Biology subjects

Shukla, M.

Publications and source records attributed to Shukla, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Leveraging collaborations between researchers, wildlife managers and citizen-volunteers to monitor endangered carnivore populations across space and time

Monitoring populations is fundamental for conserving threatened carnivores, but can be difficult to sustain in the long term across large landscapes. We present a collaborative framework for monitoring dholes (Cuon alpinus) in India's Western Ghats, combining fecal DNA-based individual identification, Spatially Explicit Capture-Recapture (SECR) models, state Forest Department participation, and trained citizen-volunteers. We surveyed across six sites between 2019-2023, encompassing three Protected Areas (Wayanad, Parambikulam, Periyar) and their adjacent Territorial Divisions. Fecal samples were genotyped using a panel of 96 Single Nucleotide Polymorphisms, and detections were analyzed using SECR models to estimate densities. In Wayanad Sanctuary, densities remained stable across 2019, 2022 and 2023, increasing marginally from 15.67 to 19.01 individuals per 100 sq.km. In 2023, densities varied substantially among six sites, from 9.73/100 sq.km in Parambikulam to 29.23 in Nemmara Territorial Division. Field implementation involved an average of 46 Forest Department personnel co-collecting data per site-year. Among former citizen-volunteers who were surveyed for project feedback (N=29), 83% went on to either work or pursue higher education in wildlife-related or -adjacent fields. We demonstrate that molecular individual identification coupled with SECR can provide a practical basis for dhole population assessments while enabling multi-stakeholder participation in field monitoring. Sustained engagement with wildlife managers and locally recruited volunteers can strengthen institutional capacity, facilitate knowledge exchange, and improve continuity of monitoring programs. We argue that integrating molecular ecology with collaborative governance and democratizing field-science offers a scalable pathway for landscape-level monitoring required to guide dhole conservation.

ecology↗

bioRxiv2026

Variable ectopic heterochromatin islands provide an alternative route to antifungal heteroresistance in Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is estimated to cause approximately 180,000 deaths annually, primarily among immunocompromised HIV patients in resource-limited countries in central and southern Africa. Economic constraints regularly restrict treatment to fluconazole monotherapy. Unstably resistant cells arise in any otherwise fluconazole-sensitive C. neoformans population, with such heteroresistance often involving chromosome 1 disomy. Here we uncover a form of heteroresistance that occurs by a mechanism distinct from aneuploidy. Transient islands of histone H3 lysine 9 methylation-dependent heterochromatin occur at several locations across the genomes of clinical isolates from African patients. Some islands dissipate in the absence of fluconazole selection but are restored upon re-exposure. Deletion of specific heterochromatin island-located genes increased fluconazole resistance in an otherwise wild-type background, suggesting that heterochromatin-mediated alterations in gene expression elicit unstable resistance. Thus, in addition to aneuploidy, transient heterochromatin islands also appear to contribute to C. neoformans fluconazole heteroresistance. Heterochromatin islands have recently been shown to confer unstable antifungal resistance in the ascomycete fungus Schizosaccharomyces pombe (fission yeast) and fungal species of the Mucor circinelloides complex. Thus, similar unstable resistant epimutations may be widespread in pathogenic fungi, however, their instability and inaccessibility to detection by direct sequencing poses challenges for monitoring this form of heteroresistance in clinical settings.

microbiology↗

bioRxiv2026

Genomic Footprints of Bottlenecks, Isolation, and Inbreeding: A Case Study of Two Vulture Cohorts in India

Genomic erosion as a manifestation of small effective population size (Ne) and consanguinity subverts long-term perpetuation of threatened species by compromising their adaptive potential; however, the integration of genomics remains limited in applied conservation efforts to guide priorities. This study combines non-invasive sampling, double-digest Restriction site-associated DNA sequencing (ddRAD), and population-genomic analyses to assess genetic health in two vulture assemblages-mixed wild enclosure and captive breeding cohorts. Both the geographical locations exhibit signs of populations in distress: low genetic diversity and abundant intermediate-length runs of homozygosity (RoH), consistent with long-term reduced Ne plus recent demographic isolation. Our demographic model runs favoured ancient migration (AM) topology characterised by an ephemeral window of gene flow, taken over by a prolonged population separation period. The mutation quantification results from approximately 59,000 outgroup-polarised SNPs reveal higher additive burden and more homozygous-derived sites in BKN. However, this was later traced to low-impact and non-coding variants rather than a surge in the loss-of-function (LoF) alleles. The data support a genomic profile that carries an elevated risk from polygenic/aggregate deleterious burden in BKN despite a scarcity of high-impact mutations. By highlighting the disconnect between genetic resilience and demographic recovery, our results accentuate the need to incorporate genomics-informed inbreeding and monitoring programs, while also focusing on reducing anthropogenic mortality with genetic augmentation.

genomics↗

bioRxiv2026

Yin Yang Interactomes Drive the Metabolic Dysfunction-Associated Liver Disease Continuum to Hepatocellular Carcinoma

The risk for hepatocellular carcinoma (HCC) arising from metabolic dysfunction- associated steatotic liver disease (MASLD)-related cirrhosis exceeds the risk for cancer developing in the kidney, heart, or lung as a consequence of end-stage disease in those organs. Both experimental and clinical studies support the existence of circulating biomarkers whose elevated expression is associated with, and supports the diagnosis of, HCC. We posit the existence of oncogenic HCC biomarkers under regulation by tumor-suppressive microRNAs (miRs), and that reduced tumor-suppressive miR expression leads to increased oncogenic biomarker expression. By interrogating the MASLD-HCC miR interactome and miRs known to regulate HCC biomarkers, we identified hsa-miR-577, hsa-miR-9500, hsa-miR-101-3p, miR-206, hsa- miR-219a-1-3p, and hsa-miR-613 as tumor-suppressive miRs whose expression is reduced across multiple cancers. Their target genes, osteopontin, Golgi protein-73, and Dickkopf-related protein 1, exhibit increased expression in HCC and display oncogenic activity. Collectively, dysregulated expression of these miRs and their downstream gene products may contribute to progression along the MASLD continuum toward HCC. HighlightsWe identified tumor suppressive miRs, hsa-miR-577, hsa-miR-9500, hsa-miR-101-3p, miR-206, hsa-miR-219a-1-3p, and hsa-miR-613 with potentially reduced expression levels in HCC. Their gene products, osteopontin, Golgi protein-73, and Dickkopf-related protein 1, exhibit oncogenic properties and increased expression levels in HCC. These miR-gene product interactions may drive the MASLD continuum to HCC. Mechanistic insights should illuminate therapies. Identification of therapeutics that interrupt these interactions may represent a therapeutic pillar for patients with MASLD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/698028v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1892140org.highwire.dtl.DTLVardef@1e61677org.highwire.dtl.DTLVardef@cd0488org.highwire.dtl.DTLVardef@1e48d8a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigureC_FLOATNO MASLD-related HCC. The intersection set between the MASLD-HCC miR interactome and the set of miRs regulating biomarkers diagnostic for HCC was interrogated and tumor suppressive miRs regulating HCC biomarkers with oncogenic activity were identified. Increased risk for HCC in patients with MASLD and MASLD-cirrhosis may arise from interactions between these dysregulated miRs and their downstream gene products. C_FIG PrologueMetabolic Dysfunction-Associated Liver Disease (MASLD), the leading cause of hepatocellular carcinoma (HCC) in Sweden, is now taking epidemic proportions in South Asia. In India, there is a relatively high prevalence of pre-diabetes, diabetes and insulin resistance; body mass indices > 35 are being observed. Each of these and/or their combinations represent risk factors for MASLD. Nevertheless, both patients and providers are largely oblivious to this disease, both from a lack of general awareness and the asymptomatic nature of MASLD, at least early within its continuum. Diagnosis is often made late, and by exclusion, when presentation may include fatigue, drowsiness, mild encephalopathy (MASLD-pituitary feedback loop), prominent hepatomegaly and asciites, and with liver-specific test demonstrating stiffness, scarring and even HCC. Societal awareness and effective management of this disease are urgent needs. A strategy akin to the 4 pillars for treatment of type 2 diabetes and chronic kidney disease (CKD) needs to be implemented for treatment of patients with MASLD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=30 SRC="FIGDIR/small/698028v1_ufig2.gif" ALT="Figure 2"> View larger version (11K): org.highwire.dtl.DTLVardef@89a77org.highwire.dtl.DTLVardef@d50c15org.highwire.dtl.DTLVardef@10c6964org.highwire.dtl.DTLVardef@1db2cfa_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigureC_FLOATNO Four-pillar Strategy for MASLD. A strategy akin to the one in place for treatment of type 2 diabetes and CKD needs to be implemented for treatment of patients with MASLD. Aspects of this strategy, viz. SGLT2i and GLP-1{+/-}GIP agonists, are already approved in MASLD. C_FIG

bioinformatics↗

bioRxiv2025

Shaping the active site from a distance: Unravelling the regulatory role of the conserved N-terminal β-flap of thiol peroxidases through structure-function characterization of the Staphylococcal ortholog

Bacterial thiol peroxidases (Tpxs) are members of atypical two-cysteine peroxiredoxin, which detoxify hydroperoxides during host-pathogen interactions. Despite extensive research on bacterial Tpxs, the precise structure-based functional characterization of Staphylococcal Tpx (SaTpx) remains elusive. Herein, we cloned and purified SaTpx along with its plausible physiological redox partners to reconstitute its electron relay system. We solved the high-resolution crystal structure of SaTpx, revealing the essential active-site catalytic triad (Cys 60, Thr 57, Arg 128). The crystal structure of SaTpx reveals the presence of the highly conserved N-terminal {beta}-flap, absent in its mammalian orthologs (Peroxiredoxin 5). To investigate the plausible role of this highly conserved {beta}-flap, we cloned and characterized a deletion mutant, N{Delta}15-SaTpx, lacking this region. N{Delta}15-SaTpx showed reduced ability to mitigate organic hydroperoxides in vitro; partially impaired to prevent ROS-mediated ds-DNA nicking and failed to protect host E. coli cells from exogenous ROS stress. The ultra-high-resolution crystal structures of N{Delta}15-SaTpx and N{Delta}15-SaTpx with substrate mimic revealed a distorted catalytic triad (with flipped Arg 128), explaining its partially impaired catalytic activity. This study, for the first time, elucidates the critical regulatory role of the N-terminal {beta}-flap in stabilizing the catalytic triad of bacterial Tpx, providing insights for future inhibitor designing. HighlightsO_LIUltra high-resolution crystal structures of SaTpx and N{Delta}15-SaTpx C_LIO_LIUltra-high resolution crystal structure of hydroperoxide substrate mimic bound N{Delta}15-SaTpx C_LIO_LI{beta}-flap deletion mediated distortion of catalytic triad in N{Delta}15-SaTpx C_LIO_LIThe novel regulatory role of the conserved N-terminal {beta}-flap of Thiol peroxidases. C_LI

biophysics↗

bioRxiv2025

Deciphering the Structure and Mechanism of SaGpx: A Non-Canonical Glutathione Peroxidase from Staphylococcus aureus

Staphylococcus aureus encounters massive oxidative stress during infection. To counter this, the bacterium developed robust antioxidative defense mechanism. Glutathione peroxidases (Gpx) are well characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. S. aureus possesses two putative Gpx genes but lacks GSH biosynthetic machinery and glutathione reductase required for canonical Gpx function, suggesting alternate electron donor system(s) may be involved. This study aimed to elucidate structure-based biochemical characterization of one of the S. aureus glutathione peroxidases homologs (SaGpx, Uniprot Id: Q2FYZ0) and identify its plausible electron donor system. Herein, we cloned, purified and determined the high-resolution crystal structure of SaGpx (1.5 [A] resolution) using X-ray diffraction crystallography. In vitro biochemical characterization of the highly conserved active site amino acid point mutants, as well as their structural disposition suggests their precise roles in the enzymes catalysis. The crystal structure of SaGpx revealed that the enzyme adopts a canonical glutathione peroxidase fold with conserved catalytic tetrad composed of C36, Q70, W124 and N125. Also, SaGpx shows similarity with mammalian Gpx4, which was previously shown to exert phospholipid hydroperoxide peroxidase activity. Furthermore, biochemical assays suggest that SaGpx utilizes Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism follows an atypical 2-cysteine peroxiredoxin-like pathway involving the formation of a sulfenic acid intermediate, followed by an intramolecular disulfide bond subsequently resolved by thioredoxin. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.

biophysics↗

bioRxiv2025

A rapid non-traditional approach for developing biologicals against drug- resistant bacteria and yeasts.

Development of effective, long-lasting antibiotics is a challenge due to the rate at which the pathogens acquire resistance, prompting the major pharma players to omit them from their portfolio. We present a non-traditional approach for rapidly developing anti-infective biologicals that is resilient to resistance. Antibody-drug conjugates were developed against three different pathogens Pseudomonas aeruginosa (ABG 14), Staphylococcus aureus (ABG 16) and Candida. albicans (ABG 07) that were extremely specific and effective in neutralising them irrespective of their drug resistance profiles. Neutralising camelid antibody fragment (VHH) were isolated from immunised camel libraries by phage display that effectively neutralised the pathogens at a MIC 99 of 125 {micro}g mL-1 in vitro. Antimicrobial peptides (AMP) were then conjugated to them by pathogen-specific cleavable linkers and the resultant molecules were 10 -20 times more effective due to a dual mode of action-the inhibitory action of the VHH on surface transporters and enzymes and the activity of the AMPs released by the pathogen surface proteases. Called AbTids, these molecules were extremely specific, stable in plasma and were activated in the presence of 104 to 105 CFU mL-1 of the pathogens and had an efficacy in the sub-micromolar range (6.25-12.5 {micro}g mL-1, 250 nM) inhibiting their growth within 2 hrs. of administration. They were economically and efficiently produced in microbial production system as a single chain fusion protein. One of the molecules ABG 14 was characterised further and found to be pathogen specific with negligible frequency of resistance, was non-toxic, had the capability to destroy biofilms, and cleared a systemic infection in a mouse with a carbapenem resistant strain of P. aeruginosa at a dose of 5 mg kg-1. This strategy can be used to generate new hits against medically important pathogens in a matter of weeks by simply shuffling the components, using different VHHs and AMPs, bypassing years of expensive drug development efforts that can potentially rejuvenate drug discovery efforts against emerging superbugs.

microbiology↗

bioRxiv2024

Evaluating the Effects of Aircraft Noise on Hearing and Physiological Indicators: A Study of Military Personnel Using iTRAQ Proteomics and Cognitive Assessments

BackgroundNoise pollution poses a significant public health risk, with prolonged exposure to high levels of noise linked to various adverse outcomes such as annoyance, sleep disturbances, cognitive impairment, hypertension, and cardiovascular diseases. Noise-induced hearing loss (NIHL) is a prominent concern in noisy occupational settings. MethodThis study investigated NIHL among 621 male Air Force soldiers aged 18-45, exposed to intense aircraft noise. Auditory assessments included pure-tone audiometry (125 Hz to 8 kHz) to categorize hearing into normal, mild, moderate, and severe impairment. Distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) were used to evaluate cochlear and auditory nerve function. Heart rate variability (HRV) provided insights into autonomic responses. Cognitive functions were assessed through computerized tests, and blood plasma was analyzed for cardiac biomarkers, oxidative stress indicators, inflammatory markers, and neurotransmitters. Proteomic analysis used iTRAQ labeling, MudPIT, and MALDI-TOF/TOF mass spectrometry for protein quantification and identification, with validation through ELISA. ResultsThe audiometric tests revealed varying degrees of hearing impairment, with significant threshold differences at 2000, 3000, 4000, and 6000 Hz, especially pronounced at 6000 Hz. The right ear showed greater impairment, and a characteristic high-frequency notch was observed, consistent with noise exposure. Proteomic analysis indicated that NIHL is associated with oxidative stress and systemic inflammation, with differential protein expression related to hearing, coagulation, and inflammation. ConclusionThis study highlights the severe impact of aircraft noise on hearing and systemic health, demonstrating correlations between hearing impairment and biochemical markers. It emphasizes the role of oxidative stress and inflammation in NIHL development and underscores the need for effective noise management and protective measures in noisy work environments.

molecular biology↗

bioRxiv2024

Data Imbalance in Drug Response Prediction - Multi-Objective Optimization Approach in Deep Learning Setting

Drug response prediction (DRP) methods tackle the complex task of associating the effectiveness of small molecules with the specific genetic makeup of the patient. Anti-cancer DRP is a particularly challenging task requiring costly experiments as underlying pathogenic mechanisms are broad and associated with multiple genomic pathways. The scientific community has exerted significant efforts to generate public drug screening datasets, giving a path to various machine learning (ML) models that attempt to reason over complex data space of small compounds and biological characteristics of tumors. However, the data depth is still lacking compared to computer vision or natural language processing domains, limiting current learning capabilities. To combat this issue and increase the generalizability of the DRP models, we are exploring strategies that explicitly address the imbalance in the DRP datasets. We reframe the problem as a multi-objective optimization across multiple drugs to maximize deep learning model performance. We implement this approach by constructing Multi-Objective Optimization Regularized by Loss Entropy (MOORLE) loss function and plugging it into a Deep Learning model. We demonstrate the utility of proposed drug discovery methods and make suggestions for further potential application of the work to promote equitable outcomes in the healthcare field. Availabilityhttps://github.com/AlexandrNP/MOORLE Contactonarykov@anl.gov

cancer biology↗

bioRxiv2023

Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4 dicarboxylate transporter

The COVID-19 pandemic has hastened the problem of nosocomial drug-resistant pathogens and is exerting a huge toll on hospitalized patients in critical care settings. Most small molecule antibiotics are susceptible to bacterial resistance mechanisms and constantly becoming ineffective leading to rapid shrinkage of the antibiotic armamentarium. Pseudomonas aeruginosa, one of the most common pathogens in hospital-born infection and considered critically dangerous by WHO and CDC, is extremely difficult to treat with frontline drugs, the carbapenems. In this study, we developed a camelid antibody fragment (VHH) library against whole P. aeruginosa and isolated a highly potent neutralizing hit (PsC23) that selectively targets P. aeruginosa . At 25 {micro}g/mL, PsC23 inhibited growth of the ATCC 27853 and a locally isolated carbapenem resistant MCC 50428 P. aeruginosa. The target of PsC23 is the C4 dicarboxylate transporter that transports C4 metabolites to the glyoxylate shunt during oxidative stress that is present in pathogens but not the human host. This ultimately results in the blockade of the shunt affecting bacterial energy transduction that leads to disruption of drug efflux. Interestingly, in a neutropenic mouse with MCC 50428 systemic infection, PsC23 in combination with meropenem completely reversed the drug resistance and eliminated the pathogens from the blood. PsC23 was stable in human serum and had no hemolytic or cytotoxic effect on human cells. Taken together, this VHH if co administered with the currently available carbapenems would reverse carbapenem resistance and could be used to effectively control P. aeruginosa in critical care settings.

molecular biology↗

bioRxiv2023

Prenatal experience with language shapes the brain

Human infants acquire language with striking ease compared to adults, but the neural basis of their remarkable brain plasticity for language remains little understood. Applying a scaling analysis of neural oscillations for the first time to address this question, we show that newborns electrophysiological activity exhibits increased long-range temporal correlations after stimulation with speech, particularly in the prenatally heard language, indicating the early emergence of brain specialization for the native language.

neuroscience↗

bioRxiv2023

Somatic chromosome pairing has a determinant impact on 3D 1 chromatin organization

In the nucleus, chromatin is intricately structured into multiple layers of 3D organization important for genome activity. How distinct layers influence each other is not well understood. In particular, the contribution of chromosome pairing to 3D chromatin organization has been largely neglected. Here, we address this question in Drosophila, an organism that shows robust chromosome pairing in interphasic somatic cells. The extent of chromosome pairing depends on the balance between pairing and anti-pairing factors, with the anti-pairing activity of the CAP-H2 condensin II subunit being the best documented. Here, we identify the zinc-finger protein Z4 as a strong anti-pairer that interacts with and mediates the chromatin binding of CAP-H2. We also report that hyperosmotic cellular stress induces fast and reversible chromosome unpairing that depends on Z4/CAP-H2. And, most important, by combining Z4 depletion and osmostress, we show that chromosome pairing reinforces intrachromosomal 3D interactions. On the one hand, pairing facilitates RNAPII occupancy that correlates with enhanced intragenic gene-loop interactions. In addition, acting at a distance, pairing reinforces chromatin-loop interactions mediated by Polycomb (Pc). In contrast, chromosome pairing does not affect which genomic intervals segregate to active (A) and inactive (B) compartments, with only minimal effects on the strength of A-A compartmental interactions. Altogether, our results unveil the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions, unraveling the interwoven relationship between different layers of chromatin organization and the essential contribution of chromosome pairing.

genomics↗

bioRxiv2022

GenSLMs: Genome-scale language models reveal SARS-CoV-2 evolutionary dynamics.

We seek to transform how new and emergent variants of pandemiccausing viruses, specifically SARS-CoV-2, are identified and classified. By adapting large language models (LLMs) for genomic data, we build genome-scale language models (GenSLMs) which can learn the evolutionary landscape of SARS-CoV-2 genomes. By pretraining on over 110 million prokaryotic gene sequences and finetuning a SARS-CoV-2-specific model on 1.5 million genomes, we show that GenSLMs can accurately and rapidly identify variants of concern. Thus, to our knowledge, GenSLMs represents one of the first whole genome scale foundation models which can generalize to other prediction tasks. We demonstrate scaling of GenSLMs on GPU-based supercomputers and AI-hardware accelerators utilizing 1.63 Zettaflops in training runs with a sustained performance of 121 PFLOPS in mixed precision and peak of 850 PFLOPS. We present initial scientific insights from examining GenSLMs in tracking evolutionary dynamics of SARS-CoV-2, paving the path to realizing this on large biological data.

bioinformatics↗

bioRxiv2022

MAP Kinase and mammalian target of rapamycin are main pathways of gallbladder carcinogenesis: Results from bioinformatic analysis of Next Generation Sequencing data from a hospital-based cohort.

BackgroundGallbladder Cancer (GBC) is one of the most common cancers of the biliary tract and the third commonest gastrointestinal (GI) malignancy worldwide. The disease is characterized by the late presentation and poor outcome despite treatment, and hence, newer therapies and targets need to be identified. MethodsThe current study investigated various functionally enriched pathways in GBC pathogenesis involving the genes identified through Next Generation Sequencing (NGS). The Pathway enrichment analysis and Gene Ontology (GO) were carried out after NGS, followed by the construction of the protein-protein interaction (PPI) network to discover associations among the genes. ResultsOf the thirty-three patients with GBC who were screened through next-generation sequencing (NGS), 27somatic mutations were identified. These mutations involved a total of 14 genes. The p53 and KRAS were commonly found to be mutated, while mutations in other genes were seen in one case each, the mean number of mutations were 1.2, and maximum mutation in a single case (eight) was seen in one case. The bioinformatics analysis identified MAP kinase, PI3K-AKT, EGF/EGFR, and Focal Adhesion PI3K-AKT-mTOR signaling pathways and cross-talk between these. ConclusionThe results suggest that the complex crosstalk between the mTOR, MAPK, and multiple interacting cell signaling cascades can promote GBC progression, and hence, mTOR - MAPK targeted treatment will be an attractive option.

cancer biology↗

bioRxiv2021

Analysis of the ARTIC version 3 and version 4 SARS-CoV-2 primers and their impact on the detection of the G142D amino acid substitution in the spike protein

The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. ImportanceARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.

bioinformatics↗

bioRxiv2021

Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation

One strategy for a functional cure of HIV-1 is "block and lock", which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. To suppress latent HIV-1 in a stable fashion, we depleted the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and were associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and induced DNA methylation at specific sites in the 5LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. Thus, epigenetic silencing by itself appears to be insufficient to permanently silence HIV-1 proviral transcription. Author SummaryThe "block and lock" strategy for a functional HIV-1 cure is based on the premise that permanent inactivation of the HIV-1 can be achieved by epigenetic silencing of the proviral DNA. For cellular genes, long-term silencing is achieved during cell differentiation by the induction of specific epigenetic modifications involving histone and DNA methylation. During HIV-1 silencing, histone methylation and DNA methylation are observed, but both sets of modifications can be reversed upon activation of T-cells through the T-cell receptor or potent latency reversing agents. In an attempt to enhance silencing of HIV-1 transcription, we used an inhibitor of H3K27 demethylases to increase H3K27 methylation. This in turn led to enhanced DNA methylation of HIV-1. Unfortunately, although the treatment effectively silenced HIV-1 and prevented viral reactivation, the silencing effects were short-lived and quickly reversed after removal of the drug.

microbiology↗

bioRxiv2021

Sex-specific multi-level 3D genome dynamics in the mouse brain

The female mammalian brain exhibits sex-hormone-driven plasticity during the reproductive period. Evidence implicates chromatin dynamics in gene regulation underlying this plasticity. However, whether ovarian hormones impact higher-order chromatin organization in post-mitotic neurons in vivo is unknown. Here, we mapped 3D genome of ventral hippocampal neurons across the estrous cycle and by sex in mice. In females, we found cycle-driven dynamism in 3D chromatin organization, including in estrogen-response-elements-enriched X-chromosome compartments, autosomal CTCF loops, and enhancer-promoter interactions. With rising estrogen levels, the female 3D genome becomes more similar to the male genome. Cyclical enhancer-promoter interactions are partially associated with gene expression and enriched for brain disorder-relevant genes. Our study reveals unique 3D genome dynamics in the female brain relevant to female-specific gene regulation, neuroplasticity, and disease risk.

neuroscience↗

bioRxiv2021

Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways

The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4+ T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4+ T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1. Author SummaryDissecting the cellular pathways through which HIV emerges from latency is a key step in the development of therapeutically viable approaches for latency reversal and eventual clearance of persistent HIV in infected individuals. The essential host transcription elongation factor P-TEFb, a heterodimer of CDK9 kinase and a regulatory cyclin T subunit, is a critical mediator of the trans-activation of latent HIV. Availability of P-TEFb for proviral transcription is highly limited due to a posttranscriptional restriction in cyclin T1 expression and dephosphorylation of CDK9 on its activation loop. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we have now defined the signaling pathways that are essential for the generation of transcriptionally active P-TEFb and, consequently, proviral reactivation. Crucial among these findings is the demonstration that protein kinase C (PKC) agonists signal through off-target activation of RasGRP1-Ras-Raf-MEK-ERK1/2 to effect the reactivation of both P-TEFb and proviral HIV. Understanding these pathways should lead to the discovery of novel highly selective activators of P-TEFb to improve the efficiency of HIV reactivation in the memory T-cell population of virally suppressed individuals.

molecular biology↗