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Riva, A.

Publications and source records attributed to Riva, A..

At least 19 recordsLinked to original sources
bioRxiv2026

Vertical Sleeve Gastrectomy inhibits 11βHSD1 and subsequently reduces IL6 secretion in Mice and Humans: A Shared Anti-Inflammatory Mechanism

BackgroundVertical sleeve gastrectomy (VSG) improves glycaemic control in type 2 diabetes (T2D) through mechanisms that extend beyond weight loss. The interaction between glucocorticoid metabolism and inflammation in this context remains unclear. MethodsWe investigated the role of 11{beta}-hydroxysteroid dehydrogenase type 1 (11{beta}HSD1) in mediating the metabolic effects of VSG in humans and mice. Subcutaneous adipose tissue biopsies were collected before and 6 months after VSG. Parallel studies were conducted in lean and high-fat diet-fed mice undergoing VSG or sham surgery, alongside 11{beta}HSD1 knockout models. Glucose tolerance and expression of 11{beta}HSD1 and interleukin-6 (IL6) were assessed. Mechanistic interactions were examined in IL6-treated human hepatocytes. ResultsVSG reduced 11{beta}HSD1 and IL6 expression in human adipose tissue and improved insulin resistance. In lean mice, VSG improved glucose tolerance and downregulated both markers independently of weight loss. 11{beta}HSD1 knockout mice exhibited improved glucose tolerance despite increased adiposity, partially recapitulating the VSG phenotype. Both interventions reduced circulating and tissue IL6 levels. IL6 stimulation increased HSD11B1 expression in hepatocytes. Conclusions11{beta}HSD1 links glucocorticoid metabolism, inflammation, and glucose homeostasis following VSG. Targeting this pathway may offer a strategy to replicate key metabolic benefits of metabolic bariatric surgery.

physiology↗
bioRxiv2024

Broad diversity of human gut bacteria accessible via a traceable strain deposition system

Numerous bacteria in the human gut microbiome remain unknown and/or have yet to be cultured. While collections of human gut bacteria have been published, few strains have been made publicly available. A major hurdle in making strains publicly available is their deposition to public culture collections. We propose a framework for the bulk-deposition of strains to culture collections, which removes many of the barriers previously identified (www.dsmz.de/bulk-deposit). Using this bulk-deposition system we have created a publicly available collection of human gut isolates. The Human intestinal Bacteria Collection (HiBC) (www.hibc.rwth-aachen.de) contains 340 strains representing 198 species within 29 families and 7 phyla, of which 29 previously unknown species are taxonomically described and named. These included two butyrate-producing species of Faecalibacterium and new dominant species associated with health and inflammatory bowel disease, Ruminococcoides intestinale and Blautia intestinihominis, respectively. Plasmids were prolific within the HiBC isolates, with almost half (46%) of strains containing plasmids, with a maximum of six within a strain. This included a broadly occurring plasmid (pBAC) that exists in three diverse forms across Bacteroidales species. Megaplasmids were identified within two strains, the pMMCAT megaplasmid is globally present within multiple Bacteroidales species. This collection of easily searchable and publicly available gut bacterial isolates will facilitate functional studies of the gut microbiome.

microbiology↗
bioRxiv2024

Effects of SGLT2 Ablation or Inhibition on Corticosterone Secretion in High-Fat-Fed Mice: Exploring a Nexus with Cytokine Levels

Despite recent therapeutic advances, achieving optimal glycaemic control remains a challenge in managing Type 2 Diabetes (T2D). Sodium-glucose co-transporter type 2 (SGLT2) inhibitors have emerged as effective treatments by promoting urinary glucose excretion. However, the full scope of their mechanisms extends beyond glycaemic control. At present, their immunometabolic effects remain elusive. To investigate the effects of SGLT2 inhibition or deletion, we compared the metabolic and immune phenotype between high fat diet-fed control, chronically dapagliflozin-treated mice and total-body SGLT2/Slc5a2 knockout mice. SGLT2 null mice exhibited superior glucose tolerance and insulin sensitivity compared to control or dapagliflozin-treated mice, independent of glycosuria and body weight. Moreover, SGLT2 null mice demonstrated physiological regulation of corticosterone secretion, with lowered morning levels compared to control mice. Systemic cytokine profiling also unveiled significant alterations in inflammatory mediators, particularly interleukin 6 (IL-6). Furthermore, unbiased proteomic analysis demonstrated downregulation of acute-phase proteins and upregulation of glutathione-related proteins, suggesting a role in the modulation of antioxidant responses. Conversely, IL-6 increased SGLT2 expression in kidney HK2 cells suggesting a role for cytokines in the effects of hyperglycemia. Collectively, our study elucidates a potential interplay between SGLT2 activity, immune modulation, and metabolic homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/590099v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@140041forg.highwire.dtl.DTLVardef@1ceb1b7org.highwire.dtl.DTLVardef@f575b1org.highwire.dtl.DTLVardef@101d5b4_HPS_FORMAT_FIGEXP M_FIG C_FIG Article HighlightsO_LIThe role of Sodium-glucose co-transporter type 2 (SGLT2) in immunity regulation remains elusive, despite extensive research in SGLT2 inhibitors. C_LIO_LIWe sought to discern the effects of SGLT2 inhibition or deletion on metabolic and immune profiles in high-fat-fed mice, focussing on corticosterone regulation and cytokine alterations. C_LIO_LISGLT2 null mice exhibit enhanced insulin sensitivity, alongside physiologically regulated corticosterone levels and significant alterations in inflammatory cytokines, and we identified changes in protein expression suggestive of antioxidant modulation. C_LIO_LIOur findings emphasize the interplay between immune responses and metabolic regulation mediated by SGLT2 activity. C_LI

cell biology↗
bioRxiv2024

KDM6A Regulates Immune Response Genes in Multiple Myeloma

The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration. Statement of significanceWe show that KDM6A participates in immune recognition of myeloma tumor cells by directly regulating the expression of the master regulators of MHC-I and II, NLRC5 and CIITA. The expression of these regulators can by rescued by the HDAC3 inhibitors in KDM6A-null cell lines.

cancer biology↗
bioRxiv2023

Patterns of subregional cerebellar atrophy across epilepsy syndromes: An ENIGMA-Epilepsy study

ObjectiveThe intricate neuroanatomical structure of the cerebellum is of longstanding interest in epilepsy, but has been poorly characterized within the current cortico-centric models of this disease. We quantified cross-sectional regional cerebellar lobule volumes using structural MRI in 1,602 adults with epilepsy and 1,022 healthy controls across twenty-two sites from the global ENIGMA-Epilepsy working group. MethodsA state-of-the-art deep learning-based approach was employed that parcellates the cerebellum into 28 neuroanatomical subregions. Linear mixed models compared total and regional cerebellar volume in i) all epilepsies; ii) temporal lobe epilepsy with hippocampal sclerosis (TLE-HS); iii) non-lesional temporal lobe epilepsy (TLE-NL); iv) genetic generalised epilepsy; and (v) extra-temporal focal epilepsy (ETLE). Relationships were examined for cerebellar volume versus age at seizure onset, duration of epilepsy, phenytoin treatment, and cerebral cortical thickness. ResultsAcross all epilepsies, reduced total cerebellar volume was observed (d=0.42). Maximum volume loss was observed in the corpus medullare (dmax=0.49) and posterior lobe grey matter regions, including bilateral lobules VIIB (dmax= 0.47), Crus I/II (dmax= 0.39), VIIIA (dmax=0.45) and VIIIB (dmax=0.40). Earlier age at seizure onset ({eta}{rho}2max=0.05) and longer epilepsy duration ({eta}{rho}2max=0.06) correlated with reduced volume in these regions. Findings were most pronounced in TLE-HS and ETLE with distinct neuroanatomical profiles observed in the posterior lobe. Phenytoin treatment was associated with reduced posterior lobe volume. Cerebellum volume correlated with cerebral cortical thinning more strongly in the epilepsy cohort than in controls. SignificanceWe provide robust evidence of deep cerebellar and posterior lobe subregional grey matter volume loss in patients with chronic epilepsy. Volume loss was maximal for posterior subregions implicated in non-motor functions, relative to motor regions of both the anterior and posterior lobe. Associations between cerebral and cerebellar changes, and variability of neuroanatomical profiles across epilepsy syndromes argue for more precise incorporation of cerebellum subregions into neurobiological models of epilepsy. Key pointsO_LICerebellar involvement in epilepsy is poorly understood within current cortico-centric models of this disease C_LIO_LIWe used a novel, deep learning segmentation tool to parcellate the cerebellum into 28 anatomical subunits using an international MRI dataset of 1,602 individuals with epilepsy (aged between 18 and 65 years old) including temporal lobe epilepsy with hippocampal sclerosis (TLE-HS, n=562), TLE non-lesional (TLE-NL, n=284), generalised genetic epilepsy (GGE, n=186) and extra temporal focal epilepsy (ETLE, n=251) and 1,022 controls. C_LIO_LIAcross all epilepsies (vs. controls) robust changes in the corpus medullare and posterior lobe "non-motor" regions were observed, with maximal differences in bilateral VIIB and Crus II lobules. Lower volume of these regions correlated with longer disease duration. Anterior "motor lobe" regions were relatively spared. C_LIO_LIFindings were most pronounced in TLE-HS and ETLE groups, with distinct neuroanatomical profiles observed. C_LIO_LICortical thinning was associated with pronounced cerebellar volume loss in TLE-HS epilepsy, relative to controls. C_LI ETHICAL PUBLICATION STATEMENTWe confirm that we have read the Journals position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

neuroscience↗
bioRxiv2023

Heterogeneous Nuclear Ribonucleoprotein C is an Indispensable Target in Acute Myeloid Leukemia

Refractory disease is the greatest challenge in acute myeloid leukaemia (AML). Previously, we found vascular-associated AML cells as a source for refractory disease. In this study we use a bioassay of AML cells co-cultured on bone marrow-derived endothelial cells (BMECs) to screen 31 million compounds within a mixture-based synthetic combinatorial library followed by deconvolution leading to the identification of a novel polyamine sulfonamide that is selectively toxic to AML. Using three distinct proteomics methods we identified heterogeneous nuclear ribonucleoprotein C (HNRNPC) as the compound target. Depleting HNRNPC reduces AML cell proliferation, viability, colony formation and fails to establish AML in mice. In contrast, no changes in cell proliferation, survival, or colony outgrowth were observed when depleting HNRNPC in normal hematopoietic or stromal cells. Clinical data show that lower HNRNPC expression independently associates with prolonged survival times in 554 adult and pediatric AML patients. We show that HNRNPC regulates DNA replication initiation and the Myc transcriptional program. Using differential isoform expression and differential alternative splicing analyses, we show significant alternative splicing of MAX after HNRNPC depletion, resulting in MAX transcript isoforms that lack Myc-interacting domains, which we term non-activating MAX isoforms. These Myc-interacting domains are necessary for the obligate heterodimerization of Myc and Max and are critical for Myc transcriptional activation. We further analyzed the alternative splicing landscapes of 538 AML patients and 33 specimens from healthy individuals and show substantial alternative splicing of mRNA in AML patients despite the absence of splicing factor gene mutations. Moreover, we found significantly high expression of non-activating MAX isoforms in healthy individuals, while AML patients highly expressed fully functional MAX isoforms, suggesting that MAX splicing may serve an important functional role in AML. Together, these data show HNRNPC as an indispensable splicing repressor in AML that is necessary for supporting deregulated Myc-Max activity and suggest HNRNPC as a potential therapeutic target for AML.

cancer biology↗
bioRxiv2023

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. AR KO reduced levels of H3.3 at enhancers, indicating feedback mechanism. In addition, HIRA KO deregulates glucocorticoid-driven transcription, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity. Key points*H3.3 at enhancers promotes acetylation of H3K27Ac and retention of AR/BRD4 complex for transcription regulation *Knockout of H3.3 chaperone HIRA suppresses PC cells growth and deregulates androgen-induced transcription *H3.3/HIRA pathway regulates both AR and GR, suggesting a common HIRA/H3.3 mechanism of nuclear receptors function

molecular biology↗
bioRxiv2022

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer with experimental CH driven by Dnmt3a+/{Delta}, we found higher tumor penetrance and increased tumor burden compared to controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency. This study provides conceptually novel insights into non-tumor-cell-autonomous effect of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies. SUMMARYA pre-clinical mouse model demonstrates that genetic alterations in the blood system characteristic of clonal hematopoiesis (CH) contribute to an aggressive solid tumor phenotype. It further identifies cancer angiogenesis as a potential therapeutic target to mitigate adverse CH effects.

cancer biology↗
bioRxiv2022

Flap-enabled next-generation capture (FENGC): precision targeted single-molecule profiling of epigenetic heterogeneity, chromatin dynamics, and genetic variation

Targeted sequencing is an increasingly sought technology. Available methods, however, are often costly and yield high proportions of off-target reads. Here, we present FENGC, a scalable, multiplexed method in which target sequences are assembled into 5' flaps for precise excision by flap endonuclease. Recovery of length-matched sequences, amplification with universal primers, and exonucleolytic removal of non-targeted genomic regions mitigate amplification biases and consistently yield [≥] 80% on-target sequencing. Furthermore, optimized sequential reagent addition and purifications minimize sample loss and facilitate rapid processing of sub-microgram quantities of DNA for detection of genetic variants and DNA methylation. Treatment of cultured human glioblastoma cells and primary murine monocytes with GC methyltransferase followed by FENGC and high-coverage enzymatic methyl sequencing provides single-molecule, long-read detection of differential endogenous CG methylation, dynamic nucleosome repositioning, and transcription factor binding. FENGC provides a versatile and cost-effective platform for targeted sequence enrichment for analysis of genetic and/or epigenetic heterogeneity.

cancer biology↗
bioRxiv2022

HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis

Melanomas are heterogeneous and adopt multiple transcriptional states that can confer an invasive phenotype and resistance to therapy. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity and tumor progression. Here we identify stress-induced HDAC8 activity as the driver of a neural crest stem cell (NCSC)-like transcriptional state that increased the formation of melanoma brain metastases (MBM). Exposure of melanocytes and melanoma cells to multiple different stresses led to HDAC8 activation, a switch to a NCSC gene expression signature and the adoption of an amoeboid, invasive phenotype. This cell state enhanced the survival of melanoma cells under shear stress conditions and increased the formation of metastases in the brain. scRNA-seq analyses showed that HDAC8 expression was correlated with the NCSC cell state in clinical MBM specimens. ATAC-Seq and ChIP-Seq analysis showed HDAC8 to alter chromatin structure by increasing H3K27ac and accessibility at c-Jun binding sites without changing global histone acetylation. The increased accessibility of Jun binding sites was paralleled by decreased H3K27ac and accessibility at MITF binding sites and loss of melanoma-lineage gene expression. Mass spectrometry-based acetylomics demonstrated that HDAC8 deacetylated the histone acetyltransferase (HAT) EP300 leading to its enzymatic inactivation. This, in turn, led to an increased binding of EP300 to Jun-transcriptional sites and decreased binding to MITF-transcriptional sites. Increased expression of EP300 decreased invasion and increased the sensitivity of melanoma cells to multiple stresses while inhibition of EP300 function increased invasion and resistance to stress. We identified HDAC8 as a novel mediator of transcriptional co-factor inactivation and chromatin accessibility that increases MBM development.

cancer biology↗
bioRxiv2022

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

immunology↗
bioRxiv2022

Phylogenetic-informed graph deep learning to classify dynamic transmission clusters in infectious disease epidemics

In the midst of an outbreak, identification of groups of individuals that represent risk for transmission of the pathogen under investigation is critical to public health efforts. Several approaches exist that utilize the evolutionary information from pathogen genomic data derived from infected individuals to distinguish these groups from the background population, comprised of primarily randomly sampled individuals with undetermined epidemiological linkage. These methods are, however, limited in their ability to characterize the dynamics of these groups, or clusters of transmission. Dynamic transmission patterns within these clusters, whether it be the result of changes at the level of the virus (e.g., infectivity) or host (e.g., vaccination implementation), are critical in strategizing public health interventions, particularly when resources are limited. Phylogenetic trees are widely used not only in the detection of transmission clusters, but the topological shape of the branches within can be useful sources of information regarding the dynamics of the represented population. We evaluate the limitation of existing tree shape statistics when dealing with smaller sub-trees containing transmission clusters and offer instead a phylogeny-based deep learning system -DeepDynaTree- for classification of transmission cluster. Comprehensive experiments carried out on a variety of simulated epidemic growth models indicate that this graph deep learning approach is effective in predicting cluster dynamics (balanced accuracy of 0.826 vs. 0.533 and Brier score of 0.234 vs. 0.466 in independent test set). Our deployment model in DeepDynaTree incorporates a primal-dual graph neural network principle using output from phylogenetic-based cluster identification tools (available from https://github.com/salemilab/DeepDynaTree).

bioinformatics↗
bioRxiv2021

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb1, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab2, S2X2593 and S2H974, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

immunology↗
bioRxiv2021

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

cancer biology↗
bioRxiv2021

A human antibody that broadly neutralizes betacoronaviruses protects against SARS-CoV-2 by blocking the fusion machinery

The repeated spillovers of {beta}-coronaviruses in humans along with the rapid emergence of SARS-CoV-2 escape variants highlight the need to develop broad coronavirus therapeutics and vaccines. Five monoclonal antibodies (mAbs) were isolated from COVID-19 convalescent individuals and found to cross-react with multiple {beta}-coronavirus spike (S) glycoproteins by targeting the stem helix. One of these mAbs, S2P6, cross-reacts with more than twenty human and animal {beta}-coronavirus S glycoproteins and broadly neutralizes SARS-CoV-2 and pseudotyped viruses from the sarbecovirus, merbecovirus and embecovirus subgenera. Structural and functional studies delineate the molecular basis of S2P6 cross-reactivity and broad neutralization and indicate that this mAb blocks viral entry by inhibiting membrane fusion. S2P6 protects hamsters challenged with SARS-CoV-2 (including the B.1.351 variant of concern) through direct viral neutralization and Fc-mediated effector functions. Serological and B cell repertoire analyses indicate that antibodies targeting the stem helix are found in some convalescent donors and vaccinees but are predominantly of narrow specificity. Germline reversion of the identified cross-reactive mAbs revealed that their unmutated ancestors are specific for the endemic OC43 or HKU1 viruses and acquired enhanced affinity and breadth through somatic mutations. These data demonstrate that conserved epitopes in the coronavirus fusion machinery can be targeted by protective antibodies and provide a framework for structure-guided design of pan-{beta}-coronavirus vaccines eliciting broad protection.

immunology↗
bioRxiv2021

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.

immunology↗
bioRxiv2020

methylscaper: an R/Shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data

Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we developed methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin landscapes. Methylscaper implements a weighted principle component analysis that orders sequencing reads, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscapers utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets. Availability and implementationMethylscaper, is available on GitHub at https://github.com/rhondabacher/methylscaper. Contactrbacher@ufl.edu

bioinformatics↗