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Quests for productive soils to close yield gaps call for innovative strategies. This study tested an off-site formation of the Amazonian Terra Preta (TP) in a potential modern analogon under coastal savannah climatic conditions of Ghana. Four Ghanaian and two Zambian soils; two types of biochar (i.e., rice husk biochar and charcoal residues); domestic wastes (i.e., kitchen leftovers, animal manures, human urine, and kitchen ash) were mixed with the soils wetted to 100% water holding capacity, and incubated under aerobic conditions for nine months. Indicators of the TP include total carbon (C), pH, base saturation, basic cations, and plant-available P, which were measured using standard methods of soil analysis. The TP formation enhanced soil pH by 0.02 to 2.9, ranging from pH 7.2 to 8.2, with charcoal residues having the highest effect on pH. The modern TP was characterized by relatively high total C, pH, K, Ca, Mg, Na, base saturation, and plant-available P. These properties reflect unique interactions between the chars, wastes, and soils, suggesting the potential for on-site TP formation. It calls for further studies, commitment, and perseverance in their formation in the future., (© 2024. The Author(s).)

Organic soil amendments (OSA) with long residence times, such as biochar, have a high potential for soil organic carbon (SOC) sequestration. The highly aromatic structure of biochar reduces microbial decomposition and explains the slow turnover of biochar, indicating long persistence in soils and thus potential SOC sequestration. However, there is a lack of data on biochar-induced SOC sequestration in the long-term and under field conditions. We sampled two long-term field experiments in Germany, where biochar was applied 12 and 14 years ago. Both locations differ in soil characteristics and in the types and amounts of biochar and other OSA. Amendments containing compost and 31.5 Mg ha -1 of biochar on a loamy soil led to a SOC stock increase of 38 Mg ha -1 after OSA addition. The additional increase is due to non-biochar co-amendments such as compost or biogas digestate. After eleven years, this SOC stock increase was still stable. High biochar amount additions of 40 Mg ha -1 combined with biogas digestate, compost or synthetic fertilizer on a sandy soil led to an increase of SOC stocks of 61 Mg ha -1 ; 38 Mg ha -1 dissipated in the following four years most likely due to lacking physical protection of the coarse soil material, and after nine years the biochar-amended soils showed only slightly higher SOC stocks (+7 Mg ha -1 ) than the control. Black carbon stocks on the same soil increased in the short- and mid-term and decreased almost to the original stock levels after nine years. Our results indicate that in most cases the long-term effect on SOC and black carbon stocks is controlled by biochar quality and amount, while non-biochar co-amendments can be neglected. This study proves that SOC sequestration through the use of biochar is possible, especially in loamy soils, while non-biochar OSA cannot sequester SOC in the long term., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Deconvolution methods infer quantitative cell type estimates from bulk measurement of mixed samples including blood and tissue. DNA methylation sequencing measures multiple CpGs per read, but few existing deconvolution methods leverage this within-read information. We develop CelFiE-ISH, which extends an existing method (CelFiE) to use within-read haplotype information. CelFiE-ISH outperforms CelFiE and other existing methods, achieving 30% better accuracy and more sensitive detection of rare cell types. We also demonstrate the importance of marker selection and of tailoring markers for haplotype-aware methods. While here we use gold-standard short-read sequencing data, haplotype-aware methods will be well-suited for long-read sequencing., (© 2024. The Author(s).)

Assessment of pancreas cell type composition is crucial to the understanding of the genesis of diabetes. Current approaches use immunodetection of protein markers, for example, insulin as a marker of β-cells. A major limitation of these methods is that protein content varies in physiological and pathological conditions, complicating the extrapolation to actual cell number. Here, we demonstrate the use of cell type-specific DNA methylation markers for determining the fraction of specific cell types in human islet and pancreas specimens. We identified genomic loci that are uniquely demethylated in specific pancreatic cell types and applied targeted PCR to assess the methylation status of these loci in tissue samples, enabling inference of cell type composition. In islet preparations, normalization of insulin secretion to β-cell DNA revealed similar β-cell function in pre-type 1 diabetes (T1D), T1D, and type 2 diabetes (T2D), which was significantly lower than in donors without diabetes. In histological pancreas specimens from recent-onset T1D, this assay showed β-cell fraction within the normal range, suggesting a significant contribution of β-cell dysfunction. In T2D pancreata, we observed increased α-cell fraction and normal β-cell fraction. Methylation-based analysis provides an accurate molecular alternative to immune detection of cell types in the human pancreas, with utility in the interpretation of insulin secretion assays and the assessment of pancreas cell composition in health and disease., (© 2024 by the American Diabetes Association.)

Objective: Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed., Design: To assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites., Results: Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92)., Conclusion: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC., Competing Interests: Competing interests: ABu has received consulting fees from Exelixis and Geistlich Pharma. KM declares research funding from Celgene and Trovagene. AJA has consulted for Oncorus, Inc., Arrakis Therapeutics, and Merck & Co., Inc, and has research funding from Mirati Therapeutics, Syros, Deerfield, Inc., and Novo Ventures that is unrelated to this work. AM is listed as an inventor on a patent that has been licensed by Johns Hopkins University to Thrive Earlier Detection and serves as a consultant for Tezcat Biosciences. BG, RS and YD have filed patents on cfDNA analysis technology, and received research funding from GRAIL. BMW declares research funding from Celgene, Eli Lilly Novartis and Revolution Medicines, and consulting for Celgene, GRAIL, Ipsen and Mirati., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: Accurate detection of graft-versus-host disease (GVHD) is a major challenge in the management of patients undergoing hematopoietic stem cell transplantation (HCT). Here, we demonstrated the use of circulating cell-free DNA (cfDNA) for detection of tissue turnover and chronic GVHD (cGVHD) in specific organs., Methods: We established a cocktail of tissue-specific DNA methylation markers and used it to determine the concentration of cfDNA molecules derived from the liver, skin, lungs, colon, and specific immune cells in 101 patients undergoing HCT., Results: Patients with active cGVHD showed elevated concentrations of cfDNA, as well as tissue-specific methylation markers that agreed with clinical scores. Strikingly, transplanted patients with no clinical symptoms had abnormally high levels of tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident clinical pathology. An integrative model taking into account total cfDNA concentration, monocyte/macrophage cfDNA levels and alanine transaminase was able to correctly identify GVHD with a specificity of 86% and precision of 89% (AUC of 0.8)., Conclusion: cfDNA markers can be used for the detection of cGVHD, opening a window into underlying tissue dynamics in patients that receive allogeneic stem cell transplants., Funding: This work was supported by grants from the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation and the Helmsley Charitable Trust (to YD).

Inflammation is consistently linked to dysmetabolism. In transgenic mice (Def +/+ ) model the neutrophilic peptide, alpha defensin, proved atherogenic. This phenotype occurred despite favorable cholesterol and glucose levels, and lower body weight and blood pressure. In this study, integration of metabolic&behavioral phenotyping system, endocrine, biochemical and mitochondrial assessment, pathological and immunohistochemical tests, and multiple challenge tests was established to explore the metabolic impact of alpha defensin. Compared to the control group, Def +/+ mice exhibited lower total energy expenditure and carbohydrate utilization, and higher fat oxidation. Their ACTH-cortisol and thyroid profiles were intact. Intriguingly, they had low levels of glucagon, with high ammonia, uric acid, triglyceride, and lactate. Mitochondrial evaluations were normal. Overall, defensin-induced hypoglucagonemia is associated with lipolysis, restricted glucose oxidation, and enhanced wasting. Def +/+ mice may be a useful model for studying the category of lean, apparently metabolically healthy, and atherosclerotic phenotype, with insight into a potential inflammatory-metabolic link., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

A major hypothesis for the etiology of type 1 diabetes (T1D) postulates initiation by viral infection, leading to double-stranded RNA (dsRNA)-mediated interferon response and inflammation; however, a causal virus has not been identified. Here, we use a mouse model, corroborated with human islet data, to demonstrate that endogenous dsRNA in beta cells can lead to a diabetogenic immune response, thus identifying a virus-independent mechanism for T1D initiation. We found that disruption of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) in beta cells triggers a massive interferon response, islet inflammation, and beta cell failure and destruction, with features bearing striking similarity to early-stage human T1D. Glycolysis via calcium enhances the interferon response, suggesting an actionable vicious cycle of inflammation and increased beta cell workload., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

While programmed cell death plays important roles during morphogenetic stages of development, post-differentiation organ growth is considered an efficient process whereby cell proliferation increases cell number. Here we demonstrate that early postnatal growth of the pancreas unexpectedly involves massive acinar cell elimination. Measurements of cell proliferation and death in the human pancreas in comparison to the actual increase in cell number predict daily elimination of 0.7% of cells, offsetting 88% of cell formation over the first year of life. Using mouse models, we show that death is associated with mitosis, through a failure of dividing cells to generate two viable daughters. In p53-deficient mice, acinar cell death and proliferation are reduced, while organ size is normal, suggesting that p53-dependent developmental apoptosis triggers compensatory proliferation. We propose that excess cell turnover during growth of the pancreas, and presumably other organs, facilitates robustness to perturbations and supports maintenance of tissue architecture., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Early diagnosis of autism spectrum disorders (ASD) offers the possibility of early intervention and, in turn, gains in adaptive behaviour, language and cognition. The aim of the present study was to analyse whether age at diagnosis of autism spectrum disorders decreased in two regions of Switzerland from 2006 to 2016 following the implementation of different screening and referral techniques. In southern Switzerland, systematic paediatric screening using the Modified Checklist for Autism (M-CHAT) in toddlers was implemented in 2013, whereas in northwestern Switzerland, periodic trainings were used to increase paediatrician awareness of ASD. We investigated which method was associated with a younger average age at diagnosis., Methods: We conducted a retrospective, two-centre study searching clinical records of children and adolescents (aged 0-16 years) diagnosed with ASD in two neuropaediatric departments at Swiss hospitals between January 2006 and December 2016. All patients were diagnosed via a standardised evaluation based on two approved diagnostic tests: the Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) and the Autism Diagnostic Interview-Revised (ADI-R)., Results: In southern Switzerland, training and subsequent widespread use of the M-CHAT among paediatricians appeared to contribute to a significantly younger age at diagnosis. Age at diagnosis did not significantly decrease during the same period in northwestern Switzerland., Conclusion: Our results point to the possibility of successfully reducing age at diagnosis in specific geographic areas through the implementation of screening questionnaires, such as the M-CHAT, at year 2 well-baby visits.

Background: Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death., Methods: In pancreas donors after brain death, we compared clinical and biochemical data in 'insulin-treated' and 'not insulin-treated donors' (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated., Results: Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)]., Conclusions: In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients., (© 2023 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Background: Radiotherapy has an important role in the treatment of brain metastases but carries risk of short and/or long-term toxicity, termed radiation-induced brain injury (RBI). As the diagnosis of RBI is crucial for correct patient management, there is an unmet need for reliable biomarkers for RBI. The aim of this proof-of concept study is to determine the utility of brain-derived circulating free DNA (BncfDNA), identified by specific methylation patterns for neurons, astrocytes, and oligodendrocytes, as biomarkers brain injury induced by radiotherapy., Methods: Twenty-four patients with brain metastases were monitored clinically and radiologically before, during and after brain radiotherapy, and blood for BncfDNA analysis (98 samples) was concurrently collected. Sixteen patients were treated with whole brain radiotherapy and eight patients with stereotactic radiosurgery., Results: During follow-up nine RBI events were detected, and all correlated with significant increase in BncfDNA levels compared to baseline. Additionally, resolution of RBI correlated with a decrease in BncfDNA. Changes in BncfDNA were independent of tumor response., Conclusions: Elevated BncfDNA levels reflects brain cell injury incurred by radiotherapy. further research is needed to establish BncfDNA as a novel plasma-based biomarker for brain injury induced by radiotherapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Makranz 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.)

Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets., (© 2023. The Author(s).)

DOES - Dataset of European scrap classes. Today, scrap is already an important raw material for industry. Due to the transformation to green steel, the secondary raw material scrap will become increasingly important in the coming years. With DOES a free dataset is presented, which represents common non-alloyed European scrap classes. Two important points were considered in this dataset. First, scrap oxidizes under normal external conditions and the visual appearance changes, which plays an important role in visual inspections. Therefore, DOES includes scrap images of different degrees of corrosion attack. Second, images of scrap metal (mostly scrap piles) usually have no intrinsic order. For this reason, a technique to extract many overlapping rectangles from raw images was used, which can be used to train deep learning algorithms without any disadvantage. This dataset is very suitable to develop industrial applications or to research classification algorithms. The dataset was validated by experts and through machine learning models., (© 2023. The Author(s).)

Aim/hypothesis: Hyperglycaemia is associated with alpha cell dysfunction, leading to dysregulated glucagon secretion in type 1 and type 2 diabetes; however, the mechanisms involved are still elusive. The nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) plays a major role in the maintenance of alpha cell mass and function. We studied the regulation of alpha cell mTORC1 by nutrients and its role in the development of hyperglucagonaemia in diabetes., Methods: Alpha cell mTORC1 activity was assessed by immunostaining for phosphorylation of its downstream target, the ribosomal protein S6, and glucagon, followed by confocal microscopy on pancreatic sections and flow cytometry on dispersed human and mouse islets and the alpha cell line, αTC1-6. Metabolomics and metabolic flux were studied by 13 C glucose labelling in 2.8 or 16.7 mmol/l glucose followed by LC-MS analysis. To study the role of mTORC1 in mediating hyperglucagonaemia in diabetes, we generated an inducible alpha cell-specific Rptor knockout in the Akita mouse model of diabetes and tested the effects on glucose tolerance by IPGTT and on glucagon secretion., Results: mTORC1 activity was increased in alpha cells from diabetic Akita mice in parallel to the development of hyperglycaemia and hyperglucagonaemia (two- to eightfold increase). Acute exposure of mouse and human islets to amino acids stimulated alpha cell mTORC1 (3.5-fold increase), whereas high glucose concentrations inhibited mTORC1 (1.4-fold decrease). The mTORC1 response to glucose was abolished in human and mouse diabetic alpha cells following prolonged islet exposure to high glucose levels, resulting in sustained activation of mTORC1, along with increased glucagon secretion. Metabolomics and metabolic flux analysis showed that exposure to high glucose levels enhanced glycolysis, glucose oxidation and the synthesis of glucose-derived amino acids. In addition, chronic exposure to high glucose levels increased the expression of Slc7a2 and Slc38a4, which encode amino acid transporters, as well as the levels of branched-chain amino acids and methionine cycle metabolites (~1.3-fold increase for both). Finally, conditional Rptor knockout in alpha cells from adult diabetic mice inhibited mTORC1, thereby inhibiting glucagon secretion (~sixfold decrease) and improving diabetes, despite persistent insulin deficiency., Conclusions/interpretation: Alpha cell exposure to hyperglycaemia enhances amino acid synthesis and transport, resulting in sustained activation of mTORC1, thereby increasing glucagon secretion. mTORC1 therefore plays a major role in mediating alpha cell dysfunction in diabetes., Data Availability: All sequencing data are available from the Gene Expression Omnibus (GEO) repository (accession no. GSE154126; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154126 )., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chronological age prediction from DNA methylation sheds light on human aging, health, and lifespan. Current clocks are mostly based on linear models and rely upon hundreds of sites across the genome. Here, we present GP-age, an epigenetic non-linear cohort-based clock for blood, based upon 11,910 methylomes. Using 30 CpG sites alone, GP-age outperforms state-of-the-art models, with a median accuracy of ∼2 years on held-out blood samples, for both array and sequencing-based data. We show that aging-related changes occur at multiple neighboring CpGs, with implications for using fragment-level analysis of sequencing data in aging research. By training three independent clocks, we show enrichment of donors with consistent deviation between predicted and actual age, suggesting individual rates of biological aging. Overall, we provide a compact yet accurate alternative to array-based clocks for blood, with applications in longitudinal aging research, forensic profiling, and monitoring epigenetic processes in transplantation medicine and cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Strenuous physical exercise causes a massive elevation in the concentration of circulating cell-free DNA (cfDNA), which correlates with effort intensity and duration. The cellular sources and physiological drivers of this phenomenon are unknown. Using methylation patterns of cfDNA and associated histones, we show that cfDNA in exercise originates mostly in extramedullary polymorphonuclear neutrophils. Strikingly, cardiomyocyte cfDNA concentration increases after a marathon, consistent with elevated troponin levels and indicating low-level, delayed cardiac cell death. Physical impact, low oxygen levels, and elevated core body temperature contribute to neutrophil cfDNA release, while muscle contraction, increased heart rate, β-adrenergic signaling, or steroid treatment fail to cause elevation of cfDNA. Physical training reduces neutrophil cfDNA release after a standard exercise, revealing an inverse relationship between exercise-induced cfDNA release and training level. We speculate that the release of cfDNA from neutrophils in exercise relates to the activation of neutrophils in the context of exercise-induced muscle damage., Competing Interests: Declaration of interests R. Sadeh, G.F., I.S., and N.F. are founders and/or employees of Senseera Inc. J.M., B.G., R. Shemer, and Y.D. have filed patents on cfDNA methylation analysis., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Vascular endothelial cells (VECs) are an essential component of each tissue, contribute to multiple pathologies, and are targeted by important drugs. Yet, there is a shortage of biomarkers to assess VEC turnover., Methods: To develop DNA methylation-based liquid biopsies for VECs, we determined the methylome of VECs isolated from freshly dissociated human tissues., Findings: A comparison with a human cell-type methylome atlas yielded thousands of loci that are uniquely unmethylated in VECs. These sites are typically gene enhancers, often residing adjacent to VEC-specific genes. We also identified hundreds of genomic loci that are differentially methylated in organotypic VECs, indicating that VECs feeding specific organs are distinct cell types with a stable epigenetic identity. We established universal and lung-specific VEC markers and evaluated their presence in circulating cell-free DNA (cfDNA). Nearly 2.5% of cfDNA in the plasma of healthy individuals originates from VECs. Sepsis, graft versus host disease, and cardiac catheterization are associated with elevated levels of VEC-derived cfDNA, indicative of vascular damage. Lung-specific VEC cfDNA is selectively elevated in patients with chronic obstructive pulmonary disease (COPD) or lung cancer, revealing tissue-specific vascular turnover., Conclusions: VEC cfDNA biomarkers inform vascular dynamics in health and disease, potentially contributing to early diagnosis and monitoring of pathologies, and assessment of drug activity., Funding: This work was supported by the Beutler Research Program, Helmsley Charitable Trust, JDRF, Grail and the DON Foundation (to Y.D.). Y.D holds the Walter & Greta Stiel Chair in heart studies. B.G., R.S., J.M., D.N., T.K., and Y.D. filed patents on cfDNA analysis., Competing Interests: Declaration of interests T.K., B.G., R.S., and Y.D. have filed patents related to DNA methylation markers. G.C., J.B., and O.V. are employees of GRAIL, LLC, a subsidiary of Illumina, LLC., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Competing Interests: The authors have no multiplicity of interest to disclose.

The use of cardiopulmonary bypass (CPB) is thought to cause delayed cardiac damage. DNA methylation-based liquid biopsies are novel biomarkers for monitoring acute cardiac cell death. We assessed cell-free DNA molecules as markers for cardiac damage after open-heart surgery. Novel cardiomyocyte-specific DNA methylation markers were applied to measure cardiac cfDNA in the plasma of 42 infants who underwent open-heart surgery. Cardiac cfDNA was elevated following surgery, reflecting direct surgery-related tissue damage, and declined thereafter in most patients. The concentration of cardiac cfDNA post-surgery correlated with the duration of CPB and aortic cross clamping. Strikingly, cardiac cfDNA at 6 h predicted duration of mechanical ventilation and maximal vasoactive-inotropic score better than did maximal troponin levels. Cardiac cfDNA reveals heart damage associated with CPB, and can be used to monitor cardiac cell death, to predict clinical outcome of surgery and to assess performance of cardioprotective interventions., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Expanding the use of environmentally friendly materials to protect the environment is one of the key factors in maintaining a sustainable ecological balance. Poly(butylene succinate-co-adipate) (PBSA) is considered among the most promising bio-based and biodegradable plastics for the future with a high number of applications in soil and agriculture. Therefore, the decomposition process of PBSA and its consequences for the carbon stored in soil require careful monitoring. For the first time, the stable isotope technique was applied in the current study to partitioning plastic- and soil-originated C in the CO 2 released during 80 days of PBSA decomposition in a Haplic Chernozem soil as dependent on nitrogen availability. The decomposition of the plastic was accompanied by the C loss from soil organic matter (SOM) through priming, which in turn was dependent on added N. Nitrogen facilitated PBSA decomposition and reduced the priming effect during the first 6 weeks of the experiment. During the 80 days of plastic decomposition, 30% and 49% of the released CO 2 were PBSA-derived, while the amount of SOM-derived CO 2 exceeded the corresponding controls by 100.2 and 132.3% in PBSA-amended soil without and with N fertilization, respectively. Finally, only 4.1% and 5.4% of the PBSA added into the soil was mineralized to CO 2 , in the treatments without and with N amendment, respectively.

DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes 1 . Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells 2-5 . Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies., (© 2023. The Author(s).)

Diabetogenic ablation of beta cells in mice triggers a regenerative response whereby surviving beta cells proliferate and euglycemia is regained. Here, we identify and characterize heterogeneity in response to beta cell ablation. Efficient beta cell elimination leading to severe hyperglycemia (>28 mmol/L), causes permanent diabetes with failed regeneration despite cell cycle engagement of surviving beta cells. Strikingly, correction of glycemia via insulin, SGLT2 inhibition, or a ketogenic diet for about 3 weeks allows partial regeneration of beta cell mass and recovery from diabetes, demonstrating regenerative potential masked by extreme glucotoxicity. We identify gene expression changes in beta cells exposed to extremely high glucose levels, pointing to metabolic stress and downregulation of key cell cycle genes, suggesting failure of cell cycle completion. These findings reconcile conflicting data on the impact of glucose on beta cell regeneration and identify a glucose threshold converting glycemic load from pro-regenerative to anti-regenerative., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Opencast coal mining results in high loss of soil organic carbon (SOC), which may be restored via recultivation. Common strategies include liming, topsoil application, and phytoremediation. It remains unclear, however, which parameters determine the effectiveness of these varying recultivation strategies especially regarding SOC sequestration. This meta-analysis analyses the effect of varying recultivation strategies on SOC sequestration under different climate and soil conditions (pH, texture, depth) as well as in relation to time, based on 404 data entries from 51 studies. All included climatic regions recorded increases in SOC stocks, with tropical soils showing the highest potential for relative gains at up to 637%. We demonstrate that loamy soils sequester twice as much newly introduced SOC than sand. Strategy-wise, the highest mean rate of SOC sequestration is achieved by forest after topsoil application (3.9 Mg ha -1 a -1 ), agriculture after topsoil application (2.3 Mg ha -1 a -1 ), and agriculture with topsoil and fertiliser application (1.9 Mg ha -1 a -1 ) with a response ratio of 304%, 281%, and 218%, respectively. Soils analysed to less then 40 cm depth show higher SOC sequestration rates (< 10 cm: 0.6 Mg ha -1 a -1 , < 20 cm: 1.0 Mg ha -1 a -1 , and 20-40 cm: 0.4 Mg ha -1 a -1 ; response ratio of 123%, 68%, and 73%, respectively) than those analysed to a depth of 41-80 cm (0.1 Mg ha -1 a -1 ; response ratio of 6%). In terms of pH, strongly acidic soils (pH < 4.5) and alkaline conditions (pH > 7) offer the most beneficial environment for SOC sequestration at 0.4 Mg ha -1 a -1 and 0.8 Mg ha -1 a -1 , respectively (185% and 273% response). Given comparable SOC sequestration potentials of forest after topsoil application, agriculture without amendments, and forest without amendments, we recommend to weigh these strategies against each other. Potentially decisive aspects are short- vs. long-term economic gains, food security concerns, and-in case of agriculture-the risk of overintensification leading to losses in SOC. Our data suggests that amendments exert considerable influence on SOC sequestration and need to be introduced under careful consideration., (© 2022. The Author(s).)

Aims: Glucokinase (GK) is expressed in the glucose-sensing cells of the islets of Langerhans and plays a critical role in glucose homeostasis. Here, we tested the hypothesis that genetic activation of GK in a small subset of β-cells is sufficient to change the glucose set-point of the whole islet., Material and Methods: Mouse models of cell-type specific GK deficiency (GKKO) and genetic enzyme activation (GKKI) in a subset of β-cells were obtained by crossing the αGSU (gonadotropin alpha subunit)-Cre transgene with the appropriate GK mutant alleles. Metabolic analyses consisted of glucose tolerance tests, perifusion of isolated islets and intracellular calcium measurements., Key Findings: The αGSU-Cre transgene produced genetically mosaic islets, as Cre was active in 15 ± 1.2 % of β-cells. While mice deficient for GK in a subset of islet cells were normal, unexpectedly, GKKI mice were chronically hypoglycemic, glucose intolerant, and had a lower threshold for glucose stimulated insulin secretion. GKKI mice exhibited an average fasting blood glucose level of 3.5 mM. GKKI islets responded with intracellular calcium signals that spread through the whole islets at 1 mM and secreted insulin at 3 mM glucose., Significance: Genetic activation of GK in a minority of β-cells is sufficient to change the glucose threshold for insulin secretion in the entire islet and thereby glucose homeostasis in the whole animal. These data support the model in which β-cells with higher GK activity function as 'hub' or 'trigger' cells and thus control insulin secretion by the β-cell collective within the islet., 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 © 2022. Published by Elsevier Inc.)

Background: Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death., Methods: We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease., Results: Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients., Conclusions: Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics., Competing Interests: Conflict of interest: G. Cann, H. Amini, P. Moradi and S. Nagaraju are employees of Grail LLC. J. Magenheim, B. Glaser, T. Kaplan, R. Shemer and Y. Dor have patents on cfDNA methylation markers and methods of analysis. All other authors have no conflict of interest., (Copyright ©The authors 2022. For reproduction rights and permissions contact permissions@ersnet.org.)

Genome-wide association studies (GWASs) have identified hundreds of loci associated with Crohn's disease (CD). However, as with all complex diseases, robust identification of the genes dysregulated by noncoding variants typically driving GWAS discoveries has been challenging. Here, to complement GWASs and better define actionable biological targets, we analyzed sequence data from more than 30,000 patients with CD and 80,000 population controls. We directly implicate ten genes in general onset CD for the first time to our knowledge via association to coding variation, four of which lie within established CD GWAS loci. In nine instances, a single coding variant is significantly associated, and in the tenth, ATG4C, we see additionally a significantly increased burden of very rare coding variants in CD cases. In addition to reiterating the central role of innate and adaptive immune cells as well as autophagy in CD pathogenesis, these newly associated genes highlight the emerging role of mesenchymal cells in the development and maintenance of intestinal inflammation., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Much remains unknown regarding the response of the immune system to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination., Methods: We employed circulating cell-free DNA (cfDNA) to assess the turnover of specific immune cell types following administration of the Pfizer/BioNTech vaccine., Findings: The levels of B cell cfDNA after the primary dose correlated with development of neutralizing antibodies and memory B cells after the booster, revealing a link between early B cell turnover-potentially reflecting affinity maturation-and later development of effective humoral response. We also observed co-elevation of B cell, T cell, and monocyte cfDNA after the booster, underscoring the involvement of innate immune cell turnover in the development of humoral and cellular adaptive immunity. Actual cell counts remained largely stable following vaccination, other than a previously demonstrated temporary reduction in neutrophil and lymphocyte counts., Conclusions: Immune cfDNA dynamics reveal the crucial role of the primary SARS-CoV-2 vaccine in shaping responses of the immune system following the booster vaccine., Funding: This work was supported by a generous gift from Shlomo Kramer. Supported by grants from Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.), Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Alex U Soyka Pancreatic Cancer Fund, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation, the Helmsley Charitable Trust, Grail, and the DON Foundation (to Y.D.). Y.D. holds the Walter and Greta Stiel Chair and Research Grant in Heart Studies. I.F.-F. received a fellowship from the Glassman Hebrew University Diabetes Center., Competing Interests: Declaration of interests I.F.-F., B.G., R.S., and Y.D. have filed patents related to DNA methylation markers., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Schizophrenia is a common, severe, and debilitating psychiatric disorder. Despite extensive research there is as yet no biological marker that can aid in its diagnosis and course prediction. This precludes early detection and intervention. Imaging studies suggest brain volume loss around the onset and over the first few years of schizophrenia, and apoptosis has been proposed as the underlying mechanism. Cell-free DNA (cfDNA) fragments are released into the bloodstream following cell death. Tissue-specific methylation patterns allow the identification of the tissue origins of cfDNA. We developed a cocktail of brain-specific DNA methylation markers, and used it to assess the presence of brain-derived cfDNA in the plasma of patients with a first psychotic episode. We detected significantly elevated neuron- (p=0.0013), astrocyte- (p=0.0016), oligodendrocyte- (p=0.0129), and whole brain-derived (p=0.0012) cfDNA in the plasma of patients during their first psychotic episode (n=29), compared with healthy controls (n=31). Increased cfDNA levels were not correlated with psychotropic medications use. Area under the curve (AUC) was 0.77, with 65% sensitivity at 90% specificity in patients with a psychotic episode. Potential interpretations of these findings include increased brain cell death, disruption of the blood-brain barrier, or a defect in clearance of material from dying brain cells. Brain-specific cfDNA methylation markers can potentially assist early detection and monitoring of schizophrenia and thus allow early intervention and adequate therapy., Competing Interests: AL, IP, RT, AS, SP, BO, KS, YK No competing interests declared, DN, HZ, BG, RS, YD has filed patents on cfDNA analysis technology (year 2019, application number 62/828,587), (© 2022, Lubotzky, Pelov et al.)

Objectives: In-depth knowledge about surgical processes is a crucial prerequisite for future systems in operating rooms and the advancement of standards and patient safety in surgery. A holistic approach is required, but research in the field of surgical instrument tables, standardized instrument setups and involved personnel, such as nurses, is sparse in general. The goal of this study is to evaluate whether there is an existing standard within clinics for an instrument table setup. We also evaluate to which extent it is known to the personnel and whether it is accepted., Materials and Methods: The study makes use of the Nosco Trainer, a scrub nurse training and simulation system developed to analyze various aspects of the workplace of scrub nurses. The system contains a virtual instrument table, which is used to perform and record instrument table setups. We introduce a metric which delivers a measurable score for the similarity of surgical instrument table setups. The study is complemented with a questionnaire covering related aspects., Results: Fifteen scrub nurses of the Otolaryngology departments at three clinics in Germany and Switzerland performed a table setup for a Functional Endoscopic Sinus Surgery intervention and completed the questionnaire. The analysis of the developed metric with a leave one out cross-validation correctly allocated 14 of the 15 participants to their clinic., Discussion: In contrast to the identified similarities of table setups within clinics with the collected data, only a third of the participants confirmed in the questionnaire that there is an existing table setup standard for Functional Endoscopic Sinus Surgery interventions in their facility, but almost three quarters would support a written standard and acknowledge its possible benefits for trainees and new entrants in the operating room., Conclusions: The structured analysis of the surgical instrument table using a data-driven metric for comparison is a novel approach to gain deeper knowledge about intra-operative processes. The insights can contribute to patient safety by improving the workflow between surgeon and scrub nurse and also open the way for goal-oriented standardization., (© 2022. The Author(s).)

We discovered a biological mechanism supporting microbial degradation of bio-based poly(butylene succinate- co -adipate) (PBSA) plastic in soils under ambient and future climates. Here, we show that nitrogen-fixing bacteria facilitate the microbial degradation of PBSA by enhancing fungal abundance, accelerating plastic-degrading enzyme activities, and shaping/interacting with plastic-degrading fungal communities.

The repeated expansion of East Asian steppe cultures was a key driver of Eurasian history, forging new social, economic, and biological links across the continent. Climate has been suggested as important driver of these poorly understood cultural expansions, but paleoclimate records from the Mongolian Plateau often suffer from poor age control or ambiguous proxy interpretation. Here, we use a combination of geochemical analyses and comprehensive radiocarbon dating to establish the first robust and detailed record of paleohydrological conditions for Lake Telmen, Mongolia, covering the past ~ 4000 years. Our record shows that humid conditions coincided with solar minima, and hydrological modeling confirms the high sensitivity of the lake to paleoclimate changes. Careful comparisons with archaeological and historical records suggest that in the vast semi-arid grasslands of eastern Eurasia, solar minima led to reduced temperatures, less evaporation, and high biomass production, expanding the power base for pastoral economies and horse cavalry. Our findings suggest a crucial link between temperature dynamics in the Eastern Steppe and key social developments, such as the emergence of pastoral empires, and fuel concerns that global warming enhances water scarcity in the semi-arid regions of interior Eurasia., (© 2022. The Author(s).)

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

Large-scale gene sequencing studies for complex traits have the potential to identify causal genes with therapeutic implications. We performed gene-based association testing of blood lipid levels with rare (minor allele frequency < 1%) predicted damaging coding variation by using sequence data from >170,000 individuals from multiple ancestries: 97,493 European, 30,025 South Asian, 16,507 African, 16,440 Hispanic/Latino, 10,420 East Asian, and 1,182 Samoan. We identified 35 genes associated with circulating lipid levels; some of these genes have not been previously associated with lipid levels when using rare coding variation from population-based samples. We prioritize 32 genes in array-based genome-wide association study (GWAS) loci based on aggregations of rare coding variants; three (EVI5, SH2B3, and PLIN1) had no prior association of rare coding variants with lipid levels. Most of our associated genes showed evidence of association among multiple ancestries. Finally, we observed an enrichment of gene-based associations for low-density lipoprotein cholesterol drug target genes and for genes closest to GWAS index single-nucleotide polymorphisms (SNPs). Our results demonstrate that gene-based associations can be beneficial for drug target development and provide evidence that the gene closest to the array-based GWAS index SNP is often the functional gene for blood lipid levels., Competing Interests: Declaration of interests The authors declare no competing interests for the present work. P.N. reports investigator-initiated grants from Amgen, Apple, and Boston Scientific; is a scientific advisor to Apple, Blackstone Life Sciences, and Novartis; and has spousal employment at Vertex, all unrelated to the present work. A.V.K. has served as a scientific advisor to Sanofi, Medicines Company, Maze Pharmaceuticals, Navitor Pharmaceuticals, Verve Therapeutics, Amgen, and Color; received speaking fees from Illumina, MedGenome, Amgen, and the Novartis Institute for Biomedical Research; received sponsored research agreements from the Novartis Institute for Biomedical Research and IBM Research; and reports a patent related to a genetic risk predictor (20190017119). C.J.W.’s spouse is employed at Regeneron. L.E.S. is currently an employee of Celgene/Bristol Myers Squibb. Celgene/Bristol Myers Squibb had no role in the funding, design, conduct, and interpretation of this study. M.E.M. receives funding from Regeneron unrelated to this work. E.E.K. has received speaker honoraria from Illumina, Inc and Regeneron Pharmaceuticals. B.M.P. serves on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. L.A.C. has consulted with the Dyslipidemia Foundation on lipid projects in the Framingham Heart Study. P.T.E. is supported by a grant from Bayer AG to the Broad Institute focused on the genetics and therapeutics of cardiovascular disease. P.T.E. has consulted for Bayer AG, Novartis, MyoKardia, and Quest Diagnostics. S.A.L. receives sponsored research support from Bristol Myers Squibb/Pfizer, Bayer AG, Boehringer Ingelheim, Fitbit, and IBM and has consulted for Bristol Myers Squibb/Pfizer, Bayer AG, and Blackstone Life Sciences. The views expressed in this article are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. M.I.M. has served on advisory panels for Pfizer, NovoNordisk, and Zoe Global and has received honoraria from Merck, Pfizer, Novo Nordisk, and Eli Lilly and research funding from Abbvie, Astra Zeneca, Boehringer Ingelheim, Eli Lilly, Janssen, Merck, NovoNordisk, Pfizer, Roche, Sanofi Aventis, Servier, and Takeda. As of June 2019, M.I.M. is an employee of Genentech and a holder of Roche stock. M.E.J. holds shares in Novo Nordisk A/S. H.M.K. is an employee of Regeneron Pharmaceuticals; he owns stock and stock options for Regeneron Pharmaceuticals. M.E.J. has received research grants form Astra Zeneca, Boehringer Ingelheim, Amgen, and Sanofi. S.K. is founder of Verve Therapeutics., (Copyright © 2021 American Society of Human Genetics. All rights reserved.)

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here, we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N = 242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N = 92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with eosinophilic esophagitis (N = 21) and B-cell lymphoma (N = 27) showed selective elevation of eosinophil and B-cell cfDNA, respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods., Competing Interests: IF, JM, JM, TK, BG, RS, YD has filed patents on cfDNA analysis technology, SP, BO, AK, AP, NL, DC, YD, NF, MM, MR, JC, MR, DL No competing interests declared, AJ, GC is an employee of GRAIL, (© 2021, Fox-Fisher et al.)

Background: The pancreatic β cell, as the sole source of the vital hormone insulin, has been under intensive study for more than a century. Given the potential of newly created insulin-producing cells as a treatment or even cure of type 1 diabetes (T1D) and possibly in severe cases of type 2 diabetes (T2D), multiple academic and commercial laboratories are working to derive surrogate glucose-responsive, insulin-producing cells., Scope of Review: The recent development of advanced phenotyping technologies, including molecular, epigenomic, histological, or functional, have greatly improved our understanding of the critical properties of human β cells. Using this information, here we summarize the salient features of normal, fully functional adult human β cells, and propose minimal criteria for what should rightfully be termed 'β cells' as opposed to insulin-producing but not fully-functional surrogates that we propose should be referred to as 'β-like' cells or insulin-producing cells., Major Conclusions: Clear criteria can be established to differentiate fully functional, mature β cells from 'β-like' surrogates. In addition, we outline important knowledge gaps that must be addressed to enable a greater understanding of the β cell., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Objective: While the molecular events controlling insulin secretion from β-cells have been documented in detail, the exact mechanisms governing glucagon release by α-cells are understood only partially. This is a critical knowledge gap, as the normal suppression of glucagon secretion by elevated glucose levels fails in type 2 diabetes (T2D) patients, contributing to hyperglycemia through stimulation of hepatic glucose production. A critical role of glycolytic flux in regulating glucagon secretion was supported by recent studies in which manipulation of the activity and expression of the glycolytic enzyme glucokinase altered the setpoint for glucose-suppression of glucagon secretion (GSGS). Given this precedent, we hypothesized that genetic activation of glucokinase specifically in α-cells would enhance GSGS and mitigate T2D hyperglucagonemia., Methods: We derived an inducible, α-cell-specific glucokinase activating mutant mouse model (Gck LoxPGck∗/LoxPGck∗ ; Gcg-CreER T2 ; henceforth referred to as "α-mutGCK") in which the wild-type glucokinase gene (GCK) is conditionally replaced with a glucokinase mutant allele containing the ins454A activating mutation (Gck∗), a mutation that increases the affinity of glucokinase for glucose by almost 7-fold. The effects of α-cell GCK activation on glucose homeostasis, hormone secretion, islet morphology, and islet numbers were assessed using both in vivo and ex vivo assays. Additionally, the effect of α-cell GCK activation on GSGS was investigated under diabetogenic conditions of high-fat diet (HFD) feeding that dysregulate glucagon secretion., Results: Our study shows that α-mutGCK mice have enhanced GSGS in vivo and ex vivo, independent of alterations in insulin levels and secretion, islet hormone content, islet morphology, or islet number. α-mutGCK mice maintained on HFD displayed improvements in glucagonemia compared to controls, which developed the expected obesity, glucose intolerance, elevated fasting blood glucose, hyperinsulinemia, and hyperglucagonemia., Conclusions: Using our novel α-cell specific activation of GCK mouse model, we have provided additional support to demonstrate that the glycolytic enzyme glucokinase is a key determinant in glucose sensing within α-cells to regulate glucagon secretion. Our results contribute to our fundamental understanding of α-cell biology by providing greater insight into the regulation of glucagon secretion through α-cell intrinsic mechanisms via glucokinase. Furthermore, our HFD results underscore the potential of glucokinase as a druggable target which, given the ongoing development of allosteric glucokinase activators (GKAs) for T2D treatment, could help mitigate hyperglucagonemia and potentially improve blood glucose homeostasis., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Hundreds of thousands of genetic variants have been reported to cause severe monogenic diseases, but the probability that a variant carrier develops the disease (termed penetrance) is unknown for virtually all of them. Additionally, the clinical utility of common polygenetic variation remains uncertain. Using exome sequencing from 77,184 adult individuals (38,618 multi-ancestral individuals from a type 2 diabetes case-control study and 38,566 participants from the UK Biobank, for whom genotype array data were also available), we apply clinical standard-of-care gene variant curation for eight monogenic metabolic conditions. Rare variants causing monogenic diabetes and dyslipidemias display effect sizes significantly larger than the top 1% of the corresponding polygenic scores. Nevertheless, penetrance estimates for monogenic variant carriers average 60% or lower for most conditions. We assess epidemiologic and genetic factors contributing to risk prediction in monogenic variant carriers, demonstrating that inclusion of polygenic variation significantly improves biomarker estimation for two monogenic dyslipidemias.

Aims/hypothesis: Acute hyperglycaemia stimulates pancreatic beta cell proliferation in the mouse whereas chronic hyperglycaemia appears to be toxic. We hypothesise that this toxic effect is mediated by increased beta cell workload, unrelated to hyperglycaemia per se., Methods: To test this hypothesis, we developed a novel mouse model of cell-autonomous increased beta cell glycolytic flux caused by a conditional heterozygous beta cell-specific mutation that activates glucokinase (GCK), mimicking key aspects of the rare human genetic disease GCK-congenital hyperinsulinism., Results: In the mutant mice, we observed random and fasting hypoglycaemia (random 4.5-5.4 mmol/l and fasting 3.6 mmol/l) that persisted for 15 months. GCK activation led to increased beta cell proliferation as measured by Ki67 expression (2.7% vs 1.5%, mutant and wild-type (WT), respectively, p < 0.01) that resulted in a 62% increase in beta cell mass in young mice. However, by 8 months of age, mutant mice developed impaired glucose tolerance, which was associated with decreased absolute beta cell mass from 2.9 mg at 1.5 months to 1.8 mg at 8 months of age, with preservation of individual beta cell function. Impaired glucose tolerance was further exacerbated by a high-fat/high-sucrose diet (AUC 1796 vs 966 mmol/l × min, mutant and WT, respectively, p < 0.05). Activation of GCK was associated with an increased DNA damage response and an elevated expression of Chop, suggesting metabolic stress as a contributor to beta cell death., Conclusions/interpretation: We propose that increased workload-driven biphasic beta cell dynamics contribute to decreased beta cell function observed in long-standing congenital hyperinsulinism and type 2 diabetes.

Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.

Background: Typical development of socio-communicative skills relies on keen observation of others. It thus follows that decreased social attention negatively impacts the subsequent development of socio-communicative abilities in children with autism spectrum disorders (ASD). In addition, studies indicate that social attention is modulated by context and that greater social difficulties are observed in more socially demanding situations. Our study aims to investigate the effect of social complexity on visual exploration of others' actions in preschoolers., Methods: To investigate the impact of social complexity, we used an eye-tracking paradigm with 26 typically developing preschoolers (TD, age = 3.60 ± 1.55) and 37 preschoolers with ASD (age = 3.55 ± 1.21). Participants were shown videos of two children engaging in socially simple play (parallel) versus socially complex play (interactive). We subsequently quantified the time spent and fixation duration on faces, objects, bodies, as well as the background and the number of spontaneous gaze shifts between socially relevant areas of interest., Results: In the ASD group, we observed decreased time spent on faces. Social complexity (interactive play) elicited changes in visual exploration patterns in both groups. From the parallel to the interactive condition, we observed a shift towards socially relevant parts of the scene, a decrease in fixation duration, as well as an increase in spontaneous gaze shifts between faces and objects though there were fewer in the ASD group., Limitations: Our results need to be interpreted cautiously due to relatively small sample sizes and may be relevant to male preschoolers, given our male-only sample and reported phenotypic differences between males and females., Conclusion: Our results suggest that similar to TD children, though to a lesser extent, visual exploration patterns in ASD are modulated by context. Children with ASD that were less sensitive to context modulation showed decreased socio-communicative skills or higher levels of symptoms. Our findings support using naturalistic designs to capture socio-communicative deficits in ASD.

Manure application to agricultural soils is widely considered as a source of nutrients and a method of maintaining levels of soil organic carbon (SOC) to mitigate climate change. At present, it is still unclear which factors are responsible for the SOC stock dynamics. Therefore, we analyzed the relationship between SOC stock changes and site characteristics, soil properties, experiment characteristics and manure characteristics. Overall, we included 101 studies with a total of 592 treatments. On average, the application of manure on agricultural soils increased SOC stocks by 35.4%, corresponding to 10.7 Mg ha -1 . Manure applications in conventional tillage systems led to higher SOC stocks (+ 2.2 Mg ha -1 ) than applications under reduced tillage. Soil organic carbon increase upon manure application was higher in soils under non-tropical climate conditions (+ 2.7 Mg ha -1 ) compared to soils under sub-tropical climate. Larger SOC increases after manure application were achieved in intermediate and shallow topsoils (in 0-15 cm by 9.5 Mg ha -1 and in 16-20 cm by 13.6 Mg ha -1 ), but SOC stocks were also increased in deeper soils (> 20 cm 4.6 Mg ha -1 ), regardless of the tillage intensity. The highest relative SOC increase (+ 48%) was achieved if the initial SOC was below 1% but the absolute SOC increased with increasing initial SOC. Clay soils showed higher SOC increase rates compared to sandy soils (+ 3.1 Mg ha -1 ). Acidic soils showed comparable relative effects but a higher stock difference than neutral (+ 5.1 Mg ha -1 ) and alkaline soils (+ 5.1 Mg ha -1 ). The application of farmyard-, cattle- and pig manure showed the highest SOC increases (50%, 32% and 41%, respectively), while green manure and straw showed only minor effects. If manure applications were combined with additional mineral fertilizer, the SOC increases were higher (+ 1.7 Mg ha -1 ) compared to manure alone. Higher applied amounts generally led to higher SOC stocks. However the annually applied amount is only important under conventional tillage, non-tropical climate conditions, and pH-neutral as well as SOC-rich or SOC-depleted soils and if no additional mineral fertilization is applied. Further studies should focus on the SOC dynamics under tropical climate conditions and factors influencing a potential carbon saturation. In both cases, the number of data was too small. For this reason, additional field studies should be conducted primarily in the tropics. On the other hand, long-term field trials should be re-assessed or newly established to specifically investigate potential saturation effects and long-term (> 20 years) fertilizer effects and carbon sequestration.

Diminished orienting to social stimuli, and particularly to faces, is a core feature of autism spectrum disorders (ASDs). Impaired face processing has been linked to atypical attention processes that trigger a cascade of pathological development contributing to impaired social communication. The aim of the present study is to explore the processing of emotional and neutral faces using an eye-tracking paradigm (the emotional faces task) with a group of 24 children with ASD aged 6 and under and a group of 22 age-matched typically developing (TD) children. We also measure habituation to faces in both groups based on the presentation of repeated facial expressions. Specifically, the task consists of 32 pairs of faces, a neutral face and an emotional face from the same identity, shown side by side on the screen. We observe differential exploration of emotional faces in preschoolers with ASD compared with TD. Participants with ASD make fewer fixations to emotional faces than their TD peers, and the duration of their first fixation on emotional faces is equivalent to their first fixation on neutral faces. These results suggest that emotional faces may be less interesting for children with ASD. We also observe a habituation process to neutral faces in both children with ASD and TD, who looked less at neutral faces during the last quarter of the task compared with the first quarter. By contrast, TD children show increased interest in emotional faces throughout the task, looking slightly more at emotional faces during the last quarter of the task than during the first quarter. Children with ASD demonstrate neither habituation nor increased interest in the changing emotional expressions over the course of the task, looking at the stimuli for equivalent time throughout the task. A lack of increased interest in emotional faces may suggest a lack of sensitivity to changes in expression in young children with ASD., 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 © 2020 Bochet, Franchini, Kojovic, Glaser and Schaer.)

Objective: Dedifferentiation of pancreatic β-cells may reduce islet function in type 2 diabetes (T2D). However, the prevalence, plasticity and functional consequences of this cellular state remain unknown., Methods: We employed single-cell RNAseq to detail the maturation program of α- and β-cells during human ontogeny. We also compared islets from non-diabetic and T2D individuals., Results: Both α- and β-cells mature in part by repressing non-endocrine genes; however, α-cells retain hallmarks of an immature state, while β-cells attain a full β-cell specific gene expression program. In islets from T2D donors, both α- and β-cells have a less mature expression profile, de-repressing the juvenile genetic program and exocrine genes and increasing expression of exocytosis, inflammation and stress response signalling pathways. These changes are consistent with the increased proportion of β-cells displaying suboptimal function observed in T2D islets., Conclusions: These findings provide new insights into the molecular program underlying islet cell maturation during human ontogeny and the loss of transcriptomic maturity that occurs in islets of type 2 diabetics., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

It has been proposed that unmethylated insulin promoter fragments in plasma derive exclusively from β cells, reflect their recent demise, and can be used to assess β cell damage in type 1 diabetes. Herein we describe an ultrasensitive assay for detection of a β cell-specific DNA methylation signature, by simultaneous assessment of 6 DNA methylation markers, that identifies β cell DNA in mixtures containing as little as 0.03% β cell DNA (less than 1 β cell genome equivalent). Based on this assay, plasma from nondiabetic individuals (N = 218, aged 4-78 years) contained on average only 1 β cell genome equivalent/mL. As expected, cell-free DNA (cfDNA) from β cells was significantly elevated in islet transplant recipients shortly after transplantation. We also detected β cell cfDNA in a patient with KATP congenital hyperinsulinism, in which substantial β cell turnover is thought to occur. Strikingly, in contrast to previous reports, we observed no elevation of β cell-derived cfDNA in autoantibody-positive subjects at risk for type 1 diabetes (N = 32), individuals with recent-onset type 1 diabetes (<4 months, N = 92), or those with long-standing disease (>4 months, N = 38). We discuss the utility of sensitive β cell cfDNA analysis and potential explanations for the lack of a β cell cfDNA signal in type 1 diabetes.

The hydrogen isotopic composition of leaf wax-derived n-alkanes (δ 2 H n-alkanes ) is a widely applied proxy for (paleo)climatic changes. It has been suggested that the coupling with the oxygen isotopic composition of hemicellulose-derived sugars (δ 18 O sugar ) - an approach dubbed 'paleohygrometer' - might allow more robust and quantitative (paleo)hydrological reconstructions. However, the paleohygrometer remains to be evaluated and tested regionally. In this study, topsoil samples from South Africa, covering extensive environmental gradients, are analysed. δ 2 H n-alkanes correlates significantly with the isotopic composition of precipitation (δ 2 H p ), whereas no significant correlation exists between δ 18 O sugar and δ 18 O p . The apparent fractionation (ε app ) is the difference between δ 2 H n-alkanes and δ 2 H p (ε app 2H ) and δ 18 O sugar and δ 18 O p (ε app 18O ), respectively, and integrates i) isotopic enrichment due to soil water evaporation, ii) leaf (and xylem) water transpiration and iii) biosynthetic fractionation. We find no correlation of ε app 18O nor for ε app 2H with temperature, and no correlation of ε app 2H with potential evapotranspiration and an aridity index. By contrast, ε app 18O correlates significantly with both potential evapotranspiration and the aridity index. This highlights the strong effect of evapotranspirative enrichment on δ 18 O sugar . In study areas without plant predominance using Crassulacean Acid Metabolism (CAM), coupling δ 18 O sugar and δ 2 H n-alkanes enables to reconstruct δ 2 H p and δ 18 O p with an offset of Δδ 2 H = 6 ± 27‰ and Δδ 18 O = 0.8 ± 3.7‰, respectively, as well as relative humidity (RH) with an offset of ΔRH = 6 ± 17%. The paleohygrometer does, however, not work well for our study areas where CAM plants prevail (reconstructed δ 18 O p , δ 2 H p and RH are off by 3.1‰, 27.2‰ and 31.7%). This probably reflects plant-specific (phenological) adaptations and/or post-photosynthetic exchange reactions related to CAM metabolism. Overall, our findings corroborate that δ 2 H n-alkanes and δ 18 O sugar are valuable proxies, and the paleohygrometer is a promising approach for paleoclimate reconstructions in southern Africa., Competing Interests: Declaration of competing interest PS, TH, MB, JS, BG, MZ and RZ declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)