Biology subjects

Singh, U.

Publications and source records attributed to Singh, U..

At least 19 recordsLinked to original sources

bioRxiv2026

An age-preserving neuronal iSpheroid platform for modeling human aging and Alzheimer disease

Aging is the strongest risk factor for Alzheimer disease (AD), yet existing human neuronal models either erase age-associated features during reprogramming or fail to recapitulate three-dimensional organization of human neuronal tissue. Here, we establish directly induced neuronal spheroids (iSpheroids), a scalable three-dimensional platform generated by direct conversion of primary fibroblasts without pluripotency. iSpheroids generate long-lived, electrophysiologically active neurons from young, unimpaired aged, and sporadic AD donors while preserving donor-specific epigenetic age. Single-nucleus RNA sequencing revealed approximately 96% of cells acquired a neuronal identity and were distributed across multiple neuronal states, including a diversity of neurons. The platform supports incorporation of astrocytes and microglia and generation from non-human primate fibroblasts. AD iSpheroids spontaneously recapitulated neuronal degeneration, amyloid-beta; accumulation, progressive Tau pathology, and metabolic dysfunction. Integrated proteomic, metabolomic, and lipidomic profiling distinguished physiological aging from AD and revealed disease-associated metabolic remodeling. Oxaliplatin shifted AD iSpheroids toward oxidative metabolism, whereas a multi-compound screen identified trametinib as a modulator of inflammatory and age-associated programs.

neuroscience↗

bioRxiv2026

Deep analysis of FANTOM CAGE data reveals hierarchical patterns of TSS co-deployment hubs and their disruption in cancers

Selective deployment of multiple transcription start sites is a major regulatory feature of human transcriptomes. FANTOM CAGE data exhibit a near-universal TSS deployment parsimony which is disrupted in cancers. We have recently shown that TSS deployment is sensitive to gene function, futile upstream transcription, and cellular biosynthetic states. Patterns in FANTOM CAGE data can reveal mechanisms underlying TSS co-deployments. We propose and test the possibility that some TSSs act like epromoters and act as co-varying hubs of transcriptional activities for multiple other promoters. Using deep analysis of CAGE data implemented through neural networks we show that non-cancers implement transcription co-deployments through cores of epromoter-like TSSs which are generally proximal to their start codons. These TSSs show enhancer-like TFBSs profiles. A comparison with cancer CAGE data shows that the concentrated epromoter core is disrupted in cancers with multiple distal TSSs replacing the proximal TSS cores. We provide evidence that the core TSSs are rich in YY1 and CTCF binding sites and associated with genes coding for transcription factors. Our findings show that covariance of TSS deployment is sensitive to transcriptional resource cost and a parsimonic design of TSS co-deployments depends on proximal TSSs in non-cancers, a mechanism grossly disrupted in cancers. HighlightsO_LIHeterogeneous FANTOM CAGE data contains universal patterns of TSSs co-deployments. C_LIO_LITSS co-deployments exhibit a parsimonious "core-covariant" scheme which is disrupted in cancers. C_LIO_LICore TSSs are enriched in transcription factor binding sites and gene functions which justify biological features of the samples. C_LIO_LIThe DL pipeline we present identifies the core-covariant TSS sets in an unbiased manner. C_LI

genomics↗

bioRxiv2025

Gene functions determine stochastic or adaptive futile transcription in cancers through deregulated start site deployments

BackgroundCancers display near-universal hallmarks, including energy addiction, aerobic glycolysis and biosynthesis. In addition to glucose (and glutamine) addiction, cancers also display futile transcription. Discretely measurable futile transcription occurs between transcription start sites (TSSs) and start codons of protein-coding genes. Multiple TSSs for each gene offer various combinations of futile transcription with no effect on the encoded proteins. The relationship between proximal versus distal TSS deployment, futile transcription and energy addiction of cancers remain unclear. MethodsBy analyzing FANTOM CAGE data we show that TSS deployment dysregulation in cancers increases the energy cost coefficients of cancer transcriptomes. We define the bases of the altered energy cost coefficients of cancer transcriptomes by comparing TSS deployment frequencies, associated entropies and futile transcription distances across heterogeneous pools of cancers and non-cancers. ResultsWe show that TSS deployment entropies differ between cancers and non-cancers. It leads to an overall higher distal TSS deployment in cancers but selectively favors an energy- economical proximal TSS deployment for cancer hallmark genes involved in cell proliferation and biosynthesis. We show that the frequency of TSS deployment is linked to the futile transcription distance and gene function differently in cancers and non-cancers. ConclusionsThis work lays out a theoretical framework describing stochasticity of TSS deployment in the context of cancerogenesis and energetics of transcription. It also suggests that the current human TSS landscape has evolved to minimize futile transcription, an outcome favored by stochastic TSS deployment. Under normal conditions of energy metabolism they form a threshold which incipient cancer cells breach. Highlights Stochastic TSS deployment favors transcription from proximal TSSs. Proximal or distal TSS deployment depends on gene function. Cancers generally deploy distal TSSs at a high resource cost. Key cancer hallmark supporting genes shift to proximal TSSs even in cancers.

cancer biology↗

bioRxiv2025

CGGBP1-regulated heterogeneous C-T transition rates relate with G-quadruplex potential of terrestrial vertebrate genomes

The formation of G-quadruplexes (G4) is fundamentally linked to GC content of the DNA. There is little evidence for evolutionary selection of G4 stabilizers; G4 formation depends strictly on inherent sequence properties. Vertebrate promoters are notable for their consistently high GC-content as well as pronounced G/C strand asymmetry. Genomic GC content has changed in the course of evolution of terrestrial vertebrates, especially the amniotes but its relationship with the potential to form G4 remains less well understood. By analyzing genomes of 101 amniotes we report that lineage-specific differences in association between GC-content and G4 formation potential (pG4) in amniotes are concentrated at 1kb promoter regions. To understand possible mechanisms underlying high GC-concentrations in pG4 of mammalian and avian promoters, we test the possibility of selective cytosine methylation restriction leading to G/C-retention by CGGBP1, a protein involved in mitigation of cytosine methylation as well as G4-formation. By analyzing promoterome-wide C-T transition rates at TFBSs in pG4s we show that mammalian and avian CGGBP1s preserve C through methylation restriction. Our approach involves a combined meta-analysis of (i) genomes and promoters of 101 amniote genomes classified into reptilian, avian and mammalian classes through PQS finder and FIMO for 1019 JASPAR vertebrate motifs, (ii) recently reported global cytosine methylation patterns affected by overexpression of amniotic or non-amniotic forms of CGGBPs, (iii) and PWM reconstruction of motifs differentially enriched in pG4s showing homeothermic specific GC-retention. Our findings suggest that cytosine methylation restriction by CGGBP1 shapes G4 forming profiles of vertebrate promoters by preserving C on the G4-complementary strand resulting in minute differences in TFBSs across amniotes. HighlightsO_LIEvolution of vertebrate genome G4-forming potential mirrors that of CGGBP1. C_LIO_LICGGBP1 prevents meC-to-T mutations at pG4 sites, preserving G4-forming potential specifically in amniote promoters. C_LIO_LIpG4-TFBS mutations reveal homeothermic GC-retention bias, mirroring CGGBP1-mediated pG4 protection patterns. C_LIO_LIThe G4-forming regions most responsive to CGGBP1 exemplify this methylation-linked defense against C-to-T substitutions, distinguishing homeotherms from poikilotherms. C_LI

genomics↗

bioRxiv2025

Probiotic and postbiotic treatment in situ during a marine heat wave improves coral health and promotes specific metabolic and microbiome changes

Microbial therapies are emerging as promising tools for coral protection against heat stress, yet such application was not tested in situ. We tested two coral-derived probiotic consortia and their heat-killed counterparts (postbiotics) on bleaching Acropora cf. valida in the Red Sea during a marine heatwave. Over 15 days, both live and one of the heat-killed treatments maintained photosynthetic efficiency (Fv/Fm), whereas placebo-treated corals exhibited significant thermal stress-induced decline. 16S rRNA gene sequencing profiling showed enrichment of putatively beneficial genera (e.g., Terasakiispira spp., Pseudoalteromonas spp.), and untargeted metabolomics resolved treatment-specific metabolic signatures that differed among consortia and between live and inactivated formulations. These molecular fingerprints illuminate specific underlying protective mechanisms and host-microbiome interactions under heat stress. Together, our results position microbial therapies (probiotics and specific postbiotics) as field-validated, mechanism-informed interventions capable of minimizing impacts on corals during real-world thermal extremes and provide design cues for scalable deployment.

microbiology↗

bioRxiv2024

CGGBP1 from higher amniotes restricts cytosine methylation and drives a GC-bias in transcription factor binding sites at repressed promoters

CGGBP1, a 20 kDa protein, has several functions associated with its DNA-binding through a C2H2 zinc finger. A range of studies have shown that GC richness, inter-strand G/C-skew and low cytosine methylation are associated with CGGBP1 occupancy. The non-preference of any sequence motif as CGGBP1 binding site suggests widespread association of CGGBP1 with DNA including at potent transcription factor binding sites (TFBSs) in promoter regions. The evolutionary advantage of such a design remains unclear. The regulatory interference by human CGGBP1 at TFBSs is supported by purifying selection in the DNA-binding domain of CGGBP1 and its requirement for gene repression as well as restriction of cytosine methylation at GC-rich TFBSs. Here we describe an evolutionary trajectory of this property of CGGBP1 by combining global gene expression and cytosine methylation analyses on human cells expressing CGGBPs from four different vertebrates (representatives of coelacanth, reptiles, aves and mammals). We discover a potent cytosine methylation restriction by human CGGBP1 at some GC-rich TFBSs in repressed promoters. Further, we combine a high-throughput analysis of GC compositional bias of these CGGBP-regulated TFBSs from available orthologous sequences from a pool of over 100 species. We show that cytosine methylation restriction by CGGBP1 is tightly linked to GC retention in a set of TFBSs. Orthology analyses demonstrate that this property of CGGBPs has evolved in higher amniotes (aves and mammals) with lineage-specific heterogeneities in lower amniotes (reptiles). CGGBP1 ChIP-seq data suggest that occupancy of CGGBP1 at these target TFBSs plays a crucial role in their low methylation, GC-biased evolution and associated functions in gene repression. HighlightsO_LIResemblances in gene repression by overexpression of CGGBP1 from higher amniotes (Homo sapiens and Gallus gallus) is enhanced upon heat stress and differs from the non-repressive effects of lower amniotic CGGBPs (Anolis carolinensis and Latimeria chalumnae). C_LIO_LIGene repression by higher amniotic CGGBP1 is associated with restriction of cytosine methylation at specific GC-rich TFBSs in 1 kb promoters of target genes. Lower amniotic CGGBPs allow TFBS cytosine methylation and C-T transitions. C_LIO_LIOrthologs of CGGBP1-repressed genes from >100 vertebrates show signs of accelerated C-T losses explicitly in the TFBSs at which higher amniotic CGGBP1 restricts cytosine methylation. Such a TFBS GC-loss difference between lower and higher amniotes is restricted to genes repressed by higher amniotic CGGBP1 at physiological temperature, not heat stress. C_LIO_LIThis higher amniote-specific cytosine methylation restriction by CGGBP1 has likely influenced the differences between GC-rich TFBS composition and their abundance in target gene promoters throughout vertebrate evolution. C_LI SummaryEvolution of transcription factor binding sites (TFBSs) depends on a variety of factors including cytosine methylation-associated C-T transition rates. Most of our understanding of TFBS evolution is based on omic-scale sequence comparisons with only circumstantial evidence for the relationship between the TFBSs and physiological adaptation. We report a TFBS landscaping function for CGGBP1 by expressing its different taxon-derived forms in human cells through profiling of global gene expression and cytosine methylation alongside a meta-analysis of C-T transition rates from over 100 vertebrae genomes. We show that CGGBP1 from higher amniotes restricts cytosine methylation and maintains GC-rich TFBSs in target gene promoters for repression. This epigenetic affection of TFBS evolution by CGGBP1 is selectively seen at genes repressed at physiological temperature only and not under heat stress when gene repression by CGGBP1 becomes largely transcription factor binding site independent. Our findings connect epigenetic mechanisms to cellular physiology through TFBS evolution linked with changes in CGGBP1.

evolutionary biology↗

bioRxiv2024

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

microbiology↗

bioRxiv2024

The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

CGGBP1 is a GC-rich DNA-binding protein which is important for genomic integrity, gene expression and epigenome maintenance through regulation of CTCF occupancy and cytosine methylation. It has remained unclear how CGGBP1 integrates multiple diverse functions with its simple architecture of only a DNA-binding domain tethered to a C-terminal tail with low structural rigidity. We have used truncated forms of CGGBP1 with or without the DNA-binding domain (DBD) to assay cytosine methylation and global gene expression. Proximal promoters of CGGBP1-repressed genes, although significantly GC-poor, contain GC-rich transcription factor binding motifs and exhibit base compositions indicative of low C-T transition rates due to prevention of cytosine methylation. Genome-wide analyses of cytosine methylation and binding of CGGBP1 DBD show that CGGBP1 restricts cytosine methylation in a manner that depends on its DBD and its DNA-binding. The CGGBP1-repressed genes show an increase in promoter cytosine methylation alongside a decrease in transcript abundance when the DBD-deficient CGGBP1 is expressed. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss and thereby regulates gene expression. By analysing orthologous promoter sequences we show that restriction of cytosine methylation is a function of CGGBP1 progressively acquired during vertebrate evolution. A superimposition of our results and evolution of CGGBP1 suggests that mitigation of cytosine methylation is majorly achieved by its N-terminal DBD. Our results position CGGBP1 DNA-binding as a major evolutionarily acquired mechanism through which it keeps cytosine methylation under check and regulates TFBS retention and gene activity.

molecular biology↗

bioRxiv2024

Sex differences and immune correlates of Long COVID development, persistence, and resolution

Sex differences have been observed in acute COVID-19 and Long COVID (LC) outcomes, with greater disease severity and mortality during acute infection in males and a greater proportion of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to the pathogenesis of LC. To investigate the immunologic underpinnings of LC development and persistence, we used single-cell transcriptomics, single-cell proteomics, and plasma proteomics on blood samples obtained during acute SARS-CoV-2 infection and at 3 and 12 months post-infection in a cohort of 45 patients who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Specifically, males who would develop LC at 3 months had widespread increases in TGF-{beta} signaling during acute infection in proliferating NK cells. Females who would develop LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, and increased IL1 signaling in monocytes at 12 months post infection. Several immune features of LC were also conserved across sexes. Both males and females with LC had reduced co-stimulatory signaling from monocytes and broad upregulation of NF-{kappa}B transcription factors. In both sexes, those with persistent LC demonstrated increased LAG3, a marker of T cell exhaustion, reduced ETS1 transcription factor expression across lymphocyte subsets, and elevated intracellular IL-4 levels in T cell subsets, suggesting that ETS1 alterations may drive an aberrantly elevated Th2-like response in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics. One Sentence SummaryThis multi-omic analysis of Long COVID reveals sex differences and immune correlates of Long COVID development, persistence, and resolution.

immunology↗

bioRxiv2024

A pan-tissue, pan-disease compendium of human orphan genes

Species-specific genes are ubiquitous in evolution, with functions ranging from prey paralysis to survival in subzero temperatures. Because they are typically expressed under limited conditions and lack canonical features, such genes may be vastly under-identified, even in humans. Here, we leverage terabytes of human RNA-Seq data to identify thousands of highly-expressed transcripts that do not correspond to any Gencode-annotated gene. Many may be novel ncRNAs although 80% of them contain ORFs that have the potential of encoding proteins unique to Homo sapiens (orphan genes). We validate our findings with independent strand-specific and single-cell RNA-seq datasets. Hundreds of these novel transcripts overlap with deleterious genomic variants; thousands show significant association with disease-specific patient survival. Most are dynamically regulated and accumulate selectively in particular tissues, cell-types, developmental stages, tumors, COVID-19, sex, and ancestries. As such, these transcripts hold potential as diagnostic biomarkers or therapeutic targets. To empower future discovery, we provide a compendium of these huge RNA-Seq expression data, and RiboSeq data, with associated metadata. Further, we supply the gene models for the novel genes as UCSC Genome Browser tracks.

evolutionary biology↗

bioRxiv2024

Proteomic analysis of gut in Labeo rohita reveals ECM as Key Player in host's Response to Aeromonas hydrophila Infection

In the aquaculture sector, one of the challenges include disease outbreaks such as bacterial infections, particularly from Aeromonas hydrophila (Ah), impacting both wild and farmed fish. In this study, we conducted a proteomic analysis of the gut tissue in Labeo rohita following Ah infection to elucidate the protein alterations and its implications for immune response. Our findings reveal significant dysregulation in extracellular matrix (ECM) associated proteins during Ah infection, with increased abundance of elastin and Collagen alpha-3(VI) contributing to matrix rigidity. Pathway and enrichment analysis of differentially expressed proteins (DEPs) highlights the involvement of ECM-related pathways, including Focal adhesions, Integrin cell surface interactions, and actin cytoskeleton organization. Focal adhesions, crucial for connecting intracellular actin bundles to the ECM, play a pivotal role in immune response during infections. Increased abundance of integrin alpha 1, integrin beta 1, and Tetraspanin suggests their involvement in the hosts response to Ah infection. Proteins associated with actin cytoskeleton reorganization, such as myosin, tropomyosin, and phosphoglucomutase, exhibit increased abundance, influencing changes in cell behavior. Additionally, upregulated proteins like LTBP1 and Fibrillin-2 contribute to TGF-{beta} signaling and focal adhesion, indicating their role in immune regulation. The study also identifies elevated levels of laminin, galectin 3, and tenascin-C, which interact with integrins and other ECM components, influencing immune cell migration and function. These proteins, along with decorin and lumican, act as immunomodulators, coordinating pro- and anti-inflammatory responses. ECM fragments released during pathogen invasion serve as "danger signals," initiating pathogen clearance and tissue repair through Toll-like receptor signaling. IMPORTANCEThe study underscores the critical role of the extracellular matrix (ECM) and its associated proteins in the immune response of aquatic organisms during bacterial infections like Aeromonas hydrophila (Ah). Understanding the intricate interplay between ECM alterations and immune response pathways provides crucial insights for developing effective disease control strategies in aquaculture. By identifying key proteins and pathways involved in host defense mechanisms, this research lays the groundwork for targeted interventions to mitigate the impact of bacterial infections on fish health and aquaculture production.

microbiology↗

bioRxiv2023

Red Fluorescent Carbon Nanoparticles Derived from Spinacia oleracea L.: A Versatile Tool for Bioimaging & Biomedical Applications

Carbon-based fluorescent quantum dots are an emerging class of nanoparticles for targeted bioimaging and biomedical applications. We present a facile microwave-assisted approach for synthesizing carbon nanoparticles with bright red fluorescence using ethanolic extracts of Spinacia oleracea leaves, with a quantum yield of 94.67%. These nanoparticles, called CNPs, ranging from 15-50 nm, demonstrated fluorescence emission in the near-infrared (NIR) region between 650 and 700 nm, independent of excitation wavelength. Upon excitation at a wavelength of 410 nm, they exhibit an emission maxima peak at 672 nm. The significant uptake of CNPs in mammalian cells and zebrafish larvae highlights their potential as bioimaging agents in diverse biomedical applications in vivo. Further, these quantum dots enhance cellular proliferation and migration as observed by wound healing assay in mammalian cells, indicating their possible application in tissue engineering and regenerative medicine. These findings suggest that biosynthesized carbon nanoparticles possess significant potential for biomedical activities, which can serve as a robust benchmark for researchers towards promoting sustainability.

bioengineering↗

bioRxiv2023

Psidium guajava derived carbon nanoparticles: A promising red emissive cellular bioimaging agent

We report a simple, cost-effective, microwave-assisted green synthesis route of red-emitting fluorescence carbon nanoparticles (CNPs) using Psidium guajava (Guava leaves). The synthesis of CNPs is a simple, affordable, and rapid method of producing carbon nanoparticles. The CNPs were characterized by various spectroscopic and microscopic techniques. Atomic force microscopy studies showed that the average size of CNPs is approximately 50 nm. The CNPs exhibited excellent photoluminescence properties with a maximum emission at 677 nm, making them suitable for bioimaging applications. The Ionic, photostability, and thermal stability of CNPs were also checked to understand their robustness. Retinal pigment epithelium (RPE) cells were exposed to these nanoparticles and showed very efficient uptake, some fraction of it also getting targeted to the nucleus, indicating that CNPs are non-toxic and biocompatible for future biological experiments. The results indicate that guava leaves can be a promising source for the synthesis of red emissive CNPs through the very simple method of synthesis and with bioimaging applications.

bioengineering↗

bioRxiv2023

Tomato brown rugose fruit virus Mo gene is a novel microbial source tracking marker

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker. ImportanceHuman exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of fecal host-associated MST markers. Here we design and test novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool, and highly abundant in human stool and wastewater samples.

microbiology↗

bioRxiv2022

Accuracy of functional gene community detection in Saccharomyces cerevisiae by maximizing Generalized Modularity Density

Identifying functionally-cohesive gene communities from large data sets of expression data for individual genes is a key approach to understanding the molecular components of biological processes. Here, we compare the accuracy of twelve different approaches to infer gene co-expression networks and then find gene communities within the networks. Among the approaches used are ones involving a recently developed clustering method that identifies communities by maximizing Generalized Modularity Density (Qg). RNA-Seq data from 691 samples of S. cerevisiae (yeast) are analyzed. These data have been obtained from organisms grown under diverse environmental and developmental conditions and encompass varied mutant lines. To assess the accuracy of different approaches, we introduce a statistical measure, the Average Adjusted Rand Index (AARI) score, which compares their results to Gene Ontology (GO) term associations. Inferring gene networks using the Context Likelihood of Relatedness (CLR) and subsequently clustering by maximizing Generalized Modularity Density is found to identify the most significant functional communities. Also, to quantify the extent to which the identified communities are biologically relevant, a GO term enrichment analysis is performed. The results indicate that many of the communities found by maximizing Generalized Modularity Density are enriched in genes with known biological functions. Furthermore, some of the communities contain genes of unknown function, enabling inference of potentially novel functional interactions involving these genes. Furthermore, some genes are species-specific orphan genes; assignment of these orphan genes to communities enriched in a particular biological process provides a method to infer the biological process in which they are involved. We focus on a few communities that are highly significantly enriched in a particular biological process, and develop experimentally-testable predictions about the orphan genes in these communities. Author summaryFinding gene communities that are of biological relevance from expression profiles of individual genes is a critical approach to understanding biological processes and their molecular components. Various computational methods have been developed to infer underlying metabolic and regulatory networks and to identify functional communities of genes. Which network inference and clustering methods works best to achieve this goal has largely remained an open question. Here, using genome-wide transcriptomic data for S. cerevisiae, we systematically compare the effectiveness of several commonly used network inference and clustering methods. We rank these methods by comparing the clusters obtained by different methods to Gene Ontology (GO) terms. We find that inferring gene networks using a method known as the Context Likelihood of Relatedness (CLR) and subsequently clustering by maximizing Generalized Modularity Density identifies the most significant functional communities.

systems biology↗

bioRxiv2022

G-quadruplex landscape and its regulation revealed by a new antibody capture method

Our understanding of DNA G-quadruplexes (G4s) from in vitro studies has been complemented by genome-wide G4 landscapes from cultured cells. Conventionally, the formation of G4s is accepted to depend on G-repeats such that they form tetrads. However, genome-wide G4s characterized through high-throughput sequencing suggest that these structures form at a large number of regions with no such canonical G4-forming signatures. Many G4-binding proteins have been described with no evidence for any protein that binds to and stabilizes G4s. It remains unknown what fraction of G4s formed in human cells are protein-bound. The G4-chromatin immunoprecipitation (G4-ChIP) method hitherto employed to describe G4 landscapes preferentially reports G4s that get crosslinked to proteins in their proximity. Our current understanding of the G4 landscape is biased against representation of G4s which escape crosslinking as they are not stabilized by protein-binding and presumably transient. We report a protocol that captures G4s from the cells efficiently without any bias as well as eliminates the detection of G4s formed artifactually on crosslinked sheared chromatin post-fixation. We discover that G4s form sparingly at SINEs. An application of this method shows that depletion of a repeat-binding protein CGGBP1 enhances net G4 capture at CGGBP1-dependent CTCF-binding sites and regions of sharp interstrand G/C-skew transitions. Thus, we present an improved method for G4 landscape determination and by applying it we show that sequence property-specific constraints of the nuclear environment mitigate G4 formation.

genomics↗

bioRxiv2022

Novel class of yellow emitting carbon dots stimulate collective cell migration and 3D uptake in vivo

We present a new class of nitrogen-doped yellow fluorescent carbon dots, synthesized using a one-step hydrothermal method. These bright fluorescent nanoparticles have excitation and emission spectra near the red region of the visible light spectrum that are quite useful for bioimaging applications. Using organic molecules like ortho- phenylenediamine (OPDA), L-ascorbic acid and urea, yellow fluorescent carbon dots (CDs) were synthesized. We obtained a scalable number of CDs having an average size of 3 nm. The CDs show significant emission spectra in the yellow fluorescence region ({lambda}em= 557 nm). The CDs show remarkable stability in their fluorescence in different pH conditions, ionic stability, photostability as well as thermal stability. These CDs are efficiently uptaken by mammalian cells through clathrin-mediated pathway. Apart from in vitro studies we have also used zebrafish larvae as a 3D in vivo model, and showed that CDs were uptaken efficiently by larvae showing maximum accumulation and fluorescence in the yolk sac region and the notochord region. The CDs also offer enhancement in cell proliferation, hence showing the application in wound healing. The fluorescence of CDs is quite robust and is not affected by most external stimuli, hence can be explored as a promising bioimaging tool for targeted bioimaging and biomedical applications.

bioengineering↗

bioRxiv2022

Hypomorphism of a novel long ERα isoform causes severe reproductive dysfunctions in female mice

Estrogen receptor alpha (ER)-mediated estrogen signaling play a pivotal role in both reproductive and non-reproductive functions. Transcriptional regulation of ER gene is highly complex, with multiple transcript variants being differentially produced across the tissues. However, tissue-specific variation and physiological specificity of the ER variants are not yet fully understood. In an attempt to generate a Cre-dependently restorable ER-null mice for functional investigation of the genetic sufficiency, we unexpectedly produced ER hypomorphic mice with biased downregulation of a previously unappreciated long ER isoform that is enriched in the female reproductive organs (uterus and ovaries) and the pituitary but minimally expressed in the brain. Female homozygous mutant mice were capable of pregnancy but displayed irregular estrus cycle and rarely maintained alive newborns without significant morphological and pathological changes in reproductive system and the disruption of body weight homeostasis, indicating the vital role of this long isoform in female reproductive function. Collectively, our results define a tissue-specifically enriched long ER isoform and its preferential role in female reproductive function over body weight homeostasis.

physiology↗