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Microwave-assisted acid dissolution of minerals and inorganics is rapid, but difficulties in controlling the temperatures of such systems present potential safety problems. This drawback can be overcome by the use of specifically designed equipment which is, however, expensive. Dissolution under conventional heating, although slower, is inherently safer, temperature control being simple and reliable, as well as inexpensive. For laboratories with high sample loads, the importance of the dissolution time can be reduced significantly simply by heating a large number of samples simultaneously. For such systems, microwave heating holds little or no advantage over conventional heating. Presented here is a vessel designed specifically for this purpose. It is constructed of polyvinylidene difluoride, has a volume of 20 mL, and can be used up to 120 [degrees] C at 4 atm. This vessel has two major advantages: the use of conventional heating obviates the need for special (and generally expensive) ancillary equipment and the small size and relatively low cost facilitate heating large numbers of such vessels simultaneously. These vessels have been in routine use in these laboratories for the past 8 years. Forty of them are often heated together in a conventional laboratory oven.

An abstract of the article "Role of the Candida Albicans Copper Transporter CaCcc2 in Iron Acquisition," by Z. Weissman, R. Shemer, and D. Kornitzer is presented.

The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies 1 . Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events 1,2 . However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischaemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterioarterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Rapid neutrophil NETosis was identified as the main source of cell-free DNA after stroke and NET-DNA as the causative agent leading to AIM2 inflammasome activation. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischaemic events in patients with atherosclerosis. The detailed mechanisms uncovered here provide clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events., (© 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.)

Objective: To present a novel method that uses an epigenetic fingerprint to measure changes in plasma concentrations of cardiac-specific cell-free DNA (CS-cfDNA) as a marker of myocardial cell death., Methods: This prospective, analytic, observational comparative study included patients with heart disease or multiple risk factors for heart disease undergoing major noncardiac, mostly vascular surgery, requiring an arterial-line, and at least 24 h hospitalization in the post anaesthesia care unit or critical care unit after surgery. Blood samples were collected at least four times per patient to measure troponin-T (via high-sensitivity troponin-T test) and CS-cfDNA pre- and postoperatively., Results: A total of 117 patients were included (group 1, 77 patients [66%] with low preoperative and postoperative troponin-T; group 2, 18 patients [15%] with low preoperative but increased postoperative troponin-T; group 3, 16 patients [14%] with high troponin-T both preoperatively and postoperatively; and group 4, six patients [5%] with elevated preoperative troponin-T that decreased postoperatively). The increase in CS-cfDNA after surgery was statistically significant only in group 2, which correlated with an increase in troponin-T in the same group., Conclusions: CS-cfDNA increased early postoperatively, particularly in patients with silent postoperative troponin elevation, and was correlated with an increase in troponin-T. These results may suggest that, in the subgroup of patients with postoperative elevated troponin, cardiomyocyte death indeed occurred., Competing Interests: Declaration of conflicting interestThe authors declare that there are no conflicts of interest.

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).

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).)

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.)

Immune and inflammatory processes occurring within tissues are often undetectable by blood cell counts, standard circulating biomarkers, or imaging, representing an unmet biomedical need. Here, we outline recent advances indicating that liquid biopsies can broadly inform human immune system dynamics. Nucleosome-size fragments of cell-free DNA (cfDNA) released from dying cells into blood contain rich epigenetic information such as methylation, fragmentation, and histone mark patterns. This information allows to infer the cfDNA cell of origin, as well as pre-cell death gene expression patterns. We propose that the analysis of epigenetic features of immune cell-derived cfDNA can shed light on immune cell turnover dynamics in healthy people, and inform the study and diagnosis of cancer, local inflammation, infectious or autoimmune diseases, as well as responses to vaccination., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. 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.)

The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P < .0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/ml, respectively; P = .013). Increased c-cfDNA after 1 week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

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.)

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).)

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.)

The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy., (© 2022. The Author(s).)

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.)

Although it is known that the mutation rate varies across the genome, previous estimates were based on averaging across various numbers of positions. Here, we describe a method to measure the origination rates of target mutations at target base positions and apply it to a 6-bp region in the human hemoglobin subunit beta ( HBB ) gene and to the identical, paralogous hemoglobin subunit delta ( HBD ) region in sperm cells from both African and European donors. The HBB region of interest (ROI) includes the site of the hemoglobin S (HbS) mutation, which protects against malaria, is common in Africa, and has served as a classic example of adaptation by random mutation and natural selection. We found a significant correspondence between de novo mutation rates and past observations of alleles in carriers, showing that mutation rates vary substantially in a mutation-specific manner that contributes to the site frequency spectrum. We also found that the overall point mutation rate is significantly higher in Africans than in Europeans in the HBB region studied. Finally, the rate of the 20A→T mutation, called the "HbS mutation" when it appears in HBB , is significantly higher than expected from the genome-wide average for this mutation type. Nine instances were observed in the African HBB ROI, where it is of adaptive significance, representing at least three independent originations; no instances were observed elsewhere. Further studies will be needed to examine mutation rates at the single-mutation resolution across these and other loci and organisms and to uncover the molecular mechanisms responsible., (© 2022 Melamed et al.; Published by Cold Spring Harbor Laboratory Press.)

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.

Two variants at the APOL1 gene, encoding apolipoprotein L1, account for more than 70% of the increased risk for chronic kidney disease in individuals of African ancestry. While the initiating event for APOL1 risk variant cell injury remains to be clarified, we explored the possibility of blocking APOL1 toxicity at a more upstream level. We demonstrate that deletion of the first six amino acids of exon 4 abrogates APOL1 cytotoxicity by impairing APOL1 translocation to the lumen of ER and splicing of the signal peptide. Likewise, in orthologous systems, APOL1 lethality was partially abrogated in yeast strains and flies with reduced dosage of genes encoding ER translocon proteins. An inhibitor of ER to Golgi trafficking reduced lethality as well. We suggest that targeting the MSALFL sequence or exon 4 skipping may serve as potential therapeutic approaches to mitigate the risk of CKD caused by APOL1 renal risk variants., Competing Interests: KS and RS are inventors on Patent No.: US 10,927,414 B2. KS is an Associate Editor for The Kidney, 11th Edition, Elsevier 2020., (© 2022 The Authors.)

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 & Aims: Mature hepatocytes have limited expansion capability in culture and rapidly loose key functions. Recently however, tissue culture conditions have been developed that permit rodent hepatocytes to proliferate and transform into progenitor-like cells with ductal characteristics in vitro. Analogous cells expressing both hepatic and duct markers can be found in human cirrhotic liver in vivo and may represent an expandable population., Methods: An in vitro culture system to expand epithelial cells from human end stage liver disease organs was developed by inhibiting the canonical TGF-β, Hedgehog and BMP pathways., Results: Human cirrhotic liver epithelial cells became highly proliferative in vitro. Both gene expression and DNA methylation site analyses revealed that cirrhosis derived epithelial liver cells were intermediate between normal hepatocytes and cholangiocytes. Mouse hepatocytes could be expanded under the same conditions and retained the ability to re-differentiate into hepatocytes upon transplantation. In contrast, human cirrhotic liver derived cells had only low re-differentiation capacity., Conclusions: Epithelial cells of intermediate ductal-hepatocytic phenotype can be isolated from human cirrhotic livers and expanded in vitro. Unlike their murine counterparts they have limited liver repopulation potential., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.