Biology subjects

Moss, J.

Publications and source records attributed to Moss, J..

At least 19 recordsLinked to original sources

bioRxiv2025

The growth-promoting protein TMEM263 is an ER resident that controls early stages of lipid droplet biogenesis

Cell and organismal growth is controlled not only by the availability of nutrients, but also the ability to dynamically sense and respond to changes in metabolic demand. We have identified a protein, TMEM263 (also known as C12orf23) as a new mechanistic link between growth and lipid metabolism. TMEM263 was discovered in a screen as an ER-resident protein, and we have characterised its role as both necessary and sufficient for lipid droplet formation. TMEM263 has two transmembrane domains that fold into a hairpin structure which are essential for its localisation to the ER and to lipid droplets. Functionally, we propose that TMEM263 can interact with and support the condensation of neutral lipids in a bilayer to promote lipid droplet formation and growth. Consistently, loss of TMEM263 in cells and in zebrafish significantly impairs lipid droplet formation. Loss of TMEM263 protein function in vivo is associated with organismal growth defects and proportional dwarfism and our study provides a mechanistic understanding linking these phenotypes to impaired lipid droplet biology.

cell biology↗

bioRxiv2025

Prominent white matter related abnormalities associated with diverse molecular alterations in a mouse model of Col4a1 cerebral small vessel disease.

White matter abnormalities are a hallmark of cerebral small vessel disease (cSVD) and are closely linked to cognitive decline and dementia. However, despite their clinical importance, underlying mechanisms remain poorly understood. Collagen IV, encoded by genes COL4A1/COL4A2, is a major component of the basement membrane, a specialised extracellular matrix (ECM) structure. Mutations in these genes cause a genetic form of cSVD. We tested the hypothesis that ECM defects caused by a Col4a1 mutation lead to white matter pathology using an established mouse model of cSVD (Col4a1+/Svc). Behavioural testing with magnetic resonance diffusion tensor imaging, pathology and ultrastructural investigations of white matter were studied. The studies revealed that Col4a1+/Svc mice have cognitive impairments, reduced myelinating oligodendrocyte pools, axonal myelination defects, and altered white matter structural integrity. Proteomic analysis, of isolated white matter from Col4a1+/Svcmice, identified extensive changes to ECM composition and endoplasmic reticulum (ER) biology including ER stress induction. We also demonstrated that targeting protein folding to promote collagen secretion and reduce ER stress, increased myelinating oligodendrocytes and axon-glial integrity in Col4a1+/Svc mice. These data provide novel insight into the pathomolecular mechanisms of white matter abnormalities in cSVD and identify a modifiable pathway as a putative therapeutic target for cSVD.

neuroscience↗

bioRxiv2025

Multiregional blood-brain barrier phenotyping identifies the prefrontal cortex as the most vulnerable region to ageing in mice

Age-associated vascular alterations make the brain more vulnerable to neuropathologies. Research in humans and rodents have demonstrated structural, molecular, and functional alterations of the aged brain vasculature that suggest blood-brain barrier (BBB) dysfunction. However, these studies focused on particular features of the BBB and specific brain regions. Thus, it remains unclear if and which BBB age-associated phenotypes are conserved across brain areas. Moreover, there is very limited information about how BBB dysfunction and cell-specific phenotypes relate to each other. In this manuscript, we use immunofluorescence, transmission electron microscopy (TEM), and permeability assays to assess how age-associated BBB molecular, structural, and functional phenotypes correlate between the BBB cell types at three brain regions (prefrontal cortex, hippocampus, and corpus callosum) during mouse early ageing. We discovered that at 18-20 months of age, the mouse prefrontal cortex BBB is the most affected region, with alterations in brain endothelial cell protein expression, BBB permeability, basement membrane thickness, and astrocyte endfoot size when compared to young mice. Here, we deliver a detailed multicellular characterisation of region-dependent BBB changes at early stages of ageing. Our data paves the way for future studies to investigate how region-specific BBB dysfunction may contribute to disease-associated regional vulnerability.

neuroscience↗

bioRxiv2025

Methylation-Based Deconvolution Unveils Glioblastoma Heterogeneity and Cell-Type Composition Linked to Patient Survival

BackgroundIDH-wildtype glioblastoma (GBM) is an aggressive, heterogeneous brain tumor with limited treatment options. DNA methylation profiling allows detailed tumor characterization. This study applies methylation-based deconvolution to define GBMs cellular composition and its association with patient outcomes. MethodsWe generated oligodendroglial precursor cells at various developmental stages from enriched human neural progenitor cultures and used their DNA methylation signatures, along with published signatures of brain tumor and tumor microenvironment-relevant cell types, to deconvolve 263 adult GBMs (Heidelberg-cohort). Tumor purity was estimated using RF_Purify. An independent cohort of 199 GBMs from TCGA and GEO, all treated with standard-of-care therapy, was similarly deconvolved, followed by Kaplan-Meier survival analysis to assess the prognostic value of neoplastic component proportions. ResultsDeconvolution uncovered distinct cellular compositions, consistent with single-cell RNA sequencing findings. Tumor purity analysis showed neoplastic fractions averaged 70% of tumor bulk, predominantly oligodendrocyte-like (43%), oligodendrocyte precursor-like (27%), astrocyte-like (19%), and mesenchymal stem cell-like (11%) populations. Non-neoplastic fractions were enriched for macrophages, vascular cells, and immune populations. A higher oligodendrocyte-like signature was linked to poorer survival (median survival 14.3 vs. 15.3 months; p = 0.017), while a higher astrocyte-like signature correlated with improved outcomes (15.3 vs. 13.4 months; p = 0.044). The astrocyte-to-oligodendrocyte ratio emerged as a strong prognostic marker, with a higher ratio predicting significantly longer survival (15.8 vs. 11.9 months; p < 0.00011). ConclusionsMethylation-based deconvolution provides insight into GBM heterogeneity, highlighting the prognostic relevance of the astrocyte-to-oligodendrocyte ratio, which may guide personalized treatment strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/640603v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@942af0org.highwire.dtl.DTLVardef@18f2626org.highwire.dtl.DTLVardef@111a756org.highwire.dtl.DTLVardef@1194b_HPS_FORMAT_FIGEXP M_FIG A. We generated oligodendroglial precursor cells (OPs) at various developmental stages from enriched human neural progenitor cultures. A reference atlas of methylation signatures from 14 normal cell types, including the in vitro-generated cells, combined with published profiles of brain tumor and tumor microenvironment-relevant cell types, was applied to deconvolve 263 GBM samples based on their methylation profiles (Heidelberg cohort). Cell type proportions were analyzed with RF_Purify to estimate tumor purity and distinguish neoplastic from non-neoplastic components. B. An independent cohort of 199 GBMs from TCGA and GEO, with available clinical data and all treated with standard-of-care therapy, was similarly deconvolved based on methylation profiles. Kaplan-Meier survival analysis assessed the prognostic impact of cell type proportions, identifying the astrocyte-to-oligodendrocyte ratio as the most significant marker. (Created with BioRender.com) C_FIG Key pointsO_LIMethylation-based deconvolution incorporating in-vitro-derived oligodendrocyte precursor differentiation data reveals GBM heterogeneity C_LIO_LIGBMs neoplastic fraction is dominated by oligodendrocyte-lineage cells C_LIO_LIAstrocyte/oligodendrocyte ratio is a strong prognostic marker for patient survival C_LI Importance of the studyThis study advances neuro-oncology research by using methylation-based deconvolution to uncover the cellular heterogeneity of glioblastoma. By constructing a comprehensive reference atlas and identifying cell-type-specific methylation signatures, it provides prognostic insights into the distinct cellular compositions of glioblastoma. Combining deconvolution results with purity analysis enables the differentiation between neoplastic and non-neoplastic tumor components. This approach complements single-cell RNA sequencing while offering greater clinical applicability, as DNA methylation profiling can be performed on FFPE or fresh frozen samples, unlike the more costly and tissue-sensitive single-cell methods. Importantly, the identification of the astrocyte-to-oligodendrocyte ratio as a strong prognostic marker highlights key cellular determinants of glioblastoma survival. These findings deepen our understanding of glioblastoma biology and offer a practical tool for patient stratification.

cancer biology↗

bioRxiv2025

Characterizing organisms from three domains of life with universal primers from throughout the global ocean

We introduce the Global rRNA Universal Metabarcoding Plankton database (GRUMP), which consists of 1194 samples that were collected from 2003-2020 and cover extensive latitudinal and longitudinal transects, as well as depth profiles in all major ocean basins. DNA from unfractionated (> 0.2{micro}m) seawater samples was amplified using the 515Y/926R universal three- domain rRNA gene primers, simultaneously quantifying the relative abundance of amplicon sequencing variants (ASVs) from bacteria, archaea, eukaryotic nuclear 18S, and eukaryotic plastid 16S. Thus, the ratio between taxa in one sample is directly comparable to the ratio in any other GRUMP sample, regardless of gene copy number differences. This obviates a problem in prior global studies that used size-fractionation and different rRNA gene primers for bacteria, archaea, and eukaryotes, precluding comparisons across size fractions or domains. On average, bacteria contributed 71%, eukaryotes 19%, and archaea 8% to rRNA gene abundance, though eukaryotes contributed 32% at latitudes > 40{degrees}. GRUMP is publicly available on the Simons Collaborative Marine Atlas Project (CMAP), promoting the global comparison of marine microbial dynamics.

ecology↗

bioRxiv2024

Presynaptic filopodia form kinapses and modulate membrane mechanics for synchronous neurotransmission and seizure generation.

The structural stability of synapses directly contrasts with their functional plasticity. This conceptual dichotomy is explained by the assumption that all synaptic plasticity is generated via either electrical and/or biochemical signaling. Here, we challenge this dogma by revealing an activity-dependent presynaptic response that is physical in nature. We show that dynamic filopodia emerge during action potential discharge and transiently deform synaptic boutons to enhance connectivity. Filopodia generation requires neuronal activity, calcium and actin, and occurs in intact brain circuits and human brain. Mechanistically, their extension preserves synchronous neurotransmitter release by increasing presynaptic membrane tension. However, filopodia generation becomes maladaptive during dysregulated brain activity, exacerbating seizures in vivo. Therefore, we provide direct evidence that presynaptic mechanical forces determine the extent and timing of synaptic signals.

neuroscience↗

bioRxiv2024

Microdroplet screening rapidly profiles a biocatalyst to enable its AI-assisted engineering

Engineering enzymes for increased efficiency is key to enabling sustainable, green biocatalytic production processes in the chemical and pharmaceutical industries. This challenge can be tackled from two angles: by directed evolution, based on labour-intensive experimental testing of enzyme variant libraries, or by computational methods, where data-dependent algorithms relating sequence and function are used to predict biocatalyst improvements. Here, we combine both approaches into a two-week, low-cost workflow, in which ultra-high throughput screening of a library of imine reductases (IREDs) in microfluidic devices provides not only selected hits, but also long-read sequence data linked to fitness scores of >17 thousand enzyme variants. We demonstrate the engineering of an IRED for chiral amine synthesis by mapping its local fitness landscape in one go, ready to be used for interpretation and extrapolation by protein engineers with the help of machine learning (ML). We calculate position-dependent mutability and combinability scores of mutations and comprehensively illuminate a complex interplay of mutations driven by synergistic, often positively epistatic effects. When interpreted by easy-to-use regression and tree-based ML algorithms designed for random whole-gene mutagenesis data, 3-fold improved hits initially obtained from experimental screening are extrapolated further to give another order of magnitude improvement (23-fold in kcat) after testing only a handful of designed mutants. Predictions succeed in >80% of cases. The catalytic features discovered in one IRED are shown to be portable and confer activity on IREDs with [~]50% homology. Our campaigns yield biocatalytically efficient IREDs and are paradigmatic for future enzyme engineering efforts that rely on large sequence-function maps, profiling how a biocatalyst responds to mutation. In the age of predictive biology, these maps will chart the way to improved function by exploiting the synergy of rapid experimental screening combined with ML evaluation and extrapolation.

synthetic biology↗

bioRxiv2023

A PARP inhibitor, rucaparib, improves cardiac dysfunction in ADP-ribose-acceptor hydrolase 3 (Arh3) deficiency

AimsPatients with ADP-ribose-acceptor hydrolase 3 (ARH3) deficiency exhibit stress-induced childhood-onset neurodegeneration with ataxia and seizures (CONDSIAS). ARH3 degrades protein-linked poly(ADP- ribose) (PAR) synthesized by poly(ADP-ribose)polymerase (PARP)-1 during oxidative stress, leading to cleavage of the ADP-ribose linked to protein. ARH3 deficiency leads to excess accumulation of PAR, resulting in PAR-dependent cell death or parthanatos. Approximately one-third of patients with homozygous mutant ARH3 die from cardiac arrest, which has been described as neurogenic, suggesting that ARH3 may play an important role in maintaining myocardial function. To address this question, cardiac function was monitored in Arh3-knockout (KO) and - heterozygous (HT) mice. Methods and resultsArh3-KO male mice displayed cardiac hypertrophy by histopathology and decreased cardiac contractility assessed by MRI. In addition, both genders of Arh3-KO and -HT mice showed decreased cardiac contractility by dobutamine stress test assessed by echocardiography. A direct role of ARH3 on myocardial function was seen with a Langendorff-perfused isolated heart model. Arh3-KO male mouse hearts showed decreased post-ischemic rate pressure products, increased size of ischemia-reperfusion (IR) infarcts, and elevated PAR levels. Consistently, in vivo IR injury showed enhanced infarct size in Arh3-KO mice in both genders. In addition, Arh3-HT male mice showed increased size of in vivo IR infarcts. Treatment with an FDA-approved PARP inhibitor, rucaparib, improved cardiac contractility during dobutamine-induced stress and exhibited reduced size of in vivo IR infarcts. To understand better the role of ARH3, CRISPR-Cas9 was used to generate different Arh3 genotypes of myoblasts and myotubes. Incubation with H2O2 decreased viability of Arh3-KO and -HT myoblasts and myotubes, resulting in PAR-dependent cell death that was reduced by PARP inhibitors or by transfection with the Arh3 gene. ConclusionARH3 regulates PAR homeostasis in myocardium to preserve function and protect against oxidative stress; PARP inhibitors reduce the myocardial dysfunction seen with Arh3 mutations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/527369v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@a76514org.highwire.dtl.DTLVardef@1dd3184org.highwire.dtl.DTLVardef@ddfd26org.highwire.dtl.DTLVardef@16e0cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

ADP-ribose-acceptor hydrolase 2 (Arh2) deficiency results in cardiac dysfunction, tumorigenesis, inflammation, and decreased survival

ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2-knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2-KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2-Het mice showed results similar to those seen in Arh2-KO mice except for cardiomegaly. Arh2-KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2-Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2-KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.

cancer biology↗

bioRxiv2023

Macrodomain Mac1 of SARS-CoV-2 Nonstructural Protein 3 Hydrolyzes Diverse ADP-ribosylated Substrates

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. ADP-ribosylation results from the transfer of the ADP-ribose moiety of NAD+ to various acceptors, e.g., proteins, DNA, RNA, contributing on a cells biological processes. ADP-ribosylation is the mechanism of action of bacterial toxins, e.g., Pseudomonas toxins, diphtheria toxin that disrupt protein biosynthetic and signaling pathways. On the other hand, some viral macrodomains cleavage ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved -NAD+, but not {beta}-NAD+, consistent with stereospecificity at the C-1" bond. In contrast to ARH1 and ARH3, Mac1 did not require Mg2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1"-phosphat, but not Mac2 and Mac3. However, Mac1 did not cleave -ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 activities may shed light on potential SARS-CoV-2 therapeutic targets. IMPORTANCESARS-CoV-2, the virus responsible for COVID-19, encodes 3 macrodomain-containing proteins, e.g., Mac1, Mac2, Mac3, within non-structural proteins 3 (Nsp3). Mac1 was shown previously to hydrolyze ADP-ribose-phosphate. Inactivation of Mac1 reduced viral proliferation. Here we report that Mac1, but not Mac2 and Mac3, has multiple activities, i.e., Mac1 hydrolyzed. -NAD+ and O-acetyl-ADP-ribose. However, Mac1 did not hydrolyze {beta}-NAD+, ADP-ribose-serine on a histone 3 peptide (aa1-21), and ADP-ribose-arginine, exhibiting substrate selectivity. These data suggest that Mac1 may have multi-function as a -NAD+ consumer for viral replication and a disruptor of host-mediated ADP-ribosylation pathways. Understanding Mac1s mechanisms of action is important to provide possible therapeutic targets for COVID-19.

biochemistry↗

bioRxiv2023

Mono-ADP-ribosyltransferase 1 (Artc1)-deficiency decreases tumorigenesis, increases inflammation, decreases cardiac contractility, and reduces survival

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD+ to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1-KO and Artc1/Arh1-double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF-. In a xenograft model using tumorigenic Arh1-KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1-KO and heterozygous recipient mice, with tumor infiltration by CD8+ T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1-double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1-KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1-KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1-KO was less than their Artc1-WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/527366v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@1cded54org.highwire.dtl.DTLVardef@9851ceorg.highwire.dtl.DTLVardef@22cd8aorg.highwire.dtl.DTLVardef@e42e7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Loss of genital epithelial barrier function is greater with depot-medroxyprogesterone acetate than intravaginal rings that release etonogestrel and ethinyl estradiol

The injectable progestin depot-medroxyprogesterone acetate (DMPA) is a popular contraceptive choice in sub-Saharan Africa although mouse models indicate it weakens genital epithelial integrity and barrier function and increases susceptibility to genital infection. The intravaginal ring NuvaRing(R) is another contraceptive option that like DMPA suppresses hypothalamic pituitary ovarian (HPO) axis function with local release of progestin (etonogestrel) and estrogen (ethinyl estradiol). As we previously reported that treating mice with DMPA and estrogen averts the loss of genital epithelial integrity and barrier function induced by DMPA alone, in the current investigation we compared genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and genital epithelial permeability in rhesus macaques (RM) treated with DMPA or a NuvaRing(R)re-sized for RM (N-IVR). While these studies demonstrated comparable inhibition of the HPO axis with DMPA or N-IVR, DMPA induced significantly lower genital DSG1 levels and greater tissue permeability to intravaginally administered low molecular mass molecules. By identifying greater compromise of genital epithelial integrity and barrier function in RM administered DMPA vs. N-IVR, our results add to the growing body of evidence that indicate DMPA weakens a fundamental mechanism of anti-pathogen host defense in the female genital tract.

immunology↗

bioRxiv2022

Multi-omic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation

Over 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, there remains scope to improve upon the therapeutic window of the current generation of AKT inhibitors. Here we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-driven phenotypes in breast cancer cell lines. A systematic growth inhibition screen across 288 cancer cell lines confirmed a substantially higher potency for INY-05-040 (median GI50adj = 1.1 {micro}M) compared to our first-generation AKT degrader (INY-03-041; median GI50adj = 3.1 {micro}M), with both compounds outperforming catalytic AKT inhibition with GDC-0068 (median GI50adj > 10 {micro}M). Using multi-omic profiling and causal network integration in breast cancer cells, we demonstrate that the enhanced efficacy of INY-05-040 is associated with sustained suppression of AKT signaling, followed by a potent induction of the stress mitogen activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low baseline JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Collectively, our study presents a systematic framework for mapping the network-wide signaling effects of therapeutically relevant compounds, and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

cancer biology↗

bioRxiv2022

Megakaryocyte and erythroblast DNA in plasma and platelets

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 determined 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 discovered 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 novel biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/510502v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@164e8borg.highwire.dtl.DTLVardef@7eb5e1org.highwire.dtl.DTLVardef@d6ff73org.highwire.dtl.DTLVardef@11cf846_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2022

Development of methylation-based biomarkers for breast cancer detection by model training and validation in synthetic cell-free DNA

Circulating tumour-derived DNA (ctDNA) carries the genetic and epigenetic characteristics of the tumour from which it is derived and can give information about the biology and tissue origins of the underlying tumour. DNA methylation is an epigenetic mark that is specific to individual tissues and, as methylation profiles are disrupted in tumours, they can indicate the tissue of origin and cancer type of ctDNA. We have developed a set of methylation biomarkers for detecting breast cancer in plasma cell-free DNA (cfDNA). First, we mined publicly available methylation datasets to create synthetic methylation profiles that were modelled to reflect cfDNA from healthy subjects and cancer patients. These profiles were restricted to the most differentially methylated CpGs between breast tumour samples and haematopoietic cells. Regularised logistic regression models were trained using 10-fold cross-validation on synthetic cfDNA datasets with distinct fractions of breast tumour DNA spiked in silico into healthy cfDNA with the addition of 10% of a mix of different tissues. Initial validation with synthetic cfDNA permitted detection of breast cancer-derived DNA with as little as 0.25% tumour DNA spiked in silico into healthy subject cfDNA with an area under ROC curve (AUC) of 0.63. Performances of classifiers increased with increased fractions of spike-in tumour DNA (AUCs 0.77 and 0.93 at tumour DNA fractions 0.5% and 1% respectively). We then combined the most discriminative CpG markers from our models with methylation markers of breast cancer that had already been published to obtain a single marker set. In vitro testing of MCF-7 breast cancer cell line DNA spiked into leukocyte DNA showed highly significant correlation for individual markers between laboratory-measured and published methylation data for MCF-7 and leukocytes (R > 0.89, P < 2.2 x 10-16). These preliminary data indicate promising results for detection of breast cancer cell line DNA using this methylation marker set, which now require testing in cfDNA from breast cancer patients and healthy controls.

cancer biology↗

bioRxiv2022

A human DNA methylation atlas reveals principles of cell type-specific methylation and identifies thousands of cell type-specific regulatory elements

DNA methylation is a fundamental epigenetic mark that governs chromatin organization, cell identity, and gene expression. 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 207 healthy tissue samples. Replicates of the same cell-type are >99.5% identical, demonstrating robustness of cell identity programs to genetic variation and environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny, and identifies methylation patterns retained since gastrulation. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hyper-methylated loci are rare and are enriched for CpG islands, polycomb targets, and CTCF binding sites, suggesting a novel role in shaping cell type-specific chromatin looping. The atlas provides an essential resource for interpretation of disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies. Summary paragraphDNA methylation, a fundamental epigenetic mark, governs chromatin organization and gene expression1, thus defining the molecular identity of cells and providing a window into developmental processes with wide-ranging physiologic and clinical ramifications. Current DNA methylation datasets have limitations, typically including only a fraction of methylation sites, many from cell lines that underwent massive changes in culture or from tissues containing unspecified mixtures of cells2-6. We present a human methylome atlas based on deep whole-genome bisulfite sequencing of 39 sorted, primary cell types and use this dataset to address fundamental questions in developmental biology, physiology and pathology. Biological replicates are >99.5% identical, demonstrating unappreciated robustness to genetic variation and environmental perturbations. Clustering recapitulates key elements of tissue ontogeny, identifying methylation patterns retained since gastrulation. Loci uniquely unmethylated in individual cell types identify novel transcriptional enhancers and are enriched for tissue-specific transcription factors binding motifs. In contrast, loci uniquely hyper-methylated in specific cell types are rare, enriched for CpG islands and polycomb targets, and overlap CTCF binding sites, suggesting a novel role in shaping cell-type-specific chromatin looping. Finally, the atlas facilitates fragment-level deconvolution of tissue and plasma methylomes across thousands of cell-type specific regions to quantify their individual components at unprecedented resolution. The human cell-type-specific methylation atlas provides an essential resource for studying gene regulation by defining cell-type-specific distal enhancers and regulators of 3D organization, for identifying pathological changes in DNA methylation, and for the interpretation of methylation-based liquid biopsies. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY O_LIA deep methylation atlas of 39 human cell types, sorted from healthy samples C_LIO_LIMethylomes record developmental history of cells C_LIO_LIThousands of novel cell type-specific methylation markers C_LIO_LIHypo-methylation uncovers cell type-specific regulatory map of distal enhancers C_LIO_LIHyper-methylation across CTCF sites C_LIO_LICell type-specific biomarkers facilitate fragment-level deconvolution of tissues and cfDNA C_LI

genomics↗

bioRxiv2021

An intrinsic endothelial dysfunction causes cerebral small vessel disease

Small Vessel Disease (SVD) is the leading cause of vascular dementia, causes a quarter of strokes, and worsens stroke outcomes(1, 2). The disease is characterised by cerebral small vessel and white matter pathology, but the underlying mechanisms are poorly understood. Classically, the microvascular and tissue damage has been considered secondary to extrinsic factors, such as hypertension, consisting of microvessel stiffening, impaired vasoreactivity and blood-brain barrier dysfunction identified in human sporadic SVDs. However, increasing evidence points to an underlying vulnerability to SVD-related brain damage, not just extrinsic factors. Here, in a novel normotensive transgenic rat model where the phospholipase flippase Atp11b is deleted, we show pathological, imaging and behavioural changes typical of those in human sporadic SVD, but that occur without hypertension. These changes are due to an intrinsic endothelial cell dysfunction, identified in vessels of the brain white matter and the retina, with pathological evidence of vasoreactivity and blood-brain barrier deficits, which precipitate a secondary maturation block in oligodendroglia and myelin disruption around the small vessels. This highlights that an intrinsic endothelial dysfunction may underlie vulnerability to human sporadic SVD, providing alternative therapeutic targets to prevent a major cause of stroke and dementia.

neuroscience↗

bioRxiv2021

ETV2 regulates PARP-1 binding protein to induce ER stress-mediated cell death in tuberin-deficient cells

Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes (Tsc1 or Tsc2), and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). The effectiveness of mTORC1 inhibitors is limited by their lack of cytotoxic effects. Here, we report that E26 transformation specific (ETS) Variant Transcription Factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. Nuclear localization of ETV2 in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP), a coregulator of transcription, mRNA and protein expression. Silencing of ETV2 or PARPBP in Tsc2-deficient cells induced ER-stress and increased cell death in vitro and in vivo. We also found ETV2 expression in human cells with loss of heterozygosity for TSC2 lending support to the translational relevance of our findings. In conclusion, we report a novel signaling axis unique to Syk-inhibition is mTORC1-independent and promotes a cytocidal response in Tsc2-deficient cells, and therefore, maybe a potential alternative therapeutic target in LAM.

cell biology↗