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One of the key tools to understand and reduce the spread of the SARS-CoV-2 virus is testing. The total number of tests, the number of positive tests, the number of negative tests, and the positivity rate are interconnected indicators and vary with time. To better understand the relationship between these indicators, against the background of an evolving pandemic, the association between the number of positive tests and the number of negative tests is studied using a joint modeling approach. All countries in the European Union, Switzerland, the United Kingdom, and Norway are included in the analysis. We propose a joint penalized spline model in which the penalized spline is reparameterized as a linear mixed model. The model allows for flexible trajectories by smoothing the country-specific deviations from the overall penalized spline and accounts for heteroscedasticity by allowing the autocorrelation parameters and residual variances to vary among countries. The association between the number of positive tests and the number of negative tests is derived from the joint distribution for the random intercepts and slopes. The correlation between the random intercepts and the correlation between the random slopes were both positive. This suggests that, when countries increase their testing capacity, both the number of positive tests and negative tests will increase. A significant correlation was found between the random intercepts, but the correlation between the random slopes was not significant due to a wide credible interval., Competing Interests: The authors declare that no competing interests exist., (Copyright: © 2024 De Witte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In September 2022, deaths of pigs manifesting pox-like lesions caused by swinepox virus were reported in Tshuapa Province, Democratic Republic of the Congo. Two human mpox cases were found concurrently in the surrounding community. Specific diagnostics and robust sequencing are needed to characterize multiple poxviruses and prevent potential poxvirus transmission.

Purpose: To develop and validate a deep learning model for distinguishing healthy vocal folds (HVF) and vocal fold polyps (VFP) on laryngoscopy videos, while demonstrating the ability of a previously developed informative frame classifier in facilitating deep learning development., Methods: Following retrospective extraction of image frames from 52 HVF and 77 unilateral VFP videos, two researchers manually labeled each frame as informative or uninformative. A previously developed informative frame classifier was used to extract informative frames from the same video set. Both sets of videos were independently divided into training (60%), validation (20%), and test (20%) by patient. Machine-labeled frames were independently verified by two researchers to assess the precision of the informative frame classifier. Two models, pre-trained on ResNet18, were trained to classify frames as containing HVF or VFP. The accuracy of the polyp classifier trained on machine-labeled frames was compared to that of the classifier trained on human-labeled frames. The performance was measured by accuracy and area under the receiver operating characteristic curve (AUROC)., Results: When evaluated on a hold-out test set, the polyp classifier trained on machine-labeled frames achieved an accuracy of 85% and AUROC of 0.84, whereas the classifier trained on human-labeled frames achieved an accuracy of 69% and AUROC of 0.66., Conclusion: An accurate deep learning classifier for vocal fold polyp identification was developed and validated with the assistance of a peer-reviewed informative frame classifier for dataset assembly. The classifier trained on machine-labeled frames demonstrates improved performance compared to the classifier trained on human-labeled frames., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Rigorous data management systems and planning are essential to successful research projects, especially for large, multicountry consortium studies involving partnerships across multiple institutions. Here we describe the development and implementation of data management systems and procedures for the Enterics For Global Health (EFGH) Shigella surveillance study-a 7-country diarrhea surveillance study that will conduct facility-based surveillance concurrent with population-based enumeration and a health care utilization survey to estimate the incidence of Shigella- -associated diarrhea in children 6 to 35 months old., Methods: The goals of EFGH data management are to utilize the knowledge and experience of consortium members to collect high-quality data and ensure equity in access and decision-making. During the planning phase before study initiation, a working group of representatives from each EFGH country site, the coordination team, and other partners met regularly to develop the data management systems for the study., Results: This resulted in the Data Management Plan, which included selecting REDCap and SurveyCTO as the primary database systems. Consequently, we laid out procedures for data processing and storage, study monitoring and reporting, data quality control and assurance activities, and data access. The data management system and associated real-time visualizations allow for rapid data cleaning activities and progress monitoring and will enable quicker time to analysis., Conclusions: Experiences from this study will contribute toward enriching the sparse landscape of data management methods publications and serve as a case study for future studies seeking to collect and manage data consistently and rigorously while maintaining equitable access to and control of data., Competing Interests: Potential conflicts of interest. All authors: no reported conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Malawi is among 7 countries participating in the Enterics for Global Health (EFGH) Shigella surveillance study, which aims to determine the incidence of medically attended diarrhea attributed to Shigella , a leading bacterial cause of diarrhea in children in low-resource settings., Methods: We describe the EFGH study site in the densely populated informal settlement of Ndirande Township, Blantyre, Malawi. We explore the site's geographical location, demographic characteristics, and the healthcare-seeking behavior of its population, particularly for childhood diarrhea. We also describe the management of childhood diarrhea at the health facility, and the associated challenges to attaining optimum adherence to local and national guidelines at the site., Conclusions: Our overarching aim is to improve global health through understanding and mitigating the impact of diarrhea attributed to Shigella ., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Comparative costs of public health interventions provide valuable data for decision making. However, the availability of comprehensive and context-specific costs is often limited. The Enterics for Global Health (EFGH) Shigella surveillance study-a facility-based diarrhea surveillance study across 7 countries-aims to generate evidence on health system and household costs associated with medically attended Shigella diarrhea in children., Methods: EFGH working groups comprising representatives from each country (Bangladesh, Kenya, Malawi, Mali, Pakistan, Peru, and The Gambia) developed the study methods. Over a 24-month surveillance period, facility-based surveys will collect data on resource use for the medical treatment of an estimated 9800 children aged 6-35 months with diarrhea. Through these surveys, we will describe and quantify medical resources used in the treatment of diarrhea (eg, medication, supplies, and provider salaries), nonmedical resources (eg, travel costs to the facility), and the amount of caregiver time lost from work to care for their sick child. To assign costs to each identified resource, we will use a combination of caregiver interviews, national medical price lists, and databases from the World Health Organization and the International Labor Organization. Our primary outcome will be the estimated cost per inpatient and outpatient episode of medically attended Shigella diarrhea treatment across countries, levels of care, and illness severity. We will conduct sensitivity and scenario analysis to determine how unit costs vary across scenarios., Conclusions: Results from this study will contribute to the existing body of literature on diarrhea costing and inform future policy decisions related to investments in preventive strategies for Shigella ., Competing Interests: Potential conflicts of interest. All authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Shigella is a leading cause of acute watery diarrhea, dysentery, and diarrhea-attributed linear growth faltering, a precursor to stunting and lifelong morbidity. Several promising Shigella vaccines are in development and field efficacy trials will require a consortium of potential vaccine trial sites with up-to-date Shigella diarrhea incidence data., Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will employ facility-based enrollment of diarrhea cases aged 6-35 months with 3 months of follow-up to establish incidence rates and document clinical, anthropometric, and financial consequences of Shigella diarrhea at 7 country sites (Mali, Kenya, The Gambia, Malawi, Bangladesh, Pakistan, and Peru). Over a 24-month period between 2022 and 2024, the EFGH study aims to enroll 9800 children (1400 per country site) between 6 and 35 months of age who present to local health facilities with diarrhea. Shigella species (spp.) will be identified and serotyped from rectal swabs by conventional microbiologic methods and quantitative polymerase chain reaction. Shigella spp. isolates will undergo serotyping and antimicrobial susceptibility testing. Incorporating population and healthcare utilization estimates from contemporaneous household sampling in the catchment areas of enrollment facilities, we will estimate Shigella diarrhea incidence rates., Conclusions: This multicountry surveillance network will provide key incidence data needed to design Shigella vaccine trials and strengthen readiness for potential trial implementation. Data collected in EFGH will inform policy makers about the relative importance of this vaccine-preventable disease, accelerating the time to vaccine availability and uptake among children in high-burden settings., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Breast cancer (BC) death rates in the USA have not significantly declined for American Indians (AIs) in comparison to Whites. Our objective was to determine whether Medicaid Expansion as part of the Affordable Care Act led to improved BC outcomes for AIs relative to Whites., Patients and Methods: Using the National Cancer Database, we conducted a retrospective cohort study. Included were BC patients who were AI and White; 40 to 64 years of age; diagnosed in 2009 to 2016; lived in states that expanded Medicaid in January 2014, and states that did not expand Medicaid. Our outcomes were stage at diagnosis, insurance status, timely treatment, and 3-year mortality., Results: There were 359,484 newly diagnosed BC patients, 99.49% White, 0.51% AI. Uninsured rates declined more in the expansion states than in the nonexpansion states (OR = 0.44, 95% CI: 0.15-0.97, P < 0.001). Lower rates of Stage I BC diagnosis was found in AIs compared to Whites (46.58% vs. 55.33%, P < .001); these differential rates did not change after Medicaid expansion. Rates of definitive treatment initiation within 30 days of diagnosis declined after Medicaid expansion (P < .001); there was a smaller decline in the expansion states (OR 1.118, 95% CI: 1.09, 1.15, P < .001). Three year mortality was not different between expansion and nonexpansion states post Medicaid expansion., Conclusions: In newly diagnosed BCs, uninsured rates declined more in the states that expanded Medicaid in January 2014. Timely treatment post Medicaid expansion declined less in states that expanded Medicaid. There was no differential benefit of Medicaid expansion in the 2 races., Competing Interests: Disclosure Ross D. Crosby is a paid statistical consultant for Health Outcomes Solutions, Winter Park, Florida. The remaining authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The fibroblast activation protein (FAP) is highly expressed on carcinoma-associated fibroblasts in the stroma of pancreatic cancer and thus is a promising target for imaging and therapy. Preliminary data on PET imaging with radiolabeled FAP inhibitors (FAPIs) demonstrate superior tumor detection. Here we assess the accuracy of FAP-directed PET in patients with pancreatic cancer. Methods: Of 64 patients with suspected or proven pancreatic cancer, 62 (97%) were included in the data analysis of the 68 Ga-FAPI PET observational trial (NCT04571086). All of these patients underwent contrast-enhanced CT, and 38 patients additionally underwent 18 F-FDG PET. The primary study endpoint was the association of 68 Ga-FAPI PET uptake intensity and histopathologic FAP expression. Secondary endpoints were detection rate, diagnostic performance, interreader reproducibility, and change in management. Datasets were interpreted by 2 masked readers. Results: The primary endpoint was met: The association between 68 Ga-FAPI SUV max and histopathologic FAP expression was significant (Spearman r , 0.48; P = 0.04). For histopathology-validated lesions, 68 Ga-FAPI PET showed high sensitivity and positive predictive values (PPVs) on per-patient (sensitivity, 100%; PPV, 96.3%) and per-region (sensitivity, 100%; PPV, 97.0%) bases. In a head-to-head comparison versus 18 F-FDG or contrast-enhanced CT, 68 Ga-FAPI detected more tumor on a per-lesion (84.7% vs. 46.5% vs. 52.9%), per-patient (97.4% vs. 73.7% vs. 92.1%), or per-region (32.6% vs. 18.8% vs. 23.7%) basis, respectively. 68 Ga-FAPI PET readers showed substantial overall agreement on the basis of the Fleiss κ: primary κ, 0.77 (range, 0.66-0.88). Minor and major changes in clinical management occurred in 5 patients (8.4%) after 68 Ga-FAPI PET. Conclusion: We confirmed an association of 68 Ga-FAPI PET SUV max and histopathologic FAP expression in pancreatic cancer patients. Additionally, we found high detection rate and diagnostic accuracy, superior to those of 18 F-FDG PET/CT. 68 Ga-FAPI might become a powerful diagnostic tool for pancreatic cancer work-up., (© 2023 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: Policymakers have struggled to maintain SARS-CoV-2 transmission at levels that are manageable to contain the COVID-19 disease burden while enabling a maximum of societal and economic activities. One of the tools that have been used to facilitate this is the so-called "COVID-19 pass". We aimed to document current evidence on the effectiveness of COVID-19 passes, distinguishing their indirect effects by improving vaccination intention and uptake from their direct effects on COVID-19 transmission measured by the incidence of cases, hospitalizations, and deaths., Methods: We performed a scoping review on the scientific literature of the proposed topic covering the period January 2021 to September 2022, in accordance with the PRISMA-ScR guidelines for scoping reviews., Results: Out of a yield of 4,693 publications, 45 studies from multiple countries were retained for full-text review. The results suggest that implementing COVID-19 passes tends to reduce the incidence of cases, hospitalizations, and deaths due to COVID-19. The use of COVID-19 passes was also shown to improve overall vaccination uptake and intention, but not in people who hold strong anti-COVID-19 vaccine beliefs., Conclusion: The evidence from the literature we reviewed tends to indicate positive direct and indirect effects from the use of COVID-19 passes. A major limitation to establishing this firmly is the entanglement of individual effects of multiple measures being implemented simultaneously., (© 2023. The Author(s).)

To monitor the COVID-19 epidemic in Cuba, data on several epidemiological indicators have been collected on a daily basis for each municipality. Studying the spatio-temporal dynamics in these indicators, and how they behave similarly, can help us better understand how COVID-19 spread across Cuba. Therefore, spatio-temporal models can be used to analyze these indicators. Univariate spatio-temporal models have been thoroughly studied, but when interest lies in studying the association between multiple outcomes, a joint model that allows for association between the spatial and temporal patterns is necessary. The purpose of our study was to develop a multivariate spatio-temporal model to study the association between the weekly number of COVID-19 deaths and the weekly number of imported COVID-19 cases in Cuba during 2021. To allow for correlation between the spatial patterns, a multivariate conditional autoregressive prior (MCAR) was used. Correlation between the temporal patterns was taken into account by using two approaches; either a multivariate random walk prior was used or a multivariate conditional autoregressive prior (MCAR) was used. All models were fitted within a Bayesian framework., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The SARS-CoV-2 Omicron variant has resulted in a high number of cases, but a relatively low incidence of severe disease and deaths, compared to the pre-Omicron variants. Therefore, we assessed the differences in symptom prevalence between Omicron and pre-Omicron infections in a sub-Saharan African population. We collected data from outpatients presenting at two primary healthcare facilities in Blantyre, Malawi, from November 2020 to March 2022. Eligible participants were aged >1month old, with signs suggestive of COVID-19, and those not suspected of COVID-19, from whom we collected nasopharyngeal swabs for SARS-CoV-2 PCR testing, and sequenced positive samples to identify infecting-variants. In addition, we calculated the risk of presenting with a given symptom in individuals testing SARS-CoV-2 PCR positive before and during the Omicron variant-dominated period. Among 5176 participants, 6.4% were under 5, and 77% were aged 18 to 50 years. SARS-CoV-2 infection prevalence peaked in January 2021 (Beta), July 2021 (Delta), and December 2021 (Omicron). We found that cough (risk ratio (RR), 1.50; 95% confidence interval (CI), 1.00 to 2.30), fatigue (RR 2.27; 95% CI, 1.29 to 3.86) and headache (RR 1.64; 95% CI, 1.15 to 2.34) were associated with a high risk of SARS-CoV-2 infection during the pre-Omicron period. In comparison, only headache (RR 1.41; 95% CI, 1.07 to 1.86) did associate with a high risk of SARS-CoV-2 infection during the Omicron-dominated period. In conclusion, clinical symptoms associated with Omicron infection differed from prior variants and were harder to identify clinically with current symptom guidelines. Our findings encourage regular review of case definitions and testing policies to ensure case ascertainment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chibwana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Vascular dysregulation seems to play a role in the pathogenesis of glaucoma, in particular normal tension glaucoma (NTG). The development of optical coherence tomography angiography (OCTA) enabled the measurement of the retinal microvasculature non-invasively and with high repeatability. Nonetheless, only a few studies transformed OCTA into a dynamic examination employing a sympathomimetic stimulus. The goal of this study was to use this dynamic OCTA exam (1) to differentiate healthy individuals from glaucoma patients and (2) to distinguish glaucoma subcategories, NTG and high-tension primary open angle glaucoma (POAG)., Methods: Retinal vessel density (VD) in NTG patients ( n = 16), POAG patients ( n = 12), and healthy controls ( n = 14) was compared before and during a hand grip test with a hydraulic dynamometer., Results: At baseline, mean peripapillary VD was lower in POAG and NTG (42.6 and 48.5%) compared to healthy controls (58.1%; p < 0.001) and higher in NTG compared to POAG ( p = 0.024) when corrected for mean arterial pressure (MAP). Peripapillary and macular (superficial and deep) VD differences were found for gender, age, and baseline MAP. No change in VD occurred (pre-/post-stimulus) in any of the groups., Conclusion: Retinal VD loss in glaucoma patients was confirmed and the necessity to correct for gender, age and especially MAP was established. Although replication in a larger population is necessary, OCTA might not be the most suitable method to dynamically evaluate the retinal microvasculature., Competing Interests: 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., (Copyright © 2023 Van Eijgen, Heintz, van der Pluijm, Delporte, De Witte, Molenberghs, Barbosa-Breda and Stalmans.)

Rationale: Research on health-related self-uniqueness beliefs suggested that these beliefs might predict adherence to precautions against COVID-19., Objective: We examined if comparative optimism (believing that one is less at less than others), self-superiority (believing that one already adheres better to precautions than others), and egocentric impact perception (believing that adverse events affect oneself more than others) predicted intended adherence to precautions., Method: We measured self-reported intentions, optimism for self and others, perceived past adherence by self and others, and perceived impact of the measures and the disease on self and others in a 5-wave longitudinal study in December 2020-May 2021 (N ≈ 5000/wave). The sample was in key respects representative for the Belgian population. We used joint models to examine the relationship between self-uniqueness beliefs and intended adherence to the precautions., Results: Believing that COVID-19 would affect one's own life more than average (egocentric impact perception) was associated with higher intentions to adhere to precautions, as was believing that the precautions affected one's life less than average (allocentric impact perception). Self-superiority concerning past adherence to precautions and comparative optimism concerning infection with COVID-19 were associated with higher intended adherence, regardless of whether their non-comparative counterparts (descriptive norm, i.e., perceived adherence to precautions by others, and personal optimism, respectively) were controlled for. Comparative optimism for severe disease and for good outcome were associated with lower intended adherence if personal optimism was not controlled for, but with higher intended adherence if it was controlled for., Conclusion: Self-uniqueness beliefs predict intended adherence to precautions against COVID-19, but do so in different directions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Rotavirus vaccines reduce rotavirus-related deaths and hospitalisations but are less effective in high child mortality countries. The human RV3-BB neonatal G3P[6] rotavirus vaccine administered in a neonatal schedule was efficacious in reducing severe rotavirus gastroenteritis in Indonesia but had not yet been evaluated in African infants., Methods: We did a phase 2, randomised, double-blind, parallel group dose-ranging study of three doses of oral RV3-BB rotavirus vaccine in infants in three primary health centres in Blantyre, Malawi. Healthy infants less than 6 days of age with a birthweight 2·5 to 4·0 kg were randomly assigned (1:1:1:1) into one of four treatment groups: neonatal vaccine group, which included high-titre (1·0 × 10 7 focus-forming unit [FFU] per mL), mid-titre (3·0 × 10 6 FFU per mL), or low-titre (1·0 × 10 6 FFU per mL); and infant vaccine group, which included high-titre (1·0 × 10 7 FFU per mL) using a computer generated code (block size of four), stratified by birth (singleton vs multiple). Neonates received their three doses at 0-5 days to 10 weeks and infants at 6-14 weeks. Investigators, participant families, and laboratory staff were masked to group allocation. Anti-rotavirus IgA seroconversion and vaccine take (IgA seroconversion and stool shedding) were evaluated. Safety was assessed in all participants who received at least one dose of vaccine or placebo. The primary outcome was the cumulative IgA seroconversion 4 weeks after three doses of RV3-BB in the neonatal schedule in the high-titre, mid-titre, and low-titre groups in the per protocol population, with its 95% CI. With the high-titre group as the active control group, we did a non-inferiority analysis of the proportion of participants with IgA seroconversion in the mid-titre and low-titre groups, using a non-inferiority margin of less than 20%. This trial is registered at ClinicalTrials.gov (NCT03483116)., Findings: Between Sept 17, 2018, and Jan 27, 2020, 711 participants recruited were randomly assigned into four treatment groups (neonatal schedule high titre n=178, mid titre n=179, low titre n=175, or infant schedule high titre n=179). In the neonatal schedule, cumulative IgA seroconversion 4 weeks after three doses of RV3-BB was observed in 79 (57%) of 139 participants in the high-titre group, 80 (57%) of 141 participants in the mid-titre group, and 57 (41%) of 138 participants in the low-titre group and at 18 weeks in 100 (72%) of 139 participants in the high-titre group, 96 (67%) of 143 participants in the mid-titre group, and 86 (62%) of 138 of participants in the low-titre. No difference in cumulative IgA seroconversion 4 weeks after three doses of RV3-BB was observed between high-titre and mid-titre groups in the neonatal schedule (difference in response rate 0·001 [95%CI -0·115 to 0·117]), fulfilling the criteria for non-inferiority. In the infant schedule group 82 (59%) of 139 participants had a cumulative IgA seroconversion 4 weeks after three doses of RV3-BB at 18 weeks. Cumulative vaccine take was detected in 483 (85%) of 565 participants at 18 weeks. Three doses of RV3-BB were well tolerated with no difference in adverse events among treatment groups: 67 (39%) of 170 participants had at least one adverse event in the high titre group, 68 (40%) of 172 participants had at least one adverse event in the mid titre group, and 69 (41%) of 169 participants had at least one adverse event in the low titre group., Interpretation: RV3-BB was well tolerated and immunogenic when co-administered with Expanded Programme on Immunisation vaccines in a neonatal or infant schedule. A lower titre (mid-titre) vaccine generated similar IgA seroconversion to the high-titre vaccine presenting an opportunity to enhance manufacturing capacity and reduce costs. Neonatal administration of the RV3-BB vaccine has the potential to improve protection against rotavirus disease in children in a high-child mortality country in Africa., Funding: Bill & Melinda Gates Foundation, Australian Tropical Medicine Commercialisation Grant., Competing Interests: Declaration of interests JEB is the lead of the Enteric Diseases group at MCRI (MCRI holds the license for RV3-BB vaccine). JEB is the Director of the Australian Rotavirus Surveillance Program funded by the Australian Government Department of Health and GlaxoSmithKline. MCRI and GLB hold the patent for the RV3-BB vaccine. NAC is affiliated to the National for Health Research Health Protection Research Unit in Gastrointestinal Infections at University of Liverpool, in partnership with Public Health England, in collaboration with University of Warwick. NAC is based at The University of Liverpool. The views expressed are those of the authors and not necessarily those of the National Institute of Health Research, the Department of Health and Social Care, or Public Health England. CMD has served on advisory boards for GSK (between 2019 and 2021) with all payments directed to an administrative fund held by MCRI., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Atypical hemolytic uremic syndrome (aHUS) is an ultra-rare orphan disease caused by dysregulated complement activation resulting in thrombotic microangiopathy. Although complement-mediated endothelial injury predominantly affects the renal microvasculature, extra-renal manifestations are present in a significant proportion of patients. While eculizumab has significantly improved the morbidity and mortality of this rare disease, optimizing therapeutic regimens of this highly expensive drug remains an active area of research in the treatment of aHUS., Case Presentation: This report describes the case of a previously healthy 4 year-old male who presented with rhabdomyolysis preceding the development of aHUS with anuric kidney injury requiring dialysis. Clinical stabilization required increased and more frequent eculizumab doses compared with the standardized weight-based guidelines. In the maintenance phase of his disease, pharmacokinetic analysis indicated adequate eculizumab levels could be maintained with an individualized dosing regimen every 3 weeks, as opposed to standard 2 week dosing, confirmed in this patient over a 4 year follow up period. Cost analyses show that weight-based maintenance dosing costs $312,000 per year, while extending the dosing interval to every 3 weeks would cost $208,000, a savings of $104,000 per year, relative to the cost of $72,000 from more frequent eculizumab dosing during his initial hospitalization to suppress his acute disease., Conclusion: This case exemplifies the potential of severe, multisystem involvement of aHUS presenting with extra-renal manifestations, including rhabdomyolysis as in this case, and highlights the possibility for improved clinical outcomes and higher value care with individualized eculizumab dosing in patients over the course of their disease., Competing Interests: BD has participated in consulting agreements with Apellis Pharmaceuticals and Alexion Pharmaceuticals. 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., (Copyright © 2022 Benoit, Fukuda, VandenHeuvel, Witte, Fuller, Willis, Dixon and Drake.)

Objective: This study aims to develop and validate a convolutional neural network (CNN)-based algorithm for automatic selection of informative frames in flexible laryngoscopic videos. The classifier has the potential to aid in the development of computer-aided diagnosis systems and reduce data processing time for clinician-computer scientist teams., Methods: A dataset of 22,132 laryngoscopic frames was extracted from 137 flexible laryngostroboscopic videos from 115 patients. 55 videos were from healthy patients with no laryngeal pathology and 82 videos were from patients with vocal fold polyps. The extracted frames were manually labeled as informative or uninformative by two independent reviewers based on vocal fold visibility, lighting, focus, and camera distance, resulting in 18,114 informative frames and 4018 uninformative frames. The dataset was split into training and test sets. A pre-trained ResNet-18 model was trained using transfer learning to classify frames as informative or uninformative. Hyperparameters were set using cross-validation. The primary outcome was precision for the informative class and secondary outcomes were precision, recall, and F1-score for all classes. The processing rate for frames between the model and a human annotator were compared., Results: The automated classifier achieved an informative frame precision, recall, and F1-score of 94.4%, 90.2%, and 92.3%, respectively, when evaluated on a hold-out test set of 4438 frames. The model processed frames 16 times faster than a human annotator., Conclusion: The CNN-based classifier demonstrates high precision for classifying informative frames in flexible laryngostroboscopic videos. This model has the potential to aid researchers with dataset creation for computer-aided diagnosis systems by automatically extracting relevant frames from laryngoscopic videos., Competing Interests: Anaïs Rameau is medical advisor for Perceptron Health, Inc. Dan Witte is co‐founder of Perceptron Health, Inc., (© 2022 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals LLC on behalf of The Triological Society.)

Aim: To analyze the association of objective and subjective sleep measures with HbA1c and insulin sensitivity in the general population., Methods: Using a cross-sectional design, data from 1028 participants in the ORISCAV-LUX-2 study from the general population in Luxembourg were analyzed. Objective sleep measures were assessed using accelerometers whereas subjective measures were assessed using the Pittsburgh Sleep Quality Index (PSQI) questionnaire. Sleep measures were defined as predictors, while HbA1c and quantitative insulin sensitivity check index (QUICKI) scores were considered outcomes. Linear and spline regression models were fitted by progressively adjusting for demographic and lifestyle variables in the total sample population as well as by stratified analyses using gender, obesity status, depressive symptoms and diabetes status., Results: In fully adjusted models, total and deep sleep durations were associated with lower HbA1c (mmol/mol) levels, whereas sleep coefficients of variation (%) and poor sleep efficiency, as measured by PSQI scores (units), were associated with higher HbA1c levels. In stratified models, such associations were observed mainly in men, and in subjects who had depressive symptoms, were overweight and no diabetes. In addition, total sleep, deep sleep, coefficients of variation and poor sleep efficiency as measured by PSQI revealed non-linear associations. Similarly, greater insulin sensitivity was associated with longer total sleep time and with PSQI-6 (use of sleep medication)., Conclusion: Associations were more frequently observed between sleep characteristics and glycaemic control with the use of objective sleep measures. Also, such associations varied within subgroups of the population. Our results highlight the relevance of measuring sleep patterns as key factors in the prevention of diabetes., (Copyright © 2021 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Introduction: 5q-associated spinal muscular atrophy is one of the most severe and common genetic diseases. In the last few years, innovative methods of therapy have been developed based on SMN2 gene modification, such as splicing, or replacement of the damaged SMN1 gene (gene replacement therapy, GRT). GRT is known to be accompanied by off target effects like temporary elevation of liver and cardiac enzymes usually without serious clinical relevance. We report a 4-year-old girl suffering from thrombotic microangiopathy (TMA) after GRT due to 5q- SMA., Case Summary: A 4-year-old girl developed TMA indicated by haemolytic anemia and thrombocytopenia in conjunction with renal failure 7 days after GRT with onasemnogene abeparvovec. The latter was characterized by a rise in serum creatinine, oliguria, hypertension, protein- and haematuria, and oedema. The patient was started on eculizumab and antihypertensives resulting in normalization of haemolytic activity, platelet count, kidney function and blood pressure within one week., Recommendation and Conclusion: SMA patients receiving GRT should undergo close monitoring for early detection of TMA. Adequate measures for TMA including eculizumab or plasmapheresis as well as renal replacement therapy should be available without delay in order to avoid progressive kidney disease or other severe complications in these patients. Careful follow-up including assessment of proteinuria and blood pressure is recommended since patients may require antihypertensive/nephroprotective treatment to avoid chronic kidney disease in later life. Therefore, GRT in SMA patients should only be performed at centers with neuropediatric and paediatric nephrology expertise., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Background: Pregnancy may be associated with adverse outcome in women with congenital heart disease (CHD). However, data regarding the outcome of pregnancy in women with CHD who receive care in cardiac-obstetric expert units are limited., Methods: We retrospectively analyzed baseline characteristics and outcome of pregnancy in 67 females with CHD who received medical care in our tertiary center for 61 singleton and 6 twin pregnancies between 2009 and 2018., Results: According to the modified World Health Organization (mWHO) risk scale for pregnancy, CHD lesions in 39 enrolled women (58%) were classified as mWHO class I or II, and in 28 females (42%) as mWHO class III or IV. Preterm births were more frequent in mWHO classes III or IV (P=0.003). Cardiac signs and complications occurred more often in mWHO classes III or IV than in women with cardiac lesions assigned to mWHO classes I or II (42.9% vs . 7.7%, P=0.002). N-terminal pro B-type natriuretic peptide (NT-proBNP) levels during pregnancy were higher in mWHO classes III or IV than in mWHO classes I or II (median 269.0 vs . 115.5 pg/mL, P=0.019). Presence of functional NYHA class III [odds ratio (OR) per standard deviation (SD) 8.8, 95% confidence interval (CI): 2.2-57.2, P=0.008] and mWHO classes III/IV (OR per SD 3.4, 95% CI: 1.2-9.9, P=0.018) prior to pregnancy were identified as independent predictors of adverse cardiac outcome of pregnancy., Conclusions: Adverse cardiac events and preterm deliveries should be anticipated in pregnant women with CHD, especially in those with mWHO classes III or IV. Therefore, these pregnancies should be under close surveillance and managed in specialized, multidisciplinary tertiary referral centers. Preconception counseling including individualized risk assessment is strongly recommended in women with CHD., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/cdt-20-650). The series “Current Management Aspects in Adult Congenital Heart Disease (ACHD): Part IV” was commissioned by the editorial office without any funding or sponsorship. YvK serves as an unpaid editorial board member of Cardiovascular Diagnosis and Therapy from February 2018 to January 2022 and served as the unpaid Guest Editor of the series. Dr. SB reports grants and personal fees from Abbott Diagnostics, grants and personal fees from Bayer, grants from SIEMENS, grants from Singulex, grants and personal fees from Thermo Fisher, personal fees from Abott, personal fees from Astra Zeneca, personal fees from AMGEN, personal fees from Medtronic, personal fees from Pfizer, personal fees from Roche, personal fees from Novartis, personal fees from Siemens Diagnostics, outside the submitted work; Dr. PK reports non-financial support from the European Union, British Heart Foundation, Leducq Foundation, Medical Research Council (UK), and German Centre for Heart Research, outside the submitted work; In addition, Dr. PK is listed as inventor on two patents held by University of Birmingham (Atrial Fibrillation Therapy WO 2015140571, Markers for Atrial Fibrillation WO 2016012783). He received research support for basic, translational and clinical research projects from several drug and device companies active in atrial fibrillation, and has received honoraria from several such companies in the past, but not in the last 3 years. The authors have no other conflicts of interest to declare., (2021 Cardiovascular Diagnosis and Therapy. All rights reserved.)

Importance: World Health Organization (WHO) guidelines do not recommend routine antibiotic use for children with acute watery diarrhea. However, recent studies suggest that a significant proportion of such episodes have a bacterial cause and are associated with mortality and growth impairment, especially among children at high risk of diarrhea-associated mortality. Expanding antibiotic use among dehydrated or undernourished children may reduce diarrhea-associated mortality and improve growth., Objective: To determine whether the addition of azithromycin to standard case management of acute nonbloody watery diarrhea for children aged 2 to 23 months who are dehydrated or undernourished could reduce mortality and improve linear growth., Design, Setting, and Participants: The Antibiotics for Children with Diarrhea (ABCD) trial was a multicountry, randomized, double-blind, clinical trial among 8266 high-risk children aged 2 to 23 months presenting with acute nonbloody diarrhea. Participants were recruited between July 1, 2017, and July 10, 2019, from 36 outpatient hospital departments or community health centers in a mixture of urban and rural settings in Bangladesh, India, Kenya, Malawi, Mali, Pakistan, and Tanzania. Each participant was followed up for 180 days. Primary analysis included all randomized participants by intention to treat., Interventions: Enrolled children were randomly assigned to receive either oral azithromycin, 10 mg/kg, or placebo once daily for 3 days in addition to standard WHO case management protocols for the management of acute watery diarrhea., Main Outcomes and Measures: Primary outcomes included all-cause mortality up to 180 days after enrollment and linear growth faltering 90 days after enrollment., Results: A total of 8266 children (4463 boys [54.0%]; mean [SD] age, 11.6 [5.3] months) were randomized. A total of 20 of 4133 children in the azithromycin group (0.5%) and 28 of 4135 children in the placebo group (0.7%) died (relative risk, 0.72; 95% CI, 0.40-1.27). The mean (SD) change in length-for-age z scores 90 days after enrollment was -0.16 (0.59) in the azithromycin group and -0.19 (0.60) in the placebo group (risk difference, 0.03; 95% CI, 0.01-0.06). Overall mortality was much lower than anticipated, and the trial was stopped for futility at the prespecified interim analysis., Conclusions and Relevance: The study did not detect a survival benefit for children from the addition of azithromycin to standard WHO case management of acute watery diarrhea in low-resource settings. There was a small reduction in linear growth faltering in the azithromycin group, although the magnitude of this effect was not likely to be clinically significant. In low-resource settings, expansion of antibiotic use is not warranted. Adherence to current WHO case management protocols for watery diarrhea remains appropriate and should be encouraged., Trial Registration: ClinicalTrials.gov Identifier: NCT03130114.

Purpose: Cyclin-dependent kinase-retinoblastoma (CDK-RB) pathway is dysregulated in some diffuse intrinsic pontine gliomas (DIPG). We evaluated safety, feasibility, and early efficacy of the CDK4/6-inhibitor ribociclib, administered following radiotherapy in newly-diagnosed DIPG patients., Methods: Following radiotherapy, eligible patients received ribociclib in 28-day cycles (350 mg/m 2 ; 21 days on/7 days off). Feasibility endpoints included tolerability for at least 6 courses, and a less than 2-week delay in restarting therapy after 1 dose reduction. Early efficacy was measured by 1-year and median overall survival (OS). Patient/parent-by-proxy reported outcomes measurement information system (PROMIS) assessments were completed prospectively., Results: The study included 10 evaluable patients, 9 DIPG and 1 diffuse midline glioma (DMG)-all 3.7 to 19.8 years of age. The median number of courses was 8 (range 3-14). Three patients required dose reduction for grade-4 neutropenia, and 1 discontinued therapy for hematological toxicity following course 4. The most common grade-3/4 toxicity was myelosuppression. After 2 courses, MRI evaluations in 4 patients revealed increased necrotic volume, associated with new neurological symptoms in 3 patients. The 1-year and median OS for DIPG was 89% and 16.1 months (range 10-30), respectively; the DMG patient died at 6 months post-diagnosis. Five patients donated brain tissue and tumor; 3 were RB+ ., Conclusions: Ribociclib administered following radiotherapy is feasible in DIPG and DMG. Increased tumor necrosis may represent a treatment effect. These data warrant further prospective volumetric analyses of tumors with necrosis. Feasibility and stabilization findings support further investigation of ribociclib in combination therapies., Trial Registration: NCT02607124.

Purpose: Metabolic syndrome in patients with morbid obesity causes a higher cardiovascular morbidity, eventually leading to left ventricular hypertrophy and decreased left ventricular ejection fraction (LVEF). Roux-en-Y gastric bypass (RYGB) is considered the gold standard modality for treatment of morbid obesity and might even lead to improved cardiac function. Our objective is to investigate whether cardiac function in patients with morbid obesity improves after RYGB., Materials and Methods: In this single center pilot study, 15 patients with an uneventful cardiac history who underwent RYGB were included from May 2015 to March 2016. Cardiac function was measured by cardiac magnetic resonance imaging (CMRI), performed preoperatively and 3, 6, and 12 months postoperative. LVEF and myocardial mass and cardiac output were measured., Results: A total of 13 patients without decreased LVEF preoperative completed follow-up (mean age 37, 48.0 ± 8.8). There was a significant decrease of cardiac output 12 months postoperative (8.3 ± 1.8 preoperative vs. 6.8 ± 1.8 after 12 months, P = 0.001). Average myocardial mass declined by 15.2% (P < 0.001). After correction for body surface area (BSA), this appeared to be non-significant (P = 0.36). There was a significant improvement of LVEF/BSA at 6 and 12 months postoperative (26.2 ± 4.1 preoperative vs. 28.4 ± 3.4 and 29.2 ± 3.6 respectively, both P = 0.002). Additionally, there was a significant improvement of stroke volume/BSA 12 months after surgery (45.8 ± 8.0 vs. 51.9 ± 10.7, P = 0.033)., Conclusion: RYGB in patients with morbid obesity with uneventful history of cardiac disease leads to improvement of cardiac function.

Background: There is no long-term evidence on the effectiveness of training for motivational interviewing in diabetes treatment., Aim: Within a trial of intensive treatment of people with screen-detected diabetes, which included training in motivational interviewing for GPs, the study examined the effect of the intervention on incident cardiovascular disease (CVD) and all-cause mortality., Design & Setting: In the ADDITION-Denmark trial, 181 general practices were cluster randomised in a 2:1:1 ratio to: (i) to screening plus routine care of individuals with screen-detected diabetes (control group); (ii) screening plus training and support in intensive multifactorial treatment of individuals with screen-detected diabetes (intensive treatment group); or (iii) screening plus training and support in intensive multifactorial treatment and motivational interviewing for individuals with screen-detected diabetes (intensive treatment plus motivational interviewing group). The study took place from 2001-2009., Method: After around 8 years follow-up, rates of first fatal and non-fatal CVD events and all-cause mortality were compared between screen-detected individuals in the three treatment groups., Results: Compared with the routine care group, the risk of CVD was similar in the intensive treatment group (hazard ratio [HR] 1.11, 95% confidence interval [CI] = 0.82 to 1.50) and the intensive treatment plus motivational interviewing group (HR 1.26, 95% CI = 0.96 to 1.64). The incidence of death was similar in all three treatment groups., Conclusion: Training of GPs in intensive multifactorial treatment, with or without motivational interviewing, was not associated with a reduction in mortality or CVD among those with screen-detected diabetes., (Copyright © 2020, The Authors.)

Detection of low-level somatic mosaicism [alternate allele fraction (AAF) ≤ 10%] in parents of affected individuals with the apparent de novo pathogenic variants enables more accurate estimate of recurrence risk. To date, only a few systematic analyses of low-level parental somatic mosaicism have been performed. Herein, highly sensitive blocker displacement amplification, droplet digital PCR, quantitative PCR, long-range PCR, and array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins. We screened 18 unrelated families with the FOXF1 variant previously determined to be apparent de novo (n = 14), of unknown parental origin (n = 1), or inherited from a parent suspected to be somatic and/or germline mosaic (n = 3). We identified four (22%) families with FOXF1 parental somatic mosaic single-nucleotide variants (n = 3) and copy number variant deletion (n = 1) detected in parental blood samples and an AAF ranging between 0.03% and 19%. In one family, mosaic allele ratio in tissues originating from three germ layers ranged between <0.03% and 0.65%. Because the ratio of parental somatic mosaicism have significant implications for the recurrence risk, this study further implies the importance of a systematic screening of parental samples for low-level and very-low-level (AAF ≤ 1%) somatic mosaicism using methods that are more sensitive than those routinely applied in diagnostics., (Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The small GTPase RAC1B functions as a powerful inhibitor of transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition, cell motility, and growth arrest in pancreatic epithelial cells. Previous work has shown that RAC1B downregulates the TGF-β type I receptor ALK5, but the molecular details of this process have remained unclear. Here, we hypothesized that RAC1B-mediated suppression of activin receptor-like kinase 5 (ALK5) involves proteinase-activated receptor 2 (PAR2), a G protein-coupled receptor encoded by F2RL1 that is crucial for sustaining ALK5 expression. We found in pancreatic carcinoma Panc1 cells that PAR2 is upregulated by TGF-β1 in an ALK5-dependent manner and that siRNA-mediated knockdown of RAC1B increased both basal and TGF-β1-induced expression of PAR2. Further, the simultaneous knockdown of PAR2 and RAC1B rescued Panc1 cells from a RAC1B knockdown-induced increase in ALK5 abundance and the ALK5-mediated increase in TGF-β1-induced migratory activity. Conversely, Panc1 cells with stable ectopic expression of RAC1B displayed reduced ALK5 expression, an impaired upregulation of PAR2, and a reduced migratory responsiveness to TGF-β1 stimulation. However, these effects could be reversed by ectopic overexpression of PAR2. Moreover, the knockdown of PAR2 alone in Panc1 cells and HaCaT keratinocytes phenocopied RAC1B's ability to suppress ALK5 abundance and TGF-β1-induced chemokinesis and growth inhibition. Lastly, we found that the RAC1B knockdown-induced increase in TGF-β1-induced PAR2 mRNA expression was sensitive to pharmacological inhibition of MEK-ERK signaling. Our data show that in pancreatic and skin epithelial cells, downregulation of ALK5 activity by RAC1B is secondary to suppression of F2RL1 /PAR2 expression. Since F2RL1 itself is a TGF-β target gene and its upregulation by TGF-β1 is mediated by ALK5 and MEK-ERK signaling, we suggest the existence of a feed-forward signaling loop involving ALK5 and PAR2 that is efficiently suppressed by RAC1B to restrict TGF-β-driven cell motility and growth inhibition.

The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition in epithelial cells, but the underlying mechanism has remained enigmatic. Using pancreatic carcinoma cells, we show that both KD and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated knockout (KO) of RAC1B increased the expression of the TGF-β type I receptor ALK5 (activin receptor-like kinase 5), but this effect was more pronounced in CRISPR-KO cells. Of note, in KO, but not KD cells, ALK5 upregulation was associated with resensitization of TGFBR1 to induction by TGF-β1 stimulation. RAC1B KO also increased TGF-β1-induced C-terminal SMAD3 phosphorylation, SMAD3 transcriptional activity, growth inhibition, and cell migration. The KD of ALK5 expression by RNA interference or inactivation of the ALK5 kinase activity by dominant-negative interference or ATP-competitive inhibition rescued the cells from the RAC1B KD/KO-mediated increase in TGF-β1-induced cell migration, whereas the ectopic expression of kinase-active ALK5 mimicked this RAC1B KD/KO effect. We conclude that RAC1B downregulates the abundance of ALK5 and SMAD3 signaling, thereby attenuating TGF-β/SMAD3-driven cellular responses, such as growth inhibition and cell motility.

Gaucher disease is caused by mutations in GBA1 encoding acid β-glucosidase (GCase). Saposin C enhances GCase activity and protects GCase from intracellular proteolysis. Structure simulations indicated that the mutant GCases, N370S (0 S), V394L (4L) and D409V(9V)/H(9H), had altered function. To investigate the in vivo function of Gba1 mutants, mouse models were generated by backcrossing the above homozygous mutant GCase mice into Saposin C deficient (C*) mice. Without saposin C, the mutant GCase activities in the resultant mouse tissues were reduced by ~50% compared with those in the presence of Saposin C. In contrast to 9H and 4L mice that have normal histology and life span, the 9H;C* and 4L;C* mice had shorter life spans. 9H;C* mice developed significant visceral glucosylceramide (GC) and glucosylsphingosine (GS) accumulation (GC»GS) and storage macrophages, but lesser GC in the brain, compared to 4L;C* mice that presents with a severe neuronopathic phenotype and accumulated GC and GS primarily in the brain. Unlike 9V mice that developed normally for over a year, 9V;C* pups had a lethal skin defect as did 0S;C* mice resembled that of 0S mice. These variant Gaucher disease mouse models presented a mutation specific phenotype and underscored the in vivo role of Saposin C in the modulation of Gaucher disease.

Objectives: We used an unbiased proteomics approach to identify candidate urine biomarkers (CUBMs) predictive of LN chronicity and pursued their validation in a larger cohort., Methods: In this cross-sectional pilot study, we selected urine collected at kidney biopsy from 20 children with varying levels of LN damage (discovery cohort) and performed proteomic analysis using isobaric tags for relative and absolute quantification (iTRAQ). We identified differentially excreted proteins based on degree of LN chronicity and sought to distinguish markers exhibiting different relative expression patterns using hierarchically clustered log10-normalized relative abundance data with linked and distinct functions by biological network analyses. For each CUBM, we performed specific ELISAs on urine from a validation cohort (n = 41) and analysis of variance to detect differences between LN chronicity, with LN activity adjustment. We evaluated for CUBM expression in LN biopsies with immunohistochemistry., Results: iTRAQ detected 112 proteins in urine from the discovery cohort, 51 quantifiable in all replicates. Simple analysis of variance revealed four differentially expressed, chronicity-correlated proteins (P-values < 0.05). Further correlation and network analyses led to selection of seven CUBMs for LN chronicity. In the validation cohort, none of the CUBMs distinguished LN chronicity degree; however, urine SERPINA3 demonstrated a moderate positive correlation with LN histological activity. Immunohistochemistry further demonstrated SERPINA3 staining in proximal tubular epithelial and endothelial cells., Conclusion: We identified SERPINA3, a known inhibitor of neutrophil cathepsin G and angiotensin II production, as a potential urine biomarker to help quantify LN activity.

Objectives: To delineate urine biomarkers that reflect kidney structural damage and predict renal functional decline in pediatric lupus nephritis (LN)., Methods: In this prospective study, we evaluated kidney biopsies and urine samples of 89 patients with pediatric LN. Urinary levels of 10 biomarkers [adiponectin, ceruloplasmin, kidney injury molecule-1, monocyte chemotactic protein-1, neutrophil gelatinase-associated lipocalin, osteopontin, transforming growth factor-ß (TGFß), vitamin-D binding protein, liver fatty acid binding protein (LFABP), and transferrin] were measured. Regression analysis was used to identify individual and combinations of biomarkers that determine LN damage status [NIH-chronicity index (NIH-CI) score ≤ 1 vs. ≥ 2] both individually and in combination, and biomarker levels were compared for patients with vs. without renal functional decline, i.e., a 20% reduction of the glomerular filtration rate (GFR) within 12 months of a kidney biopsy., Results: Adiponectin, LFABP, and osteopontin levels differed significantly with select histological damage features considered in the NIH-CI. The GFR was associated with NIH-CI scores [Pearson correlation coefficient (r) = - 0.49; p < 0.0001] but not proteinuria (r = 0.20; p > 0.05). Similar to the GFR [area under the ROC curve (AUC) = 0.72; p < 0.01], combinations of osteopontin and adiponectin levels showed moderate accuracy [AUC = 0.75; p = 0.003] in discriminating patients by LN damage status. Renal functional decline occurred more commonly with continuously higher levels of the biomarkers, especially of TGFß, transferrin, and LFABP., Conclusion: In combination, urinary levels of adiponectin and osteopontin predict chronic LN damage with similar accuracy as the GFR. Ongoing LN activity as reflected by high levels of LN activity biomarkers heralds renal functional decline., Key Messages: • Levels of osteopontin and adiponectin measured at the time of kidney biopsy are good predictors of histological damage with lupus nephritis. • Only about 20% of children with substantial kidney damage from lupus nephritis will have an abnormally low urine creatinine clearance. • Continuously high levels of biomarkers reflecting lupus nephritis activity are risk factors of declining renal function.

Objective: Latent autoimmune diabetes in adults (LADA) shares clinical features with both type 1 and type 2 diabetes; however, there is ongoing debate regarding the precise definition of LADA. Understanding its genetic basis is one potential strategy to gain insight into appropriate classification of this diabetes subtype., Research Design and Methods: We performed the first genome-wide association study of LADA in case subjects of European ancestry versus population control subjects ( n = 2,634 vs. 5,947) and compared against both case subjects with type 1 diabetes ( n = 2,454 vs. 968) and type 2 diabetes ( n = 2,779 vs. 10,396)., Results: The leading genetic signals were principally shared with type 1 diabetes, although we observed positive genetic correlations genome-wide with both type 1 and type 2 diabetes. Additionally, we observed a novel independent signal at the known type 1 diabetes locus harboring PFKFB3 , encoding a regulator of glycolysis and insulin signaling in type 2 diabetes and inflammation and autophagy in autoimmune disease, as well as an attenuation of key type 1-associated HLA haplotype frequencies in LADA, suggesting that these are factors that distinguish childhood-onset type 1 diabetes from adult autoimmune diabetes., Conclusions: Our results support the need for further investigations of the genetic factors that distinguish forms of autoimmune diabetes as well as more precise classification strategies., (© 2018 by the American Diabetes Association.)

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APC M in/+ ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APC M in/+ mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts., (© 2018 International Society on Thrombosis and Haemostasis.)

Background: Lignin is a complex, phenolic polymer found in plant cell walls that is essential for mechanical support, water and mineral transport, and defense in vascular plants. Over ten different enzymes play a role in the synthesis of lignin in plants. Suppression of any one enzyme or combinations of these enzymes may change the concentration and composition of lignin in the genetically transformed plants. Two lines of alfalfa that were downregulated for caffeoyl coenzyme A O-methyltransferase were used to assess the impact of lignin downregulation on chemical composition and fermentation rate and extent using an in vitro gas production technique. A total of 64 samples consisting of two reduced lignin (RL) and two controls (CL), four field replicates, two cutting intervals (CIs; 28 and 35 days), and two cuts (Cut-1 and Cut-3) were used., Results: No differences were detected in yield, crude protein, neutral detergent fiber (aNDF), and acid detergent fiber between the lines when harvested at the 28-day CI. The acid detergent lignin (ADL) concentration in RL alfalfa lines was significantly (P < 0.001) lower than in the CL. In alfalfa harvested at the 35-day CI, the RL alfalfa resulted in lower (P < 0.001) yield than CL. RL alfalfa lines had 24% and 22% lower (P < 0.001) ADL in Cut-1 and Cut-3 respectively than CL lines. The in vitro dry matter digestibility and aNDF digestibility (both as determined by the near-infrared reflectance method) were greater (P < 0.001) in RL than in CL lines harvested at the 35-day CI. In alfalfa harvested at the 35-day CI, extent of in vitro gas production and metabolizable energy content were greater in RL than in CL alfalfa. RL lines had 3.8% indigestible aNDF per unit ADL, whereas CL had 3.4% (P < 0.01). The positive effect of lignin downregulation was more pronounced when intervals between harvests were longer (35-day CI compared with the 28-day CI)., Conclusion: Lignin downregulation in alfalfa offers an opportunity to extend harvesting time (CI) for higher yield without compromising the nutritional quality of the alfalfa forage for dairy and livestock feeding. However, the in vitro results reported here warrant further study using in vivo methods. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Background: In a phase III trial, the RTS,S/AS01 malaria vaccine produced lower anti-circumsporozoite (CS) antibody titers when co-administered with Expanded Programme on Immunization vaccines (0-, 1- and 2-month schedule) at 6 to 12 weeks compared with 5 to 17 months at first vaccination. Alternative infant immunization schedules within the Expanded Programme on Immunization were investigated., Methods: This phase II, open, single-site (Blantyre, Malawi) trial was conducted in infants 1 to 7 days of age. Subjects were equally randomized across 7 groups to receive 3 doses of RTS,S/AS01E at time points that included ≤7 days, 6, 10, 14 and 26 weeks, and 9 months. All RTS,S/AS01E groups plus a control group (without RTS,S/AS01E) received Bacillus Calmette-Guérin + oral poliovirus vaccine at ≤7 days, diphtheria, tetanus, whole-cell pertussis, hepatitis B and Haemophilus influenzae type b vaccine + oral poliovirus vaccine at 6, 10, and 14 weeks and measles vaccine at 9 months; one RTS,S/AS01E group and the control additionally received hepatitis B vaccination at ≤7 days. Serum anti-CS antibody geometric mean concentration (GMC; enzyme-linked immunosorbent assay) and safety were assessed up to age 18 months., Results: Of the 480 infants enrolled, 391 completed the study. No causally related serious adverse event was reported. A higher frequency of fever within 7 days of RTS,S/AS01E vaccination compared with control was observed. Compared with the standard 6-, 10-, 14-week schedule, anti-CS antibody GMC ratios post-dose 3 were significantly higher in the 10-, 14- and 26-week group only (ratio 1.80; 95% confidence interval, 1.24-2.60); RTS,S/AS01E vaccination at ≤7 days and 10 and 14 weeks produced significantly lower anti-CS GMCs (ratio 0.59; 95% confidence interval, 0.38-0.92)., Conclusions: Initiation of RTS,S/AS01E vaccination above 6 weeks of age tended to improve anti-CS antibody responses. Neonatal vaccination was well tolerated but produced a comparatively lower immune response.

Myotonic dystrophy type 1 (DM1) is a progressive neuromuscular disease caused by expanded CUG repeats, which misregulate RNA metabolism through several RNA-binding proteins, including CUG-binding protein/CUGBP1 elav-like factor 1 (CUGBP1/CELF1) and muscleblind 1 protein. Mutant CUG repeats elevate CUGBP1 and alter CUGBP1 activity via a glycogen synthase kinase 3β (GSK3β)-cyclin D3-cyclin D-dependent kinase 4 (CDK4) signaling pathway. Inhibition of GSK3β corrects abnormal activity of CUGBP1 in DM1 mice [human skeletal actin mRNA, containing long repeats ( HSA LR ) model]. Here, we show that the inhibition of GSK3β in young HSA LR mice prevents development of DM1 muscle pathology. Skeletal muscle in 1-yr-old HSA LR mice, treated at 1.5 mo for 6 wk with the inhibitors of GSK3, exhibits high fiber density, corrected atrophy, normal fiber size, with reduced central nuclei and normalized grip strength. Because CUG-GSK3β-cyclin D3-CDK4 converts the active form of CUGBP1 into a form of translational repressor, we examined the contribution of CUGBP1 in myogenesis using Celf1 knockout mice. We found that a loss of CUGBP1 disrupts myogenesis, affecting genes that regulate differentiation and the extracellular matrix. Proteins of those pathways are also misregulated in young HSA LR mice and in muscle biopsies of patients with congenital DM1. These findings suggest that the correction of GSK3β-CUGBP1 pathway in young HSA LR mice might have a positive effect on the myogenesis over time.-Wei, C., Stock, L., Valanejad, L., Zalewski, Z. A., Karns, R., Puymirat, J., Nelson, D., Witte, D., Woodgett, J., Timchenko, N. A., Timchenko, L. Correction of GSK3β at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

This article focuses on the role of Rho family GTPases, particularly Rac1 and Rac1b in TGF-β-induced epithelial-mesenchymal transition (EMT) and EMT-associated responses such as cell migration, invasion, and metastasis in cancer. EMT is considered a prerequisite for cells to adopt a motile and invasive phenotype and eventually become metastatic. A major regulator of EMT and metastasis in cancer is TGF-β, and its specific functions on tumor cells are mediated beside Smad proteins and mitogen-activated protein kinases (MAPKs) by small GTPases of the Rho/Rac1 family. Available data point to extensive signaling crosstalk between TGF-β and various Rho GTPases, and in particular a synergistic role of Rho and Rac1 during EMT and cell motility in normal and neoplastic epithelial cells. In contrast, the Rac1-related isoform, Rac1b, emerges as an endogenous inhibitor of Rac1 in TGF-β signaling, at least in pancreatic carcinoma cells. Given the tumor-promoting role of TGF-β in late-stage carcinomas and the intimate crosstalk of Rho/Rac1/Rac1b and TGF-β signaling in various tumor cell responses, targeting specific Rho GTPases may allow for selective interference with prooncogenic TGF-β responses to aid in anticancer treatments. Developmental Dynamics 247:451-461, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-β1. However, it is not known whether activation of non-SMAD TGF-β signaling (e.g., RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2., Methods: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2-ALK5 physical interaction., Results: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2-AP) was strong and rapid, while it was moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 µM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2-AP- and TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a physical interaction between PAR2 and ALK5., Competing Interests: The authors declare no conflict of interest.

Prompted by earlier findings that the Rac1-related isoform Rac1b inhibits transforming growth factor (TGF)-β1-induced canonical Smad signalling, we studied here whether Rac1b also impacts TGF-β1-dependent non-Smad signalling such as the MKK6-p38 and MEK-ERK mitogen-activated protein kinase (MAPK) pathways and epithelial-mesenchymal transition (EMT). Transient depletion of Rac1b protein in pancreatic cancer cells by RNA interference increased the extent and duration of TGF-β1-induced phosphorylation of p38 MAPK in a Smad4-independent manner. Rac1b depletion also strongly increased basal ERK activation - independent of the kinase function of the TGF-β type I receptor ALK5 - and sensitised cells towards further upregulation of phospho-ERK levels by TGF-β1, while ectopic overexpression of Rac1b had the reverse effect. Rac1b depletion increased an EMT phenotype as evidenced by cell morphology, gene expression of EMT markers, cell migration and growth inhibition. Inhibition of MKK6-p38 or MEK-ERK signalling partially relieved the Rac1b depletion-dependent increase in TGF-β1-induced gene expression and cell migration. Rac1b depletion also enhanced TGF-β1 autoinduction of crucial TGF-β pathway components and decreased that of TGF-β pathway inhibitors. Our results show that Rac1b antagonises TGF-β1-dependent EMT by inhibiting MKK6-p38 and MEK-ERK signalling and by controlling gene expression in a way that favors attenuation of TGF-β signalling.

Transforming growth factor (TGF)-β promotes epithelial-mesenchymal transition and cell invasion of cancer cells in part through the small GTPase RAC1. Since RAC1 can signal through reactive oxygen species (ROS), we probed the role of the ROS-producing NADPH oxidase (NOX) and p38 mitogen-activated protein kinase (MAPK) in mediating TGF-β1/RAC1-driven random cell migration (chemokinesis). Although the NOX isoforms NOX2, 4, 5, 6, and RAC1 were readily detectable by RT-PCR in pancreatic ductal adenocarcinoma (PDAC)-derived Panc1 and Colo357 cells, only NOX4 and RAC1 were expressed at higher levels comparable to those in peripheral blood monocytes. TGF-β1 treatment resulted in upregulation of NOX4 (and NOX2) and rapid intracellular production of ROS. To analyze whether RAC1 functions through NOX and ROS to promote cell motility, we performed real-time cell migration assays with xCELLigence® technology in the presence of the ROS scavenger N-acetyl-L-cysteine (NAC) and various NOX inhibitors. NAC, the NOX4 inhibitor diphenylene iodonium or small interfering RNA (siRNA) to NOX4, and the NOX2 inhibitor apocynin all suppressed TGF-β1-induced chemokinesis of Panc1 and Colo357 cells as did various inhibitors of RAC1 used as control. In addition, we showed that blocking NOX4 or RAC1 function abrogated phosphorylation of p38 MAPK signaling by TGF-β1 and that inhibition of p38 MAPK reduced TGF-β1-induced random cell migration, while ectopic expression of a kinase-active version of the p38 activating kinase MKK6 was able to partially rescue the decline in migration after RAC1 inhibition. Our data suggest that TGF-β1-induced chemokinesis in PDAC cells is mediated through a RAC1/NOX4/ROS/p38 MAPK cascade.

The G protein-coupled receptor proteinase-activated receptor 2 (PAR2) has been implicated in various aspects of cellular physiology including inflammation, obesity and cancer. In cancer, it usually acts as a driver of cancer progression in various tumor types by promoting invasion and metastasis in response to activation by serine proteinases. Recently, we discovered another mode through which PAR2 may enhance tumorigenesis: crosstalk with transforming growth factor-β (TGF-β) signaling to promote TGF-β1-induced cell migration/invasion and invasion-associated gene expression in ductal pancreatic adenocarcinoma (PDAC) cells. In this chapter, we review what is known about the cellular TGF-β responses and signaling pathways affected by PAR2 expression, the signaling activities of PAR2 required for promoting TGF-β signaling, and the potential molecular mechanism(s) that underlie(s) the TGF-β signaling-promoting effect. Since PAR2 is activated through various serine proteinases and biased agonists, it may couple TGF-β signaling to a diverse range of other physiological processes that may or may not predispose cells to cancer development such as local inflammation, systemic coagulation and pathogen infection., Competing Interests: The authors declare no conflict of interest.

Transforming growth factor- β (TGF- β ), serine proteinases such as trypsin, and proteinase-activated receptor 2 (PAR2) promote tumor development by stimulating invasion and metastasis. Previously, we found that in cancer cells derived from pancreatic ductal adenocarcinoma (PDAC) PAR2 protein is necessary for TGF- β 1-dependent cell motility. Here, we show in the same cells that, conversely, the type I TGF- β receptor activin receptor-like kinase 5 is dispensable for trypsin and PAR2 activating peptide (PAR2-AP)-induced migration. To reveal whether G q -calcium signaling is a prerequisite for PAR2 to enhance TGF- β signaling, we investigated the effects of PAR2-APs, PAR2 mutation and PAR2 inhibitors on TGF- β 1-induced migration, reporter gene activity, and Smad activation. Stimulation of cells with PAR2-AP alone failed to enhance basal or TGF- β 1-induced C-terminal phosphorylation of Smad3, Smad-dependent activity of a luciferase reporter gene, and cell migration. Consistently, in complementary loss of function studies, abrogation of the PAR2-G q -calcium signaling arm failed to suppress TGF- β 1-induced cell migration, reporter gene activity, and Smad3 activation. Together, our findings suggest that the calcium-regulating motif is not required for PAR2 to synergize with TGF- β 1 to promote cell motility. Additional experiments in PDAC cells revealed that PAR2 and TGF- β 1 synergy may involve TGF- β 1 induction of enzymes that cause autocrine cleavage/activation of PAR2, possibly through a biased signaling function. Our results suggest that although reducing PAR2 protein expression may potentially block TGF- β 's prooncogenic function, inhibiting PAR2-G q -calcium signaling alone would not be sufficient to achieve this effect., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Improved, noninvasive biomarkers are needed to accurately detect lupus nephritis (LN) activity. The purpose of this study was to evaluate five S100 proteins (S100A4, S100A6, S100A8/9, and S100A12) in both serum and urine as potential biomarkers of global and renal system-specific disease activity in childhood-onset systemic lupus erythematosus (cSLE)., Methods: In this multicenter study, S100 proteins were measured in the serum and urine of four cSLE cohorts and healthy control subjects using commercial enzyme-linked immunosorbent assays. Patients were divided into cohorts on the basis of biospecimen availability: (1) longitudinal serum, (2) longitudinal urine, (3) cross-sectional serum, and (4) cross-sectional urine. Global and renal disease activity were defined using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and the SLEDAI-2K renal domain score. Nonparametric testing was used for statistical analysis, including the Wilcoxon signed-rank test, Kruskal-Wallis test, Mann-Whitney U test, and Spearman's rank correlation coefficient., Results: All urine S100 proteins were elevated in patients with active LN compared with patients with active extrarenal disease and healthy control subjects. All urine S100 protein levels decreased with LN improvement, with S100A4 demonstrating the most significant decrease. Urine S100A4 levels were also higher with proliferative LN than with membranous LN. S100A4 staining in the kidney localized to mononuclear cells, podocytes, and distal tubular epithelial cells. Regardless of the S100 protein tested, serum levels did not change with cSLE improvement., Conclusions: Higher urine S100 levels are associated with increased LN activity in cSLE, whereas serum S100 levels do not correlate with disease activity. Urine S100A4 shows the most promise as an LN activity biomarker, given its pronounced decrease with LN improvement, isolated elevation in urine, and positive staining in resident renal cells.

Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs-such as LINC00173 in granulocytes-and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.While micro-RNAs are known regulators of haematopoiesis and leukemogenesis, the role of long non-coding RNAs is less clear. Here the authors provide a non-coding RNA expression landscape of the human hematopoietic system, highlighting their role in the formation and maintenance of the human blood hierarchy.

Objective: To delineate urine biomarkers that forecast response to therapy of lupus nephritis (LN)., Methods: Starting from the time of kidney biopsy, patients with childhood-onset systemic lupus erythematosus who were diagnosed with LN were studied serially. Levels of 15 biomarkers were measured in random spot urine samples, including adiponectin, α-1-acid glycoprotein (AGP), ceruloplasmin, hemopexin, hepcidin, kidney injury molecule 1, monocyte chemotactic protein-1, lipocalin-like prostaglandin D synthase (LPGDS), transforming growth factor-β (TGF-β), transferrin, and vitamin D binding protein (VDBP)., Results: Among 87 patients (mean age 15.6 yrs) with LN, there were 37 treatment responders and 50 nonresponders based on the American College of Rheumatology criteria. At the time of kidney biopsy, levels of TGF-β (p < 0.0001) and ceruloplasmin (p = 0.006) were significantly lower among responders than nonresponders; less pronounced differences were present for AGP, hepcidin, LPGDS, transferrin, and VDBP (all p < 0.05). By Month 3, responders experienced marked decreases of adiponectin, AGP, transferrin, and VDBP (all p < 0.01) and mean levels of these biomarkers were all outstanding (area under the receiver-operating characteristic curve ≥ 0.9) for discriminating responders from nonresponders. Patient demographics and extrarenal disease did not influence differences in biomarker levels between response groups., Conclusion: Low urine levels of TGF-β and ceruloplasmin at baseline and marked reduction of AGP, LPGDS, transferrin, or VDBP and combinations of other select biomarkers by Month 3 are outstanding predictors for achieving remission of LN. If confirmed, these results can be used to help personalize LN therapy.

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

Genome-wide association studies have identified >50 common variants associated with kidney function, but these variants do not fully explain the variation in eGFR. We performed a two-stage meta-analysis of associations between genotypes from the Illumina exome array and eGFR on the basis of serum creatinine (eGFRcrea) among participants of European ancestry from the CKDGen Consortium ( n Stage1 : 111,666; n Stage2 : 48,343). In single-variant analyses, we identified single nucleotide polymorphisms at seven new loci associated with eGFRcrea ( PPM1J , EDEM3, ACP1, SPEG, EYA4, CYP1A1 , and ATXN2L ; P Stage1 <3.7×10 -7 ), of which most were common and annotated as nonsynonymous variants. Gene-based analysis identified associations of functional rare variants in three genes with eGFRcrea, including a novel association with the SOS Ras/Rho guanine nucleotide exchange factor 2 gene, SOS2 ( P =5.4×10 -8 by sequence kernel association test). Experimental follow-up in zebrafish embryos revealed changes in glomerular gene expression and renal tubule morphology in the embryonic kidney of acp1- and sos2 -knockdowns. These developmental abnormalities associated with altered blood clearance rate and heightened prevalence of edema. This study expands the number of loci associated with kidney function and identifies novel genes with potential roles in kidney formation., (Copyright © 2017 by the American Society of Nephrology.)

TGF-β has a dual role in tumorigenesis, acting as a tumor suppressor in normal cells and in the early stages of tumor development while promoting carcinogenesis and metastasis in advanced tumor stages. The final outcome of the TGF-β response is determined by cell-autonomous mechanisms and genetic alterations such as genomic instability and somatic mutations, but also by a plethora of external signals derived from the tumor microenvironment, such as cell-to-cell interactions, growth factors and extracellular matrix proteins and proteolytic enzymes. Serine proteinases mediate their cellular effects via activation of proteinase-activated receptors (PARs), a subclass of G protein-coupled receptors that are activated by proteolytic cleavage. We have recently identified PAR2 as a factor required for TGF-β1-dependent cell motility in ductal pancreatic adenocarcinoma (PDAC) cells. In this article, we review what is known on the TGF-β-PAR2 signaling crosstalk and its relevance for tumor growth and metastasis. Since PAR2 is activated through various serine proteinases, it may couple TGF-β signaling to a diverse range of other physiological processes, such as local inflammation, systemic coagulation or pathogen infection. Moreover, since PAR2 controls expression of the TGF-β type I receptor ALK5, PAR2 may also impact signaling by other TGF-β superfamily members that signal through ALK5, such as myostatin and GDF15/MIC-1. If so, PAR2 could represent a molecular linker between PDAC development and cancer-related cachexia., Competing Interests: The author declares no conflict of interest. The founding sponsors had no role in the writing of the manuscript, nor in the decision to publish the results.

The results of simultaneous liver-kidney transplants in highly sensitized recipients have been controversial in terms of antibody-mediated rejection and kidney allograft outcomes. This case report provides a detailed and sophisticated documentation of histocompatibility and pathologic data in a simultaneous liver-kidney transplant performed in a recipient with multiple high-titered class I and II antidonor HLA antibodies and a strongly positive cytotoxic crossmatch. Patient received induction with steroids, rituximab, and eculizumab without lymphocyte depleting agents. The kidney transplant was delayed by 6 hours after the liver transplant to allow more time to the liver allograft to "absorb" donor-specific antibodies (DSA). Interestingly, the liver allograft did not prevent immediate antibody-mediated injury to the kidney allograft in this highly sensitized recipient. Anti-HLA single antigen bead analysis of liver and kidney allograft biopsy eluates revealed deposition of both class I and II DSA in both liver and kidney transplants during the first 2 weeks after transplant. Afterward, both liver and kidney allograft functions improved and remained normal after a year with progressive reduction in serum DSA values., Competing Interests: The authors declare no funding or conflicts of interest.

Background: After hematopoietic cell transplantation (HCT), polyoma-BK virus is associated with hemorrhagic cystitis and also with polyomavirus nephropathy (PVN). However, the true burden of post-HCT PVN is unknown because kidney biopsies are avoided due to their bleeding risk. The novel, noninvasive urinary PV-Haufen test detects PVN in kidney transplant recipients with greater than 95% positive/negative predictive values. We hypothesized that the detection of PV-Haufen in voided urine samples-a positive PV-Haufen test-was also clinically significant after HCT., Methods: We examined 21 suitable urine samples from 14 patients (median age, 15 years; 71.4% male) who were selected from repositories for having varying degrees of BK viremia (range, 0-1.0 × 10 copies/mL), hemorrhagic cystitis (present/absent), and data on kidney function. Urine samples were obtained at a median of 88 days post-HCT., Results: The PV-Haufen were detected in 5 of 14 patients (35.7%) and 7 of 21 (33.3%) urine samples, with histologic confirmation of PVN in 1 autopsy specimen. After a median of 285 days post-HCT, patients with PV-Haufen had an increased risk of dialysis-dependent renal failure (P < 0.05). All 3 dialysis-dependent patients had PV-Haufen and died. The presence of urinary PV-Haufen was not significantly correlated with hemorrhagic cystitis. From the 16 urines collected during BK viremia, 43.8% were PV-Haufen-positive, and 56.2% were negative. The PV-Haufen were not present in the 5 urines from patients without concomitant BK-viremia., Conclusions: In this proof-of-concept study, a positive PV-Haufen test was only seen in some patients with BK viremia and was not associated with hemorrhagic cystitis. The detection of PV-Haufen suggests underlying PVN with an increased risk of kidney failure and dialysis.