Biology subjects

Kumar, P.

Publications and source records attributed to Kumar, P..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Essential role of hsa-miR-203a-3p in type I Interferons immune homeostasis during Influenza and NDV infection

MicroRNAs (miRNAs) are small, non-coding molecules that act as essential post-transcriptional regulators in various biological processes. Many studies suggest that miRNAs may modulate the hosts immune response during virus infections. We analyzed publicly available transcriptomics data involving infection with different RNA viruses and identified the most prominent candidate, miR-203a-3p. This miRNA is upregulated during H7N9, HCV, and DENV2 infections. Interestingly, pathway analysis of microRNA-targeted genes shows that miR-203a-3p targets multiple genes of the type-I interferon and JAK-STAT pathway. In this study, we reported the novel role of miR-203a-3p as it is elevated in response to polyinosinic-polycytidylic acid [poly(I:C)] transfection and infection with various RNA viruses such as Newcastle Disease Virus (NDV) and A/PR8/H1N1 influenza virus. We found that miR-203a-3p promotes the A/PR8/H1N1 virus replication by suppressing the hosts type-I interferons and interferon-stimulated genes. We demonstrated that the miR-203a-3p overexpression reduced the expression of ISGs and is attributed to the direct binding of miR-203a-3p to 3 UTRs of Janus-activated kinase 1 (JAK1), STAT1, SOCS3, and multiple IFNA transcripts. Altogether, these findings strongly suggest that miR-203a-3p plays a pivotal role in immune homeostasis by regulation of type I IFN and supports A/PR8/H1N1 and NDV infection by targeting multiple genes of the hosts type I IFNs and JAK-STAT signaling pathways.

immunology↗

bioRxiv2025

Discovery and therapeutic exploitation of Master Regulatory miRNAs in Glioblastoma

Glioblastoma is a fatal primary malignant brain tumor, with an average survival of only 15 months despite surgical resection, chemotherapy, and radiation therapy. Due to the concurrent deregulation of numerous genes in glioblastoma, molecular monotherapies have not improved clinical outcomes. Evidence suggests that effectively targeting multiple deregulated molecules is essential for better therapies; however, this is limited by the lack of suitable drugs and the increased toxicity of combination therapies. To address this, we hypothesized that miRNAs, small gene-regulatory RNAs that suppress multiple target genes via sequence complementarity, could be developed to inhibit multiple deregulated genes simultaneously, leading to more effective treatments. We identified master regulatory miRNAs--those that target several deregulated genes in glioblastoma--using PAR-CLIP screenings in glioblastoma cells and analyzed TCGA tumor data to find which targets were deregulated. An algorithm ranked these targets based on their significance in glioblastoma malignancy. We selected two tumor suppressor master regulatory miRNAs, miR-340 and miR-382, and one oncogenic miRNA, miR-17. Validation showed that these miRNAs target critical glioblastoma pathways and significantly inhibit cell growth, survival, invasion, and tumor growth in vivo. We developed an innovative therapeutic delivery approach using Brain Penetrating Nanoparticles in combination with MRI-guided focused ultrasound and microbubbles, resulting in reduced tumor volume and extended survival in glioblastoma-bearing mice. This strategy offers a promising pathway for translating miRNA-based therapies into clinical trials for glioblastoma and other cancers. One Sentence SummaryWe developed and used new computational, experimental, and therapeutic approaches to identify and therapeutically deliver master regulatory miRNAs to inhibit the growth of glioblastoma, the most common and deadly primary brain tumor.

cancer biology↗

bioRxiv2025

Simultaneous profiling of native-state proteomes and transcriptomes of neural cell types using proximity labeling

Deep molecular phenotyping of cells at transcriptomic and proteomic levels is an essential first step to understanding cellular contributions to development, aging, injury, and disease. Since proteome and transcriptome level abundances only modestly correlate with each other, complementary profiling of both is needed. We report a novel method called simultaneous protein and RNA -omics (SPARO) to capture the cell type-specific transcriptome and proteome simultaneously from both in vitro and in vivo experimental model systems. This method leverages the ability of biotin ligase, TurboID, to biotinylate cytosolic proteins including ribosomal and RNA-binding proteins, which allows enrichment of biotinylated proteins for proteomics as well as protein-associated RNA for transcriptomics. We validated this approach first using well-controlled in vitro systems to verify that the proteomes and transcriptomes obtained reflect the ground truth, bulk proteomes and transcriptomes. We also show that the effect of a biological stimulus (e.g., neuroinflammatory activation by lipopolysaccharide) can be faithfully captured. We also applied this approach to obtain native-state proteomes and transcriptomes from two key neural cell types, astrocytes and neurons, thereby validating the in vivo application of SPARO. Next, we used these data to interrogate protein-mRNA concordance and discordance across these cell types, providing insights into groups of molecular processes that exhibit uniform or cell type-specific patterns of mRNA-protein discordance.

neuroscience↗

bioRxiv2025

Resilience to Endoplasmic Reticulum Stress Mitigates Calcium-Dependent Membrane Hyperexcitability Underlying Late Disease Onset in SCA6

An enduring puzzle in many inherited neurological disorders is the late onset of symptoms despite expression of function-impairing mutant protein early in life. We examined the basis for onset of impairment in Spinocerebellar ataxia type 6 (SCA6), a canonical late-onset neurodegenerative ataxia which results from a polyglutamine expansion in the voltage gated calcium channel, Cav2.1. Cerebellar Purkinje cell spiking abnormalities are seen concurrent with motor impairment in SCA6 mice but the basis for these changes in spiking is unknown. We identify endoplasmic reticulum (ER) calcium depletion as the cause for Purkinje cell spiking abnormalities and that the impairments in Purkinje cell spiking are unrelated to Cav2.1 ion-flux function. Further, intact inhibitory neurotransmission in the cerebellar cortex is necessary for Purkinje neurons to exhibit spiking abnormalities in SCA6 mice. Based on serial cerebellar transcriptome analysis, we define a mechanism of disease that is related to ER stress. Further, our studies support a model whereby proteotoxicity from misfolded mutant Cav2.1 is mitigated by a HSP90-dependent unfolded protein response (UPR) and that age-related breakdown of this response causes motor dysfunction and aberrant Purkinje cell spiking. Redundant pathways of the UPR mediate this resilience to ER stress. These studies elucidate a mechanism of resilience connecting aberrant proteostasis and calcium-dependent intrinsic membrane hyperexcitability to explain delayed disease onset more widely in age-dependent neurodegenerative disease. Significance StatementAdvancing age is the single most important risk factor for neurodegenerative disease. Understanding how age intersects with genetic risk is therefore a critical challenge for neurodegenerative disease research. SCA6, a canonical late-onset degenerative cerebellar ataxia, results from a polyQ expansion in the voltage gated calcium channel, Cav2.1, encoded by CACNA1A. We define a mechanism of disease in SCA6 that is related to ER stress and unrelated to impaired calcium flux function of Cav2.1. Age-related decompensation of a HSP90-dependent unfolded protein response leads to disease onset. Mutant Cav2.1 misfolding as the basis for disease in SCA6 provides insight into a novel role for channelopathies to behave as proteinopathies and helps understand resilience to proteotoxicity more widely in adult-onset neurodegenerative disease.

neuroscience↗

bioRxiv2025

Assembly, Annotation, and Comparative Analysis of the Mitochondrial Genome of Winged Bean (Psophocarpus tetragonolobus): Insights into Evolutionary Adaptation and Codon Usage Bias

The winged bean (Psophocarpus tetragonolobus) is an underutilized, nutritionally dense legume with significant agricultural potential due to its adaptability to tropical environments and nitrogen-fixing capabilities. This study presents the assembly, annotation, and comparative analysis of its complete mitochondrial genome, spanning 366,925 bp. The genome comprises 64 genes, including 38 protein-coding genes, 12 tRNA genes, and 6 rRNA genes. Codon usage analysis revealed moderate bias in mitochondrial genes, primarily shaped by mutational pressures, while tRNA gene content aligns with codon preferences, optimizing translational efficiency. Phylogenetic analysis placed P. tetragonolobus within the Phaseoleae tribe, clustering closely with Glycine max and Vigna radiata, while synteny analysis highlighted high genomic conservation with related legumes. Ka/Ks ratio analysis underscored strong purifying selection in core mitochondrial genes, reflecting functional constraints essential for energy metabolism. Comparative analyses of mitochondrial and chloroplast genomes revealed distinct evolutionary pressures, with chloroplast genes exhibiting more significant variability influenced by functional roles in photosynthesis. These findings provide valuable insights into the evolution and adaptation of P. tetragonolobus, establishing a foundation for its potential application in sustainable agriculture and crop improvement programs.

genomics↗

bioRxiv2025

Identification of novel Kv1.3 channel-interacting proteins using proximity labelling in T-cells

Potassium channels regulate membrane potential, calcium flux, cellular activation and effector functions of adaptive and innate immune cells. The voltage-activated Kv1.3 channel is an important regulator of T cell-mediated autoimmunity and microglia-mediated neuroinflammation. Kv1.3 channels, via protein-protein interactions, are localized with key immune proteins and pathways, enabling functional coupling between K+ efflux and immune mechanisms. To gain insights into proteins and pathways that interact with Kv1.3 channels, we applied a proximity-labeling proteomics approach to characterize protein interactors of the Kv1.3 channel in activated T-cells. Biotin ligase TurboID was fused to either N or C termini of Kv1.3, stably expressed in Jurkat T cells and biotinylated proteins in proximity to Kv1.3 were enriched and quantified by mass spectrometry. We identified over 1,800 Kv1.3 interactors including known interactors (beta-integrins, Stat1) although majority were novel. We found that the N-terminus of Kv1.3 preferentially interacts with protein synthesis and protein trafficking machinery, while the C-terminus interacts with immune signaling and cell junction proteins. T- cell Kv1.3 interactors included 335 cell surface, T-cell receptor complex, mitochondrial, calcium and cytokine-mediated signaling pathway and lymphocyte migration proteins. 178 Kv1.3 interactors in T-cells also represent genetic risk factors of T cell-mediated autoimmunity, including STIM1, which was further validated using co-immunoprecipitation. Our studies reveal novel proteins and molecular pathways that interact with Kv1.3 channels in adaptive (T-cell) and innate immune (microglia), providing a foundation for how Kv1.3 channels may regulate immune mechanisms in autoimmune and neurological diseases.

cell biology↗

bioRxiv2025

Genetic mapping in Collaborative Cross mouse strains identifies loci that affect initial sensitivity to cocaine

We identified two Collaborative Cross (CC) strains, CC004/TauUncJ (CC004) and CC041/TauUncJ (CC041), that differ significantly for locomotor response and self-administration of cocaine. In the current study, we crossed each of these strains to C57BL/6NJ (B6N) mice to produce two F2 populations and identify genetic loci that influence locomotor response to cocaine. We identified three significant loci on chromosomes 7, 11 and 14 in the CC041 F2 mapping cross that collectively explain 14% of the phenotypic variance for locomotor response to cocaine. We used a bioinformatic approach to identify high quality candidate genes that are genetically plausible, have functional relevance and are suitable for further exploration. Our study is the first to use CC strains to perform QTL mapping for addiction-related phenotypes and proposes several candidate genes for follow-up analyses.

genetics↗

bioRxiv2024

The adaptive state determines the impact of mutations on evolving populations

Darwinian evolution results from an interplay between stochastic diversification of heritable phenotypes, impacting the chance of survival and reproduction, and fitness-based selection. The ability of populations to evolve and adapt to environmental changes depends on rates of mutational diversification and the distribution of fitness effects of random mutations. In turn, the distribution of fitness effects of stochastic mutations can be expected to depend on the adaptive state of a population. To systematically study the impact of the interplay between the adaptive state of a population on the ability of asexual populations to adapt, we used a spatial agent-based model of a neoplastic population adapting to a selection pressure of continuous exposure to targeted therapy. We found favorable mutations were overrepresented at the extinction bottleneck but depleted at the adaptive peak. The model-based predictions were tested using an experimental cancer model of an evolution of resistance to a targeted therapy. Consistent with the models prediction, we found that enhancement of the mutation rate was highly beneficial under therapy but moderately detrimental under the baseline conditions. Our results highlight the importance of considering population fitness in evaluating the fitness distribution of random mutations and support the potential therapeutic utility of restricting mutational variability. SIGNIFICANCE STATEMENTThe ability of a population to adapt and evolve is heavily influenced by the effects of random mutations on individuals. However, these effects can vary depending on the existing fitness level of the population. Using the development of cancer treatment resistance as an example, our research shows that populations nearing extinction can benefit from an increased rate of mutation. In contrast, mutations have a neutral or harmful effect on well-adapted populations. These findings suggest that new therapeutic strategies that manipulate mutation rates based on a populations current state of adaptation could be effective in preventing cancer and antimicrobial resistance.

evolutionary biology↗

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

Microglia modulate cerebral blood flow and neurovascular coupling through ectonucleotidase CD39

Microglia and the border-associated macrophages (BAMs) contribute to the modulation of cerebral blood flow (CBF), but the mechanisms have remained ill-defined. Here, we show that microglia regulate the CBF baseline and upsurges after whisker stimulation or intracisternal magna injection of adenosine triphosphate (ATP). Genetic or pharmacological depletion of microglia reduces the activity-dependent hyperemia but not the cerebrovascular responses to adenosine stimulation. Notably, microglia repopulation corrects these CBF reactivity deficits. The microglial-dependent regulation of CBF requires the ATP-sensing P2ry12 receptor and the ectonucleotidase CD39 that initiates the breakdown of extracellular ATP. Pharmacological inhibition or microglia-specific deletion of CD39 simulates the CBF anomalies detected in microglia-deficient mice and reduces the rise of extracellular adenosine after whisker stimulation. Together, these results suggest that the microglial CD39-initiated conversion of extracellular ATP to adenosine is an important step in neurovascular coupling and the regulation of cerebrovascular reactivity.

neuroscience↗

bioRxiv2024

Disruption of molecular interactions between G3BP1 stress granule host protein and nucleocapsid (NTD-N) protein impedes SARS-CoV-2 virus replication

The Ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a formidable barrier to viral replication by generating stress granules (SGs) in response to viral infections. Interestingly, viruses, including SARS-CoV-2, have evolved defensive mechanisms to hijack SG proteins like G3BP1 for the dissipation of SGs that lead to the evasion of hosts immune responses. Previous research has demonstrated that the interaction between the NTF2-like domain of G3BP1 (G3BP1NTF-2) and the intrinsically disordered N-terminal domain (NTD-N1-25) of the N protein plays a crucial role in regulating viral replication and pathogenicity. Interestingly, the current study identified an additional upstream stretch of residues (128KDGIIWVATEG138) (N128-138) within the N-terminal domain of the N protein (NTD-N41-174) that also forms molecular contacts with the G3BP1 protein, as revealed through in silico analysis, site-directed mutagenesis and biochemical analysis. Remarkably, WIN-62577, and fluspirilene, the small molecules targeting the conserved peptide binding pocket in G3BP1NTF-2, not only disrupted the protein-protein interactions (PPIs) between the NTD-N41-174 and G3BP1NTF-2 but also exhibited significant antiviral efficacy against SARS-CoV-2 replication with EC50 values of [~]1.8 {micro}M and [~]1.3 {micro}M, respectively. The findings of this study, validated by biophysical thermodynamics and biochemical investigations, advance the potential of developing therapeutics targeting the SG host protein against SARS-CoV-2, which may also serve as a broad-spectrum antiviral target.

biochemistry↗

bioRxiv2024

Antibody Profiles Elicited by Potent and Subpotent Whole Cell Pertussis Vaccines in Mice

Inactivated, whole cell pertussis (wP) vaccines remain at the frontline in the global fight against the resurgence of whooping cough, especially in low- and middle-income countries. However, the reliance on the intracerebral mouse potency test (ic-MPT or Kendrick assay) as the standard batch release assay is challenging for the production of commercial wP vaccines. The ic-MPT is technically challenging, labor intensive and, arguably, incongruous with modern animal welfare guidelines. Replacing the ic-MPT with a whole cell Bordetella pertussis ELISA, the so-called pertussis serology potency test (PSPT), has shown promise, but has been difficult to implement in practice. In this report, we tested the hypothesis that potent and subpotent wP vaccines have distinct serologic profiles in mice that could be developed as a substitute for the ic-MPT. We first established an accelerated decay (thermal stress) protocol in which wP, in the context of DTwP, was rendered >10-fold less effective than unstressed vaccine when evaluated in a mouse model of B. pertussis lung clearance following intranasal challenge. We then screened immune sera on a limited B. pertussis Tahoma I proteome array and identified >30 antigens whose antibody reactivity profiles increased, decreased or were unchanged as a function of wP potency. Interestingly, virtually all the "indicator" antigens identified are known virulence factors or reactive with human convalescent sera, thereby establishing a potential link between wP potency and pertussis infection and immunity. These results bode well for the development of a limited B. pertussis antigen array as a stability-indicating surrogate potency assay for the ic-MPT.

immunology↗

bioRxiv2024

The cyclin-G associated kinase (GAK) is a novel mitotic kinase and therapeutic target in diffuse large B-cell lymphoma

New drug targets are needed for diffuse large B-cell lymphoma (DLBCL), the most common lymphoma subtype, to enable enable development better treatments for patients not cured by standard care. We conducted a phenotypic screen of kinase inhibitors and identified the cyclin G-associated kinase (GAK) as a tumor-selective target. Though GAK is previously described primarily as a participant in membrane trafficking, we found its kinase activity is a key mitotic regulator in DLBCL. Inhibition caused G2/M-phase arrest, chromosome misalignment, and spindle distortion, effects absent in non-malignant controls. Transcriptomics data from clinical samples showed increased GAK expression associates with RB1 deficiency in DLBCL cases, suggesting dependency on GAK linked to retinoblastoma associated protein (RB) loss of function, a common DLBCL driver. RB-deficient DLBCL cells treated with a selective GAK tool compound showed complete arrest at G2/M, pronounced distortion of mitotic spindles, and widespread chromosomal damage. High-content live-cell imaging revealed onset of mitotic catastrophe in response to GAK inhibition, which was more rapid and severe in isogenic cells with RB1 deletion. No GAK-selective inhibitors suitable for clinical development are currently available, but several drugs approved or under development inhibit GAK activity even more potently than thier intended clinical targets. For instance, OTS167, developed against MELK for use in solid tumors, has particularly potent anti-GAK potency and has achieved single-agent tumor-burden reduction in vivo against a DLBCL patient-derived xenograft. GAK is therefore a novel mitotic kinase in DLBCL, linked to the common, undruggable RB loss of function biomarker, and suitable for rapid clinical translation through drug repurposing. SignificanceWe identify cyclin-G associated kinase (GAK) as a novel therapeutic vulnerability in diffuse large B-cell lymphoma. Clinical kinase inhibitors with GAK activity create an opportunity for rapid therapeutic translation through drug repurposing.

cancer biology↗

bioRxiv2024

Mechanistic insights into the inhibition of dengue virus NS5 methyltransferase by herbacetin

Herbacetin (HC) is a naturally occurring flavonoid compound with a dual antiviral mechanism. It inhibits the polyamine biosynthetic pathway and targets the methyltransferase (MTase) enzyme of the dengue and chikungunya virus. However, understanding the detailed inhibition mechanism of DENV MTase by HC remains unclear. The crystal structure of DENV3 nonstructural protein 5 (NS5) MTase in complex with HC and S-adenosyl-L-homocysteine presented in this study gives structural insights into the inhibition mechanism. Structural analysis revealed that HC binds to the cap 0 RNA site near the GTP binding site in the DENV3 NS5 MTase, and fluorescence polarization assay demonstrated HC inhibits GTP binding with an inhibition constant Ki value of [~] 0.43 {micro}M. This is the first study that identified an inhibitor that targets the conserved RNA-binding region of NS5 MTase, suggesting its potential as a highly effective scaffold for broad-spectrum antiviral agents against orthoflaviviruses. ImportanceDengue virus (DENV) is a vector-borne disease affecting global health, and no antiviral treatment is available for Dengue fever. DENV NS5 methyltransferase (DENV NS5 MTase) is a viral RNA capping enzyme essential for the virus replication. This study demonstrated that herbacetin (HC), a natural flavonoid, blocked the binding of GTP to NS5 MTase and inhibited the enzyme. In addition to DENV3, HC effectively inhibited the replication of DENV1,2, and 4 serotypes. The crystal structure of the DENV3 NS5 MTase in complex with HC revealed that HC binds at the cap 0 RNA binding site, which is close to the GTP binding site and thereby perturbs the spatial arrangement of GTP binding site residues. HC, as the first identified inhibitor that targets the conserved RNA-binding site of DENV3 NS5 MTase, presents a potential scaffold for the development of new antiviral drugs.

microbiology↗

bioRxiv2024

Dissecting compounded hepatocyte injury in a model of MASLD progression from human induced pluripotent stem cells

Drug discovery for multifactorial diseases like metabolic dysfunction-associated steatotic liver disease (MASLD) remains challenging due to inadequate models and untargeted drug screenings. We combined stem-cell-based modeling with computational drug predictions identifying flavin pathways as therapeutic targets in MASLD. For disease stage-specific discovery, we established a MASLD testing model, compounding metabolic triggers to intensify mitochondrial crisis. In vitro injuries included adipo- and myokines, immune cell co-culture, and genomic risk factors. Benchmarking experiments revealed similarities with advanced human MASLD. To query therapeutic compounds, protein-protein-interaction networks, weighted gene co-expression, and knowledge graph-based analyses independently predicted flavin adenine dinucleotide (FAD) as an anti-MASLD factor. Dysregulated flavoproteomes in vitro and in vivo-in pediatric and adult MASLD patients- supported our flavin network-focused strategy. We established therapeutic FAD concentrations to mitigate metabolic injury and fibro-inflammation in human multicellular liver organoids and other assays. We enhanced therapeutic FAD effects through genetic mitochondrial biogenic augmentation and identified orally available flavo-active compounds--including Aspirin--restoring mitochondrial respiration. Our study demonstrates how integrating stem cell-derived disease modeling with computed drug predictions can expedite therapeutic discovery.

cell biology↗

bioRxiv2024

Structural insights into the RNA binding inhibitors of the C-terminal domain of the SARS-CoV-2 nucleocapsid

The SARS-CoV-2 nucleocapsid (N) protein is an essential structural element of the virion, playing a crucial role in enclosing the viral genome into a ribonucleoprotein (RNP) assembly, as well as viral replication and transmission. The C-terminal domain of the N-protein (N-CTD) is essential for encapsidation, contributing to the stabilization of the RNP complex. In a previous study, three inhibitors (ceftriaxone, cefuroxime, and ampicillin) were screened for their potential to disrupt the RNA packaging process by targeting the N-protein. However, the binding efficacy, mechanism of RNA binding inhibition, and molecular insights of binding with N-CTD remain unclear. In this study, we evaluated the binding efficacy of these inhibitors using isothermal titration calorimetry (ITC), revealing the affinity of ceftriaxone (18 {+/-} 1.3 M), cefuroxime (55 {+/-} 4.2 M), and ampicillin (28 {+/-} 1.2 M) with the N-CTD. Further inhibition assay and fluorescence polarisation assay demonstrated RNA binding inhibition, with IC50 ranging from 10.4 to 12.4 M and KD values between 24 and 32 M for the inhibitors. Additionally, we also determined the inhibitor-bound complex crystal structures of N-CTD-Ceftriaxone (2.0 [A]) and N-CTD-Ampicillin (2.2 [A]), along with the structure of apo N-CTD (1.4 [A]). These crystal structures revealed previously unobserved interaction sites involving residues K261, K266, R293, Q294, and W301 at the oligomerization interface and the predicted RNA-binding region of N-CTD. These findings provide valuable molecular insights into the inhibition of N-CTD, highlighting its potential as an underexplored but promising target for the development of novel antiviral agents against coronaviruses. HighlightsO_LIThe inhibitors ceftriaxone, cefuroxime, and ampicillin-demonstrated high-affinity binding to the C-terminal domain (N-CTD) of the SARS-CoV-2 nucleocapsid (N) protein, effectively disrupting the formation of the N-CTD-RNA complex. C_LIO_LIComplex crystal structures of N-CTD with ceftriaxone and ampicillin revealed previously unobserved distinct binding sites. C_LIO_LIStructures reveal how the selected inhibitors disrupt the oligomerization of N-CTD and hinder the RNA packaging process of the virus. C_LI

biophysics↗

bioRxiv2024

Elucidation of α-glucosidase inhibitory activity and UHPLC-ESI-QTOF-MS based metabolic profiling of endophytic fungi Alternaria sp. BRN05. isolated from seeds of Swietenia macrophylla King.

There is a growing demand for new diabetes drugs with fewer side effects to replace current medications known for their adverse effects. Inhibition of -glucosidase responsible for postprandial hyperglycaemia among diabetes patients is a promising strategy for managing the disease. This study aims to explore and identify novel bioactive molecules with anti-diabetes potential from Alternaria sp. BRN05, an endophytic fungus isolated from a well-known medicinal plant Swietenia macrophylla King. Ethyl acetate extracts of Alternaria sp. BRN05 grown in full-strength (EFS) and quarter-strength (EQS) media respectively were evaluated for their -glucosidase inhibitory activities. Based on IC50 values, EQS exhibited significantly greater inhibitory activity (0.01482 {+/-} 1.809 mg/mL) as compared to EFS (1.16 {+/-} 0.173 mg/mL) as well as acarbose control (0.494 {+/-} 0.009 mg/mL). EFS and EQS were subjected to metabolic profiling using Ultra-High-Performance Liquid Chromatography - Electrospray Ionization - Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-ESI-QTOF-MS). A total of nineteen molecules from EFS and twenty from EQS were tentatively identified based on MS/MS fragmentation. Molecular docking analysis revealed that twelve among these exhibited greater binding energies than that of acarbose (-6.6 kcal/mol). Two molecules from EQS, 3,4,7-trihydroxyisoflavanone (-7.5 kcal/mol) and alternariol 9-methyl ether (-7 kcal/mol), were subjected to ensemble docking studies, which further strengthened their promise as good -glucosidase inhibitors for treating diabetes.

microbiology↗

bioRxiv2024

Concurrent large-scale brain dynamics during the emotional face matching task and their relation to behavior and mental health

Prior investigations of emotion processings neural underpinnings rely on a priori models of brain response, obscuring detection of task-relevant neurobiological processes with complex temporal dynamics. To overcome this limitation, we applied unsupervised machine learning to functional magnetic resonance imaging data acquired during the emotional face matching task (EFMT) in healthy young adults from the Human Connectome Project (n=413; n=416 replication). Tensorial independent component analysis showed that the EFMT engages 10 large-scale brain networks - each recruiting visual association cortex in distinct temporal fashions and in tandem with diverse non-visual regions - that collectively recruit 74% of cortex, posterior cerebellum, and amygdala. Despite prominent use of the EFMT to probe negative affect and related psychopathology, EFMT-recruited networks strongly reflected individual differences in cognition but not internalizing/negative affect. Overall, we characterize a richer-than-expected tapestry of concurrent EFMT-recruited brain processes, their diverse activation dynamics, and their relations to task performance and latent mental health phenotypes.

neuroscience↗