a participating Canadian university or college will be eligible to participate.English-speaking instructors who are employed within an early childhood education program allocated to the experimental group will be eligible to participate.2 = 0.053 or R2 = 0.05), with 2 groups (intervention and control) and 3 time-points , 80% of the time, with an alpha of 0.025. As we will target college/university programs as units (clusters), the sample size will be adjusted to account for the clustering effect, where: D = design effect; k = anticipated cluster size (class size in this case); and ρ = the intra-cluster correlation coefficient, a measure of the degree of homogeneity among cluster subjects for a particular outcome investigated.We used the ‘pwr’ package in R ,39 to esWe estimate an average class size of 164 pre-service ECEs . Based At colleges/universities assigned to the experimental condition, participating pre-service ECEs will be required to complete the 4-module e-Learning course  within a 4-week timeframe.Colleges/universities assigned to the comparison group will continue their typical curriculum for the duration of the study. Upon completion of the study, students in the comparison group may opt to offer the e-Learning course to their pre-service ECEs. Pre-service ECEs in the comparison group will complete the same study instruments as those in the experimental group .Various tools will be used to assess the impact of the TEACH study on pre-service ECEs’ physical-activity and sedentary behaviour-related self-efficacy and knowledge. See ECE-COMPASS) [Our research team systematically reviewed physical activity and sedentary behaviour-related self-efficacy tools for ECEs; based on the findings from this review, no such tool emerged from the literature to meet the specific needs of this project . ConsequCOMPASS) . This quCOMPASS) , assesseCOMPASS) . This suAs no validated tool exists to examine pre-service ECEs’ physical activity or sedentary behaviour-specific knowledge, our team developed a tool to assess this outcome in our study participants by creating questions based upon e-Learning course content . This 22Participant demographics, including age, gender, ethnicity, college/university, year of study, and program type and delivery method, will be captured via an online survey at baseline. Participants’ self-reported physical activity and recreational screen-viewing levels, previous professional learning courses/webinars in physical activity or sedentary behaviour , interest in physical activity and sedentary behaviour education , and previous experience with e-Learning (yes or no) will also be captured at both baseline and post-intervention.E-Learning course completion rates (percent), usage data (completion time per module), and pre-service ECE attrition will be explored within the secure module platform to examine program fidelity. Student answers for module knowledge assessments  will also be examined to measure short-term retention of material. Colleges/universities will be asked to share whether they required students to complete the modules as a course deliverable (produce a certificate of completion). The ECEs’ Movement Behavioural Intention and Perceived Control (ECE-MBIPC) questionnaire will be completed by pre-service ECEs at baseline, post-intervention, and 3-month follow-up to gather their perspectives regarding their attitudes toward promoting physical activity and minimizing sedentary behaviour in childcare. This 56-item tool, adapted from Gagné and Harnois , is baseAn online process evaluation survey (38 items) will be completed by participants in the experimental group to gather their perspectives on the acceptability , compatibility , usability of the e-Learning platform, complexity of the content, and perspectives regarding suggestions for improvement. The survey will be informed by the Evaluating e-Learning System Success (EESS) model  to captuAn online survey (23 items) will be completed by ECE instructors at each institution in the experimental group to gather their current teaching of physical activity and sedentary behaviour content (including which specific concepts are covered), their perspectives on the course design, content, and implementation, and the feasibility of integrating the e-Learning course into their curricula.n = ~24) and instructors (n = ~12) will be interviewed to gain in-depth feedback on the e-Learning course’s implementation, content, and delivery. All interviews will last 30–45 min and will be digitally recorded and transcribed verbatim. A semi-structured interview guide, with probes, will be used to direct the conversation , a checklist, created from the 39 constructs of the CFIR, will be completed by research staff . The resTo ensure the e-Learning course is functional, easy to use, and has a reasonable time commitment, we will pilot test the course, first, with ~150 in-service ECEs , and second, with pre-service ECEs attending college/university in two provinces and one territory (~50 students). While the course is designed for the pre-service ECE population, pilot testing in a sample of in-service ECEs will be undertaken to ensure the course provides them with relevant and useful education to support ECEs’ programming of active opportunities in a variety of childcare settings . Participants will complete the e-Learning course and will be asked to complete an online survey post-intervention to gather specific feedback on the course’s functionality and pre- and in-service ECEs’ satisfaction with the course content and design. Semi-structured interviews with pre-service (~8) and in-service ECEs (~8) will be conducted post-intervention to gather more in-depth information on their experiences with the course. Additionally, to explore preliminary efficacy of the intervention, a secondary objective of the pilot study will be to measure changes in participants’ physical activity and sedentary behaviour-related: 1. self-efficacy; 2. knowledge; and 3. behavioural intention and perceived behavioural control (via online survey at pre- and post-intervention). After identifying strengths and weaknesses of the e-Learning course, appropriate changes will be made prior to full-scale implementation. Pilot testing is currently underway, with full scale implementation targeted to begin in September 2022.t-tests or chi-square tests, as appropriate. If applicable, group differences on baseline measures will be statistically controlled in subsequent analyses  of physical activity and screen-viewing training provided by the college/university). Baseline characteristics of the pre-service ECEs and their physical activity programming offerings (based on the curriculum review) will be summarized descriptively. Group differences at baseline will be assessed by independent sample Pre-service ECEs’ physical activity and sedentary behaviour-related self-efficacy and knowledge will be evaluated using two  linear mixed effects models wherein group and time are our primary fixed effects. Demographic variables will be used as predictors of pre-service ECEs’ self-efficacy, knowledge, and behavioural intention and perceived behavioural control in our analyses. Specifically, we will explore the extent to which province/territory predicts the dependent variables, and whether there is a significant effect of previous training, early childhood education program type, and mode of delivery. Based on previous research, the pre-service ECE population is largely female , so gendBehavioural intention and perceived behavioural control will be evaluated using a linear mixed effects model, with group and time as fixed effects. As was the case in our primary outcome, we will evaluate the extent to which early childhood education program type, and the province or territory in which the program is situated, impact on our prediction models—and will also include student age, physical activity, and sedentary time as model covariates, if appliable. For the process evaluation, website metrics, and CFIR checklist, data will be explored using descriptive statistics. Thematic analysis  will be A large body of research supports that the childcare environment, inclusive of ECEs’ programming, practices, and facilitation behaviours, can substantially influence children’s activity levels in this setting ,48,49,50In the interest of creating an evidence-informed physical activity intervention, the TEACH study was developed on the basis of Bandura’s Social Cognitive Theory  and Mayen = 17) showed that they reported higher knowledge and self-efficacy to lead physical activity opportunities following the intervention, while also communicating that they would use the knowledge they gained in future programming [Other interventions have been developed to better support ECEs in promoting active behaviours among young children ,29; howegramming . As suchUtilizing e-Learning platforms to complement pre-service ECEs’ post-secondary education is a forward-thinking approach to ensure future ECEs receive necessary health promotion education. If successful, ECE graduates will enter their profession with greater self-efficacy to engage young children in physical activity and reduce sedentary behaviours during childcare hours, which is likely to increase the quantity and quality of programming offered to children in their care. Moreover, offering pre-service ECEs this training online improves reach. If effective, we will translate the e-Learning course into French to easily be employed by colleges/universities across Canada, and will support its adoption in countries where movement guidelines for the early years are similar to Canada’s . Thi