Biology subjects

George, B.

Publications and source records attributed to George, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Chemical Proteomics of Residual Acute Myeloid Leukemia Cells Reveals Therapeutic Vulnerabilities

Venetoclax combined with azacitidine has improved treatment outcomes in acute myeloid leukemia (AML), yet relapse and treatment persistence remain major clinical challenges. To investigate proteomic mechanisms associated with venetoclax-azacitidine response and adaptation, we applied a multi-layer combinatorial proteome integral solubility/stability alteration analysis (CoPISA) strategy in SKM-1 AML cells. Cells were treated with venetoclax, azacitidine, their combination, or vehicle control and profiled across four orthogonal layers: short-term lysate CoPISA, short-term intact-cell CoPISA, long-term intact-cell CoPISA after 5 days of treatment, and long-term expression proteomics of surviving cells. The venetoclax-azacitidine combination induced treatment-specific protein solubility and abundance changes that were not fully reproduced by either single agent. Long-term surviving cells displayed extensive proteomic remodeling, consistent with the emergence of an adaptive drug-tolerant state, although contributions from pre-existing resilient cell populations cannot be excluded. Integration of short- and long-term solubility changes with abundance remodeling revealed distinct adaptive regimes, including retained biochemical targets, dosage-compensated targets, sensitive-state-specific targets, and remodeled adaptive targets. This framework prioritized candidate resistance-associated proteins, including NRP2, RPL18, PLP2, RPS28, and NOTCH1. NRP2 emerged as a top candidate across all proteomics layers, consistently showing increased abundance. Overall, this study shows that combining CoPISA with expression proteomics can resolve temporally distinct proteomic states of venetoclax-azacitidine response and identify candidate adaptive vulnerabilities in AML.

cancer biology↗

bioRxiv2026

Temporal transcriptomic remodeling after controlled cortical impact reveals delayed AQP4/SNTA1 expression imbalance associated with ion-homeostatic remodeling

Traumatic brain injury (TBI) is often viewed as a progressively evolving molecular response, yet the molecular and cellular responses that dictate acute TBI may differ fundamentally from those that define the chronically remodeled brain. We integrated temporally-resolved transcriptomic analyses with independent mouse, human, proteomic, and spatial datasets to determine how neural-circuit and astrocyte-homeostatic transcriptional responses reorganize after controlled cortical impact (CCI). In GSE269748, a mouse CCI transcriptomic dataset spanning acute to chronic post-injury timepoints, transcriptomic remodeling was non-monotonic. The number of genes showing large expression changes was greatest at 7 days, with extensive transcriptional changes persisting at 6 months. A more selective astrocytic phenotype emerged at 7 days and persisted at 6 months, with Aqp4 increasing disproportionately relative to Snta1, producing a sustained AQP4/SNTA1 expression imbalance. This imbalance occurred alongside heterogeneous endfoot remodeling and could not be explained by inflammatory stress alone. Among 14 prespecified biological processes, potassium and ion homeostasis showed the most specific and robust association with the AQP4/SNTA1 imbalance after adjustment for broader reactivity and AP-1 activity; this observation remained stable to gene, sample, and injury-model perturbation. Circuit-associated analyses provided complementary context, showing that dopamine recipient changes occurred within broader neurotransmitter system remodeling rather than as a unique signal. Integrative temporal analysis distinguished acute stress-dominant remodeling from delayed astrocyte-homeostatic changes, with AQP4/SNTA1 imbalance and potassium and ion homeostatic remodeling becoming most prominent at later stages. External datasets provided limited contextual support while defining clear limits to generalization. Together, these findings identify delayed AQP4/SNTA1 expression imbalance within potassium and ion homeostatic remodeling as a testable feature of chronic post-traumatic astrocyte biology.

neuroscience↗

bioRxiv2026

MICRON learns outcome-associated representations of spatial immune microenvironments

Spatial imaging proteomics modalities, such as imaging mass cytometry, enable comprehensive identification of immune microenvironments driving disease outcomes. Identifying outcome-associated immune microenvironments from these data has proven to be complex, as it requires segmenting cells with complex shapes and reconciling spatial signatures across many heterogeneous samples. We present MICRON, a segmentation-free, fully automated multiple-instance learning based tool for automatic identification of outcome-linked immune microenvironments. MICRON learns representations of samples profiled with spatial imaging proteomics modalities, enabling more accurate prognostic and diagnostic prediction over existing approaches. As a case study, we show that MICRON generates a comprehensive importance map that reveals key outcome-associated immune microenvironments in brain cancer, uncovering coordinated cell-cell communication between astrocytes, NK cells, and macrophages linked to survival outcomes. MICRON is provided as open source software for broad use by clinicians and biologists at https://github.com/ChenCookie/micron.

bioinformatics↗

bioRxiv2026

Myelin-Free Nuclei Isolation from Mouse Hippocampus and Cerebellum for snRNA-Seq with Benchtop Gradient Centrifugation

Nuclei isolation from myelin-rich adult mouse brain regions remains challenging for single-nucleus RNA sequencing because myelin and debris can reduce nuclei quality. We describe an optimized protocol for mouse hippocampi and cerebella using tube-and-pestle homogenization, low-volume sucrose-gradient pelleting with a standard benchtop centrifuge, and magnetic enrichment as the recommended final cleanup step to reduce debris/non-nuclear carryover. Under the tested conditions, the workflow produces intact, debris-reduced nuclei and supports downstream 10x Genomics Flex and PARSE WT library preparation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/716374v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f75556org.highwire.dtl.DTLVardef@103bb8eorg.highwire.dtl.DTLVardef@e7a9eorg.highwire.dtl.DTLVardef@942768_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBenchtop sucrose-gradient pelleting enables rapid nuclei purification from myelin-rich adult mouse brain C_LIO_LIScales across tissue inputs (e.g., hippocampus [~]15-20 mg; cerebellum [~]50-70 mg) without ultracentrifugation or 15 mL gradients C_LIO_LIMagnetic enrichment as the recommended final cleanup step further reduces myelin/debris carryover and is compatible with 10x Flex and PARSE WT workflows. C_LI

neuroscience↗

bioRxiv2025

Spatial Conformation of Pancreatic Cancer Is Associated with Disease Recurrence after Curative-Intent Total Neoadjuvant Therapy

Pancreatic ductal adenocarcinoma (PDAC) frequently recurs after total neoadjuvant therapy (TNT) and curative-intent resection. Traditional histopathologic response assessments demonstrate that major pathologic response (<5-10% viable residual cancer) is associated with favorable outcomes. However, most TNT cases achieve only a minor response. We investigated whether the spatial organization of residual PDAC encodes clinically meaningful biology beyond residual tumor burden. In a retrospective cohort of 203 resected PDAC patients, all with minor pathologic response, H&E whole-slide images were segmented into cancer and stroma to subsequently quantify spatial composition (e.g., patch size/density, edge density) and configuration (e.g., patch complexity/compactness, spatial intermixing) to model treatment-resistant tumor architecture. Non-response was associated with a more fragmented interface-rich ecology, higher edge density and diversity, and reduced homotypic aggregation, independent of conventional clinicopathologic features. This demonstrates a link between emergent tissue architecture of treated PDAC and therapeutic resistance. Further, two multivariable, spatial risk models were independently associated with disease-free survival (DFS): (1) cancer mean shape index and stromal shape-index variability (high-risk median DFS 7.23 vs 11.57 months; adjusted HR 1.75, p=0.007) and (2) mean stromal area with edge density (high- vs low-risk adjusted HR 1.94, p=0.006), outperforming traditional treatment response assessments. Quantifying residual cancer-stroma topology thus yields independent, prognostic signals in post-TNT PDAC and motivates prospective, spatially informed adjuvant strategies and mechanistic studies of edge habitats and mixing as therapeutic vulnerabilities.

cancer biology↗

bioRxiv2025

Moving Beyond Binary Biomarkers: Machine Learning Model Resolves Concurrent and Molecularly Heterogeneous Mismatch Repair and Homologous Recombination Deficiencies in Prostate Cancer

Current DNA damage repair (DDR) biomarkers employ binary classifications that fail to capture the molecular complexity of tumors with concurrent repair deficiencies. We used genomics analysis to stratify 672 metastatic prostate cancer patients into 11 DDR subgroups, identifying 51 molecular signatures with weighted roles in class identity. We identified a tumor-mutational-burden very-high subset, characterized by 19 mutations/Mb or more, as a molecularly distinct group characterized by preserved genomic integrity and enhanced immunogenicity. Critically, 2.3 percent of tumors exhibited concurrent TMB-High and HRR mutant phenotypes, while 1.5 percent harbored MMR bi-allelic loss without MMRd (mismatch-repair-deficiency) signatures. Clinical validation in 130 patients demonstrated superior immunotherapy responses in tumors with very high TMB levels. We developed CHIMERA DDR, a probabilistic machine learning tool that integrates these 51 genomic features using a nested Random Forest architecture to infer seven clinically relevant DDR subgroups. After negating model overfit concerns, CHIMERA-DDR showed exceptional classification performance (AUCs 0.919-0.999) to accurately detect MMRd and HRR mutant molecular subtypes with or without concurrent DDR deficiencies, resolving admixed phenotypes to enable precision therapeutic stratification beyond binary methods.

genomics↗

bioRxiv2024

Divergent Immediate and Delayed Effects of Juvenile Exposure to Doxorubicin on the Thymus in C57BL/6 Mice

BackgroundThe understanding of alterations within the immune system following doxorubicin (DOX) chemotherapy, and subsequent restoration, in childhood cancer survivors remains limited. This investigation endeavors to elucidate the immediate and delayed changes in thymic immune cell populations and their phenotypes in response to clinically relevant low doses of DOX in a juvenile mouse model. MethodsMale mice underwent a regimen of repeated low-dose DOX intraperitoneal injections at 4 mg/kg/week for three consecutive weeks. One week after the last dose of DOX, a subset of mice was euthanized to assess the immediate effects of DOX administration. A second subset of mice was euthanized five weeks after the last DOX dose to evaluate the delayed effects. Thymic samples were collected for multiparameter flow cytometry analysis to evaluate alterations in immune cell composition and phenotype. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure gene expression of- cytokines and senescence markers. ResultsOne week following DOX administration, DOX treatment resulted in significant decline in thymus weight, with notable alterations in immune cell subpopulations. Reduced frequencies of mature CD3+CD4+ and CD3+CD8+ T cells were observed, along with changes in proliferation and exhaustion markers. Gene expression analysis revealed upregulation of Foxn, Pax1, Ifn{gamma}, and Il7 alongside decreased Il6 and Il17 expression. Furthermore, Cdkn1a (p21Cip1) expression was elevated, suggesting immunosenescence. Five weeks following DOX administration, delayed effects of DOX treatment manifested in rebound increase in thymus weight and altered frequencies of CD4+ and CD8+ T cell subsets, with distinct patterns of proliferation and exhaustion observed. Notably, central memory CD4+ T cells exhibited significant decrease in frequency, while naive and effector memory CD4+ T cells showed reduced proliferation (Ki67+) and PD1 expression. Similar trends were observed in CD8+ T cell subsets, indicating selective effects of DOX on T cell differentiation and function. Although expression of thymus-related genes was normalized, p21Cip1 gene expression remained elevated. ConclusionDOX treatment elicits a multifaceted influence on immune cell subsets and thymic weight. Immediate effects included thymic atrophy and reductions in mature T cell populations, while delayed effects showed rebound thymic hyperplasia and selective changes in CD4+ and CD8+ T cell subsets. Notably, both central memory and effector memory T cells exhibited reduced proliferation and exhaustion, suggesting unique impacts of DOX on immune cell function. The enduring elevation in p21Cip1 gene expression 5 weeks after DOX treatment suggests an immunosenescent phenotype. These observations collectively illuminate the formidable task of preserving immune competence and overall well-being in childhood cancer survivors subjected to DOX therapy.

pharmacology and toxicology↗

bioRxiv2024

Investigating the influence of iron parameters in regulating platelet recovery in Apheresis Platelet Donors

IntroductionThe present study investigates changes in haematological parameters in the donor during apheresis and following donation. Effect of iron parameters in influencing platelet recovery was studied. MethodsBlood samples were collected in EDTA tubes for analyses of haematological parameters. Serum samples were isolated for estimation of iron parameters. ResultsHb, RBC count and Hct increased during apheresis, but no significant changes were observed after 48 hrs post donation. Platelet recovery rates increased from 78 % after 48 hours to > 100% on day 10 (p < 0.001). Ferritin, serum iron & Transferrin saturation initially increased and subsequently declined from day 7 post donation, which may influence platelet recovery. Recovery rates were also higher in donors with lower iron stores. ConclusionsChanges in haematological parameters during plateletpheresis are transient and optimal time required for platelet recovery is dependent on pre-donation counts. Recovery of platelet counts may be positively regulated by donor iron parameters.

molecular biology↗

bioRxiv2024

Synaptogyrin-3 Prevents Cocaine Addiction and Dopamine Deficits

Synaptogyrin-3, a functionally obscure synaptic vesicle protein, interacts with vesicular monoamine and dopamine transporters, bringing together dopamine release and reuptake sites. Synaptogyrin-3 was reduced by chronic cocaine exposure in both humans and rats, and synaptogyrin-3 levels inversely correlated with motivation to take cocaine in rats. Synaptogyrin-3 overexpression in dopamine neurons reduced cocaine self-administration, decreased anxiety-like behavior, and enhanced cognitive flexibility. Overexpression also enhanced nucleus accumbens dopamine signaling and prevented cocaine-induced deficits, suggesting a putative therapeutic role for synaptogyrin-3 in cocaine use disorder.

neuroscience↗

bioRxiv2024

Targeting Metabolic Dependencies in Acute Myeloid Leukemia: A Dual Strategy using Arsenic Trioxide and Artesunate.

Acute myeloid leukemia (AML) remains a difficult disease to cure despite recent advances. Off-target side effects of therapy, especially prolonged cytopenia, lead to significant morbidity and mortality. There is an increased recognition that in AML, there is a potentially vulnerable dependence on OXPHOS metabolism, more so in the leukemia stem cell compartment (AML-LSC) that could be exploited. Drug re-purposing screens have suggested the possible role of artesunate (ART) in inhibiting mitochondrial respiration and arsenic trioxide (ATO) in inhibiting glycolysis. Here, we explore the potential of using a combination of ART and ATO to treat AML. Through in-vitro and in-vivo studies, we demonstrate the potential of this combination along with hypo-methylating agents and other mitocans, such as BCL-2 inhibitor venetoclax, to be used in the treatment of AML with minimal off-target effect on normal hematopoietic stem cells (HSC). These observations warrant further exploration of such novel combinations in clinical trials.

cancer biology↗

bioRxiv2023

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

We recently identified glycoRNA--a previously undescribed glycoconjugate--which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.

molecular biology↗

bioRxiv2023

microRNA-22 displaces ITAFs from the 5'UTR and inhibit the translation of Coxsackievirus B3 RNA

microRNAs play an essential role in gene regulation during virus infections and have major consequences on viral pathogenesis. During RNA virus infections, the host miRNAs can target both host mRNAs and the virus genomic RNA. Using the CVB3 virus as a model, we have investigated how a host miRNA can target viral genomic RNA and act as an antiviral factor limiting the growth of the virus. CVB3 is an RNA virus whose infection causes myocarditis and, eventually, dilated cardiomyopathy. We shortlisted miRNAs with a potential binding site in the CVB3 genomic RNA. Among these, miR-22 was picked for further studies as its binding site was putatively located in a region in the CVB3 5 UTR, important for recruiting ITAFs and ribosomes for IRES-mediated translation. Using mutational analysis and pull-down assays, we first confirmed the binding of miR-22 on the 5UTR. This binding negatively regulated the translation of CVB3 RNA. However, miR-22 binding-defective mutant of CVB3 RNA had no effect of miR-22 overexpression and could translate normally. Moreover, cells from which miR-22 was knocked out, showed a higher level of CVB3 infection as compared to the wild type. We have further demonstrated that the binding of miR-22 interferes with the recruitment of several ITAFs (La, PSF, and PTB) on viral mRNA. This abrogates the spatial structure necessary for ribosome recruitment on the CVB3 RNA, ultimately inhibiting its translation. Also, the level of miR-22 increases 4 hours post-infection, presumably after the synthesis of viral 2A protease, to regulate infection in the host cell more effectively. Along with the direct effect on viral RNA, the altered level of miR-22 affects the level of its cellular targets which might contribute to CVB3 infection. To identify the possible players, we obtained a list of miR-22 targets and performed pathway analysis. Several targets were shortlisted among the top hits and their levels upon CVB3 infection were checked. Protocadherin-1 (PCDH-1), a single-pass transmembrane protein, followed an expected trend, and its levels were significantly downregulated upon CVB3 infection in miR-22 dependent manner. miR-22 mediated suppression of PCDH1 levels during CVB3 infection points towards the possible role of miR-22 in either modulating antiviral signaling or in virus entry, in addition to regulating the viral translation.

molecular biology↗

bioRxiv2023

Human antigen R differentially modulates the translation of SARS-CoV-2 genomic and sub-genomic RNAs

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) viral RNA associates with different RNA-binding host proteins at each stage of its life cycle. We found sequence dependent binding of one such important protein, human antigen R (HuR) to SARS-CoV-2 5'UTR and studied its potential role in virus life cycle. The knockdown and knockout studies revealed importance of such binding in viral translation. We identified 5'-UTR mutations in SARS-CoV-2 variants of concern that altered the HuR-binding affinity. Interestingly, HuR enhanced non-structural protein translation through the genomic 5'-UTR, by promoting polypyrimidine tract-binding protein binding to the 5'-UTR. However, HuR suppressed the structural protein translation from sub genomic 5UTR. HuR knockout increased the sensitivity to remdesivir treatment by decreasing its half-maximal inhibitory concentration. An antisense oligonucleotide (whose binding site overlapped the HuR-binding site) reduced viral RNA levels in wild-type cells but not HuR-knockout cells. Our results indicate that HuR regulates the balance between SARS-CoV-2 structural and non-structural proteins and guides the infection of viral variants, implying that HuR can potentially be targeted for therapeutic interventions. Author SummaryViruses interacts with various host proteins throughout their life cycle. One significant protein is HuR, an RNA-binding protein that regulates RNA stability and translation. HuR binds to viral RNAs at the 5UTR or 3UTR, impacting their translation and replication. We identified conserved HuR binding sites in the SARS-CoV-2 5UTR across different beta coronaviruses. This binding enhanced the initiation of translation from the genomic 5 UTR, increasing the production of non-structural proteins essential for viral replication. Additionally, we discovered that another host protein, PTB, promotes HuR binding to the viral 5 UTR, facilitating its loading onto ribosomes. Conversely, HuR plays an antagonistic role concerning subgenomic RNAs (sgRNAs), which code for structural proteins, by regulating and limiting their levels. This dual regulation indicates that the virus exploits HuR for its benefit while the host employs it to control viral spread. Targeting HuR may help manipulate the SARS-CoV-2 life cycle. We found that HuR knockout increased sensitivity to the antiviral drug Remdesivir. Using an antisense oligonucleotide to block HuR binding effectively reduced viral RNA levels. Our findings highlight the critical role of HuR in regulating viral protein production and its potential as a therapeutic target.

microbiology↗

bioRxiv2022

Hepatitis C virus induces HuR relocalization by coordinating PKC-δ and AMPK- function

Host protein HuR translocation from nucleus to cytoplasm following infection is crucial for the life cycle of several RNA viruses including hepatitis C virus (HCV), a major causative agent of hepatocellular carcinoma. HuR assists the assembly of replication-complex on the viral-3'UTR, and its depletion hampers viral replication. Although cytoplasmic HuR is crucial for HCV replication, little is known about how the virus orchestrates the mobilization of HuR into the cytoplasm from the nucleus. We show that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-{delta}, which in-turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-1. Cytoplasmic retention or entry of HuR can be reversed by an AMPK activator or a PKC-{delta} inhibitor. Our findings suggest that efforts should be made to develop inhibitors of PKC-{delta} and AMPK, either separately or in combination, to inhibit HCV infection. Author summaryHepatitis C virus is a major human pathogen, which exploits cellular machinery for its propagation in liver cells. The cytoplasmic availability of cellular components is crucial for their direct influence on processes involving the viral RNA, which lacks any nuclear history. Our results establish the involvement of viral proteins, NS3 and NS5A in achieving increased cytoplasmic abundance of a host factor HuR, an RNA binding protein (RBP) critical for HCV replication. This is achieved via direct post-translational modification of HuR and indirect regulation of its nuclear carrier by coercing two host kinases, PKC-{delta} and AMPK-. RBPs are emerging as novel targetable candidates for gene regulation. Similar studies with other RBPs and targeting protein modifications, in place of whole protein knockdown, could usher in a revolutionary strategy to neutralize emerging RNA virus-based diseases, while preserving their cellular functions.

microbiology↗

bioRxiv2022

Impact of maternal iron deficiency anaemia on fetal iron status and placental iron transporters in human pregnancy

Iron deficiency anaemia is associated with maternal morbidity and poor pregnancy outcomes. Placenta expresses both haem and non-haem iron transport proteins. The aim of the study is to examine the expression of placental iron trafficking molecules and associate them with maternal and neonatal iron status. Pregnant women who received prenatal care at the department of community health and development, Christian Medical College, Vellore, India for childbirth were recruited between 2016-2018. Pregnant women who were 18-35 years old with gestational age (GA) of [≥]36weeks were eligible to participate in the study. In a prospective cohort of pregnant women, 22% were iron deficiency anaemia (IDA) and 42% were iron replete. Pregnant women in the different groups were mutually exclusive. Samples were collected (Maternal blood, placental tissue, and cord blood) from pregnant women with gestational age of [≥]38 weeks at the time of delivery. Mean gestational age at first visit and delivery was 12.8{+/-}2.72 weeks and 39{+/-}1.65 weeks, respectively. Hemoglobin (9.3{+/-}0.9g/dl) and ferritin (15.4(0.8-28.3) ng/ml) levels at delivery were significantly decreased in IDA as compared to other groups. The foetal haemoglobin and ferritin levels were in the normal range in all groups. We further analysed the expression of iron transport genes in the placenta in the iron replete controls and the IDA group. Under maternal iron insufficiency, the expression of placental iron transporters DMT1 and FPN1 were upregulated at the transcriptional level. There was no correlation of maternal and cord blood hepcidin with foetal iron status in IDA. Thus, placental iron traffickers respond to maternal iron deficiency by increasing their expression and allowing sufficient iron to pass to the foetus.

molecular biology↗

bioRxiv2020

Evidence of a dysregulated Vitamin D pathway in SARS-CoV-2 infected patient's lung cells

Although a defective vitamin D pathway has been widely suspected to be associated in SARS-CoV-2 pathobiology, the status of the vitamin D pathway and vitamin D-modulated genes in lung cells of patients infected with SARS-CoV-2 remains unknown. To understand the significance of the vitamin D pathway in SARS-CoV-2 pathobiology, computational approaches were applied to transcriptomic datasets from bronchoalveolar lavage fluid (BALF) cells of such patients or healthy individuals. Levels of vitamin D receptor, retinoid X receptor, and CYP27A1 in BALF cells of patients infected with SARS-CoV-2 were found to be reduced. Additionally, 107 differentially expressed, predominantly downregulated genes modulated by vitamin D were identified in transcriptomic datasets from patients cells. Further analysis of differentially expressed genes provided eight novel genes with a conserved motif with vitamin D-responsive elements, implying the role of both direct and indirect mechanisms of gene expression by the dysregulated vitamin D pathway in SARS-CoV-2-infected cells. Network analysis of differentially expressed vitamin D-modulated genes identified pathways in the immune system, NF-KB/cytokine signaling, and cell cycle regulation as top predicted pathways that might be affected in the cells of such patients. In brief, the results provided computational evidence to implicate a dysregulated vitamin D pathway in the pathobiology of SARS-CoV-2 infection.

bioinformatics↗

bioRxiv2020

Mosaic antimicrobial resistance/virulence plasmid in hypervirulent ST2096 Klebsiella pneumoniae in India: The rise of a new superbug?

Hypervirulent K. pneumoniae (HvKp) is typically associated with ST23 clone; however, hvKp is also emerging from clones ST11, ST15 and ST147, which are also multi-drug resistant (MDR). Here, we aimed to characterise nine novel MDR hvKp isolates harbouring mosaic plasmids simultaneously carrying antimicrobial resistance (AMR) and virulence genes. Nine HvKp isolates obtained from hospitalised patients in southern India were characterized for antimicrobial susceptibility and hypervirulence phenotypes. All nine hvKp isolates were subjected to whole genome sequencing (WGS) using Ilumina HiSeq2500 and a subset of four were sequenced using Oxford Nanopore MinION. Among the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The virulence determinants were encoded in a mosaic plasmid ([~]320 Kbp) that formed as a result of its insertion in a IncFIB-IncHI1B plasmid co-integrate. The mosaic plasmid carried AMR genes (aadA2, armA, blaOXA-1, msrE, mphE, sul1 and dfrA14) in addition to rmpA2, iutA and iucABCD virulence genes. Interestingly the mosaic plasmid carried its own type IV-A3 CRISPR-cas system that is likely able to target the acquisition of IncF plasmid with the help of a traL spacer. The convergence of virulence and AMR is the biggest threat among invasive K. pneumoniae infections. However, increasing reports of the presence of mosaic plasmid carrying both AMR and virulence genes suggests MDR-hvKp isolates are no longer confined to selected clones and the containment of such isolates is very challenging. IMPORTANCEKlebsiella pneumoniae is an opportunistic pathogen that commonly associated with hospital-acquired infections in the urinary tract, respiratory tract, lung, wound sites. The organism has gained notoriety by acquiring additional genetic traits to become either hypervirulent (HV) phenotype or multidrug resistant (MDR) phenotype. Though the infections by both these phenotypes were very challenging to treat, the MDR K. pneumonia (MDR-Kp) were remained in the hospital settings while HV K. pneumonia (hvKp) strains were mostly originated from the community settings. In a recent turn of events, the evolution of MDR-Kp and hvKp has converged as both clones found to carry both MDR plasmids and virulence plasmid. These convergent strains are challenging to treat and is associated with higher mortality rate. As the recent hvKp isolates harbour mosaic plasmid encoding both AMR and virulence determinants there is a need to investigate the evolution of these pathogens. The significance of our research is in characterising the novel mosaic plasmid identified in MDR-hvKp isolates that belong sequence type (ST) 2096. Tracking the possible evolution pathway of MDR-hvKPs would greatly help in the proper surveillance and management of this superbugs. RepositoriesThe whole genome sequences of the present study isolates have been deposited in GenBank, NCBI, with accession numbers CP053765 - CP053770, CP053771 - CP053780, CP058798-CP058806, JAARNO010000001.1 - JAARNO010000005.1, JAAQSG000000000, JAARNJ000000000, JAARMH000000000 and JAAQTC000000000

genomics↗

bioRxiv2020

CASSPER: A Semantic Segmentation based Particle Picking Algorithm for Single Particle Cryo-Electron Microscopy

Single-particle cryo-electron microscopy has emerged as the method of choice for structure determination of proteins and protein complexes. However, particle identification and selection which is a prerequisite for achieving high-resolution still poses a major bottleneck for automating the steps of structure determination. Here, we present a generalised deep learning tool, CASSPER, for the automated detection and isolation of protein particles in transmission microscope images. This deep learning tool uses Semantic Segmentation and a collection of visually prepared training samples to capture the differences in the transmission intensities of protein, ice, carbon and other impurities found in the micrograph. CASSPER is the first method to do pixel level classification and completely eliminates the need of manual particle picking. Integration of Contrast Limited Adaptive Histogram Equalization (CLAHE) in CASSPER enables high-fidelity particle detection even in micrographs with variable ice thickness and contrast. In addition, our generalized model for cross molecule picking works with high efficiency on unseen datasets and can potentially pick particles on-the-fly, thereby, enabling automation of data processing.

biophysics↗