Biology subjects

Sharma, M.

Publications and source records attributed to Sharma, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Differential modulation of Interferon and Cell Death Responses defines Human vs Avian Influenza A Virus Strain-Specific Virulence and guides Combination Therapy.

Influenza A virus (IAV) poses a significant global health risk, with highly pathogenic strains like H5N1 (CFR [~]52%) causing severe disease compared to less lethal but more transmissible strains like H1N1 (CFR 0.01-0.03%). Although IAV primarily infects lung epithelial cells, causing cell death and tissue damage, the molecular basis of strain-specific pathogenesis remains poorly understood. Here we show that in cell culture, H5N1 induced more rapid and extensive cell death than H1N1. Since Interferon (IFN) signaling is key to innate immunity, we examined its role in virus-induced cell death using STAT1-knockout A549 cells and JAK/STAT pathway inhibitors like Baricitinib. Both approaches reduced cell death across various IAV strains, including H1N1, H5N1, H7N9, and H3N2. However, inhibition increased viral titers, raising concerns about its clinical use in isolation. To overcome this, we tested a combination of Oseltamivir (antiviral) and Baricitinib (anti-inflammatory). Post-infection treatment in a murine model reduced lung inflammation and improved survival. Given that both drugs are FDA-approved, this approach has strong translational potential for clinical IAV treatment.

microbiology↗

bioRxiv2025

Convolvulus pluricaulis mediates its pharmacological effects via sod1, rdl, glut1, GABA-B-R1 and CG6293 orthologs in Drosophila melanogaster

Convolvulus pluricaulis, commonly known as Shankhpushpi, has been extensively used in the management of disorders of the nervous system, including depression and anxiety. The plant extract has also been demonstrated to function as an antioxidant. However, the molecular effectors underlying the beneficial effects of Shankpushpi are yet to be identified. In this study, we have utilized the fruit fly Drosophila melanogaster to identify the metabolic and molecular targets that mediate the beneficial effects of dietary Shankpushpi intake. Metabolomic analysis revealed changes in Ascorbic acid, glucose, and Adenosine monophosphate in the head tissue of fruit flies that were fed Shankpushpi for 20 days. Subsequent gene expression analysis revealed significant changes in expression of glut 1 (Glucose transporter 1), CG6293 (Ascorbate transporter), rdl (Resistant to dieldrin), GABA-B-R1 (GABA-B receptor subtype 1) and sod 1 (Superoxide dismutase 1) in the head tissue of adult flies that were exposed to different doses of Shankpushpi. Consistent with the expression data, knockdown of sod 1, glut1, GABA-B-R1, and CG6293 but not sod 2 abolished the Shankpushpi-mediated resistance to paraquat-induced oxidative stress. To uncover downstream effectors that are responsible for the antidepressant and anxiolytic effects of Shankpushpi, we examined the effects of dietary intake of Shankpushpi in a stress-induced model of depression. Administration of Shankpushpi during development alleviated depression-like behavior in Drosophila. Wild-type adult flies that were fed Shankpushpi accumulated high levels of Ascorbate in the head tissue, and knockdown of Ascorbate transporter abolished the antidepressant activity of Shankpushpi in the stress-induced model. Thus, indicating that Shankpushpi-induced antidepressant effects are associated with increased Ascorbate transport. Taken together, our analysis using Drosophila melanogaster as a model has uncovered five conserved downstream effectors responsible for the antioxidant activity and one conserved effector responsible for the antidepressant activity of Convolvulus pluricaulis.

molecular biology↗

bioRxiv2025

Genomic analysis uncovers unique haplotype signatures from subspecies and agronomic types associated with blanchability in groundnut

Blanchability, defined as the ease of seed coat removal after roasting, is a vital trait for enhancing processing efficiency and product quality in groundnut (Arachis hypogaea L.). To enable a comprehensive haplotype-level genetic dissection of this trait, SNPs derived from whole-genome resequencing (WGRS) were used to perform genome-wide association study (GWAS) using multi-locus models (BLINK and FarmCPU) on a diverse groundnut mini-core collection phenotyped across two crop seasons. A total of 26 significant SNP-trait associations (STAs) were identified across multiple chromosomes, with major loci on chromosomes Ah05, Ah06, and Ah17, some of which were further validated using KASP (Kompetitive Allele-Specific PCR) markers. Candidate genes, such as those involved in cell wall biosynthesis (e.g., galactoside 2-alpha-L-fucosyltransferase-like protein, protein kinase superfamily members, and glycerophosphoryl diester phosphodiesterase 3), were found to be within linkage disequilibrium (LD) of the identified STAs, suggesting their plausible association with blanchability. Haplo-pheno analyses identified superior high-blanchability haplotypes; Ah05HapBL3, Ah06HapBL5, Ah06HapBL10, and Ah17HapBL6, which were predominantly found in the fastigiata subspecies (including Valencia and Spanish bunch agronomic types) from South Asia and South America, while the low-blanchability haplotypes were from hypogaea subspecies (including Virginia runner and Virginia bunch agronomic types) from Africa. Overall, this study provides valuable insights for customizing blanchability through haplotype-based breeding of processing-grade cultivars thereby improving groundnut value chains to meet diverse industrial demands.

genomics↗

bioRxiv2025

Mapping Fusarium Wilt and Sterility Mosaic Disease Resistance-Associated Genomic Regions and Haplotype Variants in Pigeonpea

The occurrence of fusarium wilt and sterility mosaic disease in genial conditions causes significant yield losses in pigeonpea. The present genome-wide association study (GWAS) was employed on 176-genotype panel to identify candidate genomic regions associated with FW and SMD resistance in pigeonpea. A total of 869,447 filtered SNP markers were used for GWAS analysis. GWAS analysis identified significant genomic regions for FW on chromosomes 05, 10, and 11 and for SMD on chromosome 09. Four resistant sources, namely, ICP20096, ICP20097, ICPL87119 and ICP13304 were identified as a resistance source for both FW and SMD. Total 12 markers from chromosome 11 for FW and 4 markers from chromosome 09 for SMD were validated using the KASP genotyping approach. We have identified resistant haplotypes for FW on chromosome 10 and 11 and for SMD on chromosome 09. Moreover, we have identified four InDels for FW on chromosome 5 in Two component response regulator gene (Cc_11098) and four InDels for SMD on chromosome 9 in genes, namely, LRR and NB-ARC domain disease resistance protein (Cc_21069), Flavonoid 3, 5-hydroxylase (Cc_21070) respectively. For FW, Cc_10507586 region from chromosome 5 is identified in ICPL87119 and ICPL20096 donors. A region Cc_21637656 from chromosome 11 is identified in the ICPL87119 but not in the ICPL20096. Similarly, a region Cc_4438493 from chromosome 10 is identified in the ICPL20096 but not in the ICPL87119. For SMD, Cc_24131177 region is identified in both the SMD resistance donors (ICPL20096 and ICPL87119). Ccsmd04 region from chromosome 4 has been identified in ICPL87119 but not in the ICPL20096. These genomic regions can be brought together into single elite genetic background with the help of the markers identified in this study. The validated markers and donor lines in this study offer valuable tools and resources for the genetic improvement of pigeonpea cultivars with enhanced FW and SMD resistance.

genomics↗

bioRxiv2025

InDels in an intronic region of gene Ccsmd04 coding for dormancy/auxin-associated protein controls sterility mosaic disease resistance in pigeonpea

Sterility mosaic disease presents a significant challenge to pigeonpea cultivation in the Indian subcontinent, potentially leading to total crop failure. The development of diagnostic molecular markers for SMD resistance can aid in improving SMD-resistant varieties. In this context, a QTL-seq approach identified genomic regions associated with SMD resistance using a recombinant inbred line generated from ICP8863 x ICPL87119. In total, 6,105 high-confidence variants were identified in the genomic region, namely, smdCc04 based on the delta SNP index. A genomic region smdCc04 on chromosome Cc04 spans 3.2 Mb (9.3 - 12.5 Mb) comprised of 6 missense variants and eight indels. A total of 211 candidate genes were identified from this region. 1 bp insertion, 21 bp insertion, 9 bp deletion, and 3 bp insertion at different intronic positions in 22 susceptible line leads to downregulation of Dormancy/auxin associated protein (Ccsmd04) resulting in loss of signaling in disease resistance pathways. The identified sites recognize four important disease and plant growth related transcription factors. A total of 4 Indels and 8 SNPs were validated from smdCc04 genomic regions using whole genome re-sequencing data and KASP genotyping on resistant and susceptible pigeonpea lines respectively. These markers will be used in pigeonpea breeding programs.

genomics↗

bioRxiv2025

LSD1 serine 166 is a phosphorylation switch for chromatinlandscaping, gene activation, and tissue remodeling

LSD1 is a histone 3 (H3) demethylase that can either repress or activate gene expression. We discover here that the so far enigmatic balance between these two activities in non-hormonal cancer cells is regulated by phosphorylation of serine 166 (S166) on LSD1. SET-mediated Protein Phosphatase 2A (PP2A) inhibition in KRAS mutant cells promotes S166 phosphorylation. Endogenous LSD1 S166 alanine mutant (S166A) cells display H3 lysine 9 demethylation and acetylation, euchromatin, and gene activation. Mechanistically this is explained by the impaired interaction of S166A mutant LSD1 with repressor proteins SNAI2 and MYBP1. Functionally LSD1 S166A mutant cells display augmented beta1 integrin activity and stress fiber formation, and the mutant xenograft tumors have altered tumor microenvironment associated with increased macrophage recruitment. Collectively, PP2A-regulated S166 of LSD1 is a phosphorylation switch for epigenetic gene activation in non-hormonal cancer cells. Conceptually we demonstrate how dephosphorylation of one amino acid on a non-histone protein shapes chromatin landscape in cancer cells, and modify tumor stroma, and immune cell content. Highlights* Mechanism for gene activation by LSD1 in non-hormonal cancers * Single phosphorylation switch in a non-histone protein controls epigenetic landscape * Epigenetic protein phosphorylation in cancer cells shapes tumour immune microenvironment * Novel function for Protein Phosphatase 2A (PP2A) in epigenome regulation via LSD1 Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/653937v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@19548b3org.highwire.dtl.DTLVardef@1d7fd4dorg.highwire.dtl.DTLVardef@13707b3org.highwire.dtl.DTLVardef@1da7b71_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Modulating populational variance of methyl-guanine methyl transferase expression through miR-181d degradation: a novel mechanism of temozolomide resistance

Intratumoral heterogeneity plays a pivotal role in cancer evolution, providing the substrate for adaptation to selective pressures, including treatment with chemotherapy. Here, we show that micro-RNA regulation of variance in the expression of the DNA repair protein methyl-guanine methyl transferase (MGMT) contributes to this heterogeneity and acquired therapeutic resistance. In cell lines derived from glioblastomas, the most common form of primary brain tumor, treatment with standard-of-care temozolomide chemotherapy triggers a feed-forward loop between polyribonucleotide nucleotidyltransferase 1 (PNPT1) and miR-181d, an MGMT regulating miRNA, expediting miR-181d degradation. This degradation requires the activation of Ataxia Telangiectasia and Rad3-related (ATR) kinase. The degradation of miR-181d in glioblastoma cells increased both the mean and the variance of MGMT expression in the cell population. Subclone reconstituted cell populations with similar populational mean MGMT levels but with differences in the variance of MGMT expression exhibited differential temozolomide sensitivity, with the higher MGMT variance population showing increased resistance. This resistance is suppressed by exogenously transfected miR-181d. These findings suggest a key role for miRNA in regulating intra-tumoral heterogeneity through modulation of key DNA repair enzymes and provide a compelling rationale for miRNA delivery as a platform for glioblastoma therapy. Significance StatementThis study demonstrates a mechanistic link between a feed-forward loop mediating microRNA degradation and cell-to-cell variance in gene expression, and the contribution of this mechanism to intratumoral heterogeneity and therapeutic resistance. We show that when glioblastoma, the most common form of adult primary brain tumor, is treated with standard-of-care chemotherapy, temozolomide, a feed-forward loop between miR-181d and PNPT1 is initiated, causing rapid degradation of miR-181d. This degradation increases the cell-to-cell variability in methyl-guanine methyl transferase (MGMT) expression, expanding intra-tumoral heterogeneity and contributing to acquired temozolomide resistance. This process can be suppressed by therapeutic delivery of microRNA, providing compelling considerations for clinical translation.

cancer biology↗

bioRxiv2025

Identifying Networks within an fMRI Multivariate Searchlight Analysis

There is great interest in understanding how different brain regions represent information across space and time. Information-based searchlight analyses systematically examine the information encoded within clusters of functional magnetic resonance imaging (fMRI) voxels across the brain. Significant searchlights contain information that can be used to decode conditions of interest, but significant discriminability can be achieved in a variety of ways. We have developed and report on a new analysis method that can identify sub-networks of searchlights. Notably, unlike methods that collapse trials by condition, such as Representational Similarity Analysis, our method groups searchlights based on them having similar temporal changes in information. We present this method and apply it to fMRI data collected as participants viewed words, faces, shapes, and numbers. After running a searchlight analysis with a 4-way Gaussian Naive Bayes (GNB) classifier, the accuracy vector was submitted to a multi-subject Independent Component Analysis (ICA) to group searchlights based on their decoding timeseries. The ICA identified seven components (sub-networks) of searchlights. These networks identified sets of brain areas that have been commonly associated with the processing of faces, words, shapes and numbers. For instance, two of the components drew strongly on the face-processing network, including fusiform cortex. Switching the classification scheme to faces versus non-faces reconfigured the observed network to reflected face-related systems. These results demonstrate that this method can divide searchlight maps into meaningful components.

neuroscience↗

bioRxiv2025

Engineering IspH for Enhanced Terpenoid Yield: Computational and Molecular Dynamics Studies

Terpenoids play a vital role in pharmaceuticals, biofuels, and various industries, and they are produced through the methylerythritol phosphate (MEP) pathway, with IspH catalyzing the final step. This study explores IspH homologs in Bacillus and other bacteria to aid in enzyme engineering. Sequence analysis showed a conservation range of 46-79%. Phylogenetic analysis (log-likelihood -71,581.08, bootstrap 80-100) validated the evolutionary relationships. Structural modeling revealed conserved functional motifs. Molecular docking indicated HMBPP binding affinities between -4.9 and -6.4 kcal/mol, while molecular dynamics simulations confirmed the stability of the complex (RMSD 2.02-3.45 [A], Rg 21.56-22.06 [A], with a decrease in SASA upon binding). Significant hydrogen bonds were noted with His131, Asn227, and Ser271. This thorough analysis of IspH conservation, structure, and stability, along with co-evolving residue networks, underscores the potential of Bacillus IspH variants for improved terpenoid production and lays the groundwork for targeted enzyme engineering.

bioengineering↗

bioRxiv2025

Diet induced insulin resistance is due to induction of PTEN expression

Insulin resistance is a condition associated with obesity, type 2 diabetes(T2D), hyperinsulinemia, hyperglycemia and defined by reduced sensitivity to insulin signaling. Molecular causes and early signaling events underlying insulin resistance are not well understood. Here we show that insulin activation of PI3K/AKT/mTOR signaling in insulin target tissues, causes mTORC1 induction of PTEN translation, a negative regulator of PI3K signaling. We hypothesized that insulin resistance is due to insulin dependent induction of PTEN that prevents further increases in PI3K signaling. In a diet induced animal model of obesity and insulin resistance, we show that PTEN levels are increased in fat, muscle, and liver. Hyperinsulinemia and PTEN induction are followed by hyperglycemia, severe glucose intolerance, and hepatic steatosis. In response to chronic hyperinsulinemia, PTEN remains increased, while AKT activity is induced transiently before settling down to a PTEN-high and AKT-low state in the tissues, predicted by computational modeling of the PTEN-AKT feedback loop. Treatment with PTEN and mTORC1 inhibitors prevent and reverse the effect of PTEN induction, rescue insulin resistance and increase PI3K/AKT signaling. Thus, we show that PTEN induction by increased insulin levels elevates feedback inhibition of the pathway causing insulin resistance, its associated phenotypes, and is a potential therapeutic target.

cell biology↗

bioRxiv2025

Agriculturally sourced multidrug-resistant Escherichia coli for use as control strains

The environment plays a crucial role in the spread of antibiotic resistance, however environmental surveillance efforts lag far behind clinical surveillance. Natural and human- impacted soil and water are considered major sources of antibiotic resistance in medical and veterinary diagnostics, and the lack of environmental and agricultural surveillance data hinders efforts to assess management impacts or estimate risk. Here we introduce two agriculturally sourced, fully characterized, and genetically sequenced control strains for use in surveillance of extended-spectrum {beta}-lactamase producing (ESBL) and tetracycline-resistant Escherichia coli in the environment, available via publicly accessible culture collections. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/641915v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@34cf46org.highwire.dtl.DTLVardef@11403f3org.highwire.dtl.DTLVardef@10f5da3org.highwire.dtl.DTLVardef@1ccb879_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Dynamic regulation of NeuroD1 expression level by a novel viral construct during astrocyte-to-neuron reprogramming

Astrocyte-to-neuron reprogramming presents a viable approach for regenerative medicine. The reprogramming factor NeuroD1 has demonstrated capability of neuronal reprogramming with high efficiency both in culture and in the injured central nervous system. High level of NeuroD1 expression is required to break down the cellular identity barrier for a successful reprogramming, and yet persistence of this high level drives the reprogrammed neurons primarily to glutamatergic subtype. This is consistent with the critical role of NeuroD1 in determination of glutamatergic neuronal lineage during development. However, diversified neuronal subtypes are needed to establish appropriate neuronal connectivity in disease/injury conditions. We reason that continuously high level of NeuroD1 expression forces the reprogrammed neurons into glutamatergic subtype, and that reducing NeuroD1 level after reprogramming may allow generation of neurons with diversified subtypes. For this purpose, we engineered a novel viral expression vector by which NeuroD1 expression can be dynamically regulated during the reprogramming process. Specifically, the target site of a neuron-specific microRNA (miR-124) is incorporated in the expression system. Therefore, this novel construct would still achieve a high NeuroD1 expression level in astrocytes for reprogramming to occur and yet reduce its level in the reprogrammed neurons by suppression of endogenous miR-124. In this study, we demonstrated that this construct elicits a dynamic gene expression pattern with much reduced level of NeuroD1 at later stages of neuronal reprogramming. We also showed that this construct still retains relatively high reprogramming efficiency and can generate mature neurons with an enhanced GABAergic neuronal phenotype.

neuroscience↗

bioRxiv2025

Chalcone isomerase-like impedes the lactone shunt and enhances flux partitioning in a bifurcated pathway towards isoflavonoid biosynthesis

The reconstitution of biosynthetic pathways in heterologous hosts is often challenged by the switch to a foreign cellular environment, lacking compatible structural or regulatory features. Auxiliary or non-catalytic proteins can play a critical role in modulating metabolic flux and pathway efficiency. Chalcone isomerase-like (CHIL) is a non-catalytic protein known to serve as a partner to chalcone synthase (CHS) in flavonoid biosynthesis, rectifying its promiscuous activity and preventing by-product formation, such as the aberrant p-coumaroyltriacetic acid lactone (CTAL). Here, we extended the characterization of CHILs to the legume-characteristic isoflavonoid pathway. We assessed four CHIL orthologs from diverse plant lineages: Glycine max (GmCHIL), Oryza sativa (OsCHIL), Selaginella moellendorffii (SmCHIL), and Marchantia polymorpha (MpCHIL). Structural modelling suggested that naringenin (flavanone) entry into the CHIL binding cleft may be sterically hindered compared to catalytic CHIs. Moreover, legume CHIL isoforms possess an additional bulky residue, Tyr48, that is expected to impose further constraints on ligand binding. In vitro, CHS produced up to 60% lactone CTAL instead of its desired output; however, CHIL suppressed this aberrant activity to 10%, concomitantly increasing target compound titers. Combinatorial enzyme and yeast biotransformation assays revealed a critical role for CHIL in conducting flux through chalcone, flavanone, and isoflavone biosynthesis. The inclusion of CHIL in our engineered yeast strains enhanced overall titers and, unexpectedly, promoted carbon flux toward the so-called deoxy-branch (isoliquiritigenin, liquiritigenin, and daidzein) by up to 67%, with a 33% increase in final daidzein titers. By extending CHIL characterization to the isoflavonoid pathway, we have revealed an expanded role for this auxiliary protein and underscored its utility in engineered metabolic contexts. Our findings reiterate the often-overlooked impact of non-catalytic proteins in shaping specialized metabolism. HIGHLIGHTSO_LICombinatorial enzyme assays reveal a species-dependent preference for CHILs from more closely related plant phyla by soybean CHS, which improved chalcone and downstream flavanone output by suppressing aberrant lactone formation. C_LIO_LIYeast co-expressing CHIL with the components of the isoflavonoid metabolon exhibited a 67% increase in flux through a legume-characteristic branch of the pathway, resulting in a 33% increase in titers of the major isoflavone, daidzein. C_LIO_LICHIL proteins can be included to engineer the output of phenylpropanoid-derived intermediates preferentially toward isoflavonoid biosynthesis. C_LIO_LIAuxiliary components, such as CHIL, can be designed to refine metabolic composition in bifurcated pathways, as well as enhancing general flux through pathways. C_LI

synthetic biology↗

bioRxiv2025

Females use multiple cues to assess competition for egg-laying decisions

Animals deploy a wide suite of behavioural responses to reduce fitness costs related to competition. One common strategy is spatial avoidance of resource patches associated with high competition and this involves a need to accurately assess the level of competition associated with patches. Although well-studied in the context of predation risk (alarm cues vs kairomones), we lack in our understanding of how inputs from multiple types of cues are used for assessing the level of conspecific competition. We tested how Aedes aegypti females select sites for oviposition when they encounter multiple types of cues from varying densities of conspecifics. We hypothesised that females perceive conspecific young within these sites as potential competition for their offspring, and tailor their response to the type and level of competition. Through binary choice trials, we assessed female oviposition behaviour in response to a gradient in two types of conspecific cues - egg and larvae. Our results indicate that female response was sensitive to the type of cue. Females showed attraction towards pools with low densities of both egg and larval conspecifics indicating conspecific cueing and the strength of response was stronger towards egg cues. However, this attraction response disappeared at high densities of conspecifics (both egg and larvae) suggesting that the resulting trade-off between benefits from conspecific cueing and costs of competition likely shapes oviposition site selection responses. The broad nature of the oviposition response was similar toward both egg and larval cues, but there were fine level differences in responses towards egg and larvae that depended on the conspecific density level. Our study shows that animals are under selection to use multiple cues to assess conspecific competition to minimise the costs of competition.

animal behavior and cognition↗

bioRxiv2025

Matrix stiffness modulated release of spheroid-derived extracellular vesicles and discovery of Piezo1 cargo

Augmented extracellular matrix (ECM) stiffness is a mechanical hallmark of cancer. Mechanotransduction studies have extensively probed the mechanisms by which ECM stiffness regulates intracellular communication. However, the influence of stiffness on intercellular communication aiding tumor progression in three-dimensional microenvironments remains unknown. Small extracellular vesicles (EVs) are communicators of altered biophysical cues to distant sites through EV-ECM interactions and EV-mediated recipient cell-ECM interactions. Here we demonstrate stiffness-mediated modulation of small EVs secretion and cargo from three-dimensional oral squamous cell carcinoma spheroids. Using a spheroid culture platform with varying matrix stiffness properties, we show that small EVs carry parental biomolecular cargo, including mechanosensitive Piezo1 ion channel and adhesion molecule CD44. We comprehensively validate the presence of both markers in our EV populations using proteomic and genetic analysis. Transcriptomic analysis of microRNA and long non-coding RNA cargo of small EVs released from soft and stiff ECM spheroids revealed enrichment of tumorigenic and metastatic profiles in EVs from stiff ECM cultures compared to that of soft ones. Gene set enrichment analysis of a comparative dataset obtained by overlaying spheroid mRNA and EV miRNA profiles identified key oncogenic pathways involved in cell-EV crosstalk in the spheroid model.

cancer biology↗

bioRxiv2025

Maturase K forms a plastidial splicing complex with a neofunctionalized branching enzyme

Chloroplast group IIA introns derive from bacterial ribozymes. Their splicing likely requires Maturase K (MatK), which has been largely inaccessible to functional analyses being itself a chloroplast intron-encoded protein. Here we show that MatK physically interacts with a conserved, essential plastid-localized homolog of starch-branching enzymes (BEs), dubbed MATURASE K INTERACTING PROTEIN1 (MKIP1). We demonstrate that MKIP1 proteins have lost BE activity and acquired an insertion enabling direct interaction with the N-terminal region of MatK. Arabidopsis MKIP1 specifically co-precipitates all known intron targets of MatK. Induced MKIP1 silencing results in pale newly emerging leaves, in which the splicing of these intron targets is strongly reduced. Our data suggest that MKIP1 functionally diverged from canonical BEs to facilitate splicing in conjunction with MatK. We propose that the N-terminus of MatK, in turn, has evolved from an RNA-binding domain into a platform for protein interaction, helping its transition towards a general splicing factor.

plant biology↗

bioRxiv2024

4-Phenylbutyric acid modulates Connexin 43 expression restricting murine-β-coronavirus infectivity and virus-induced demyelination

Gap junction intercellular communication, particularly involving Connexin 43 and Connexin 47, plays a critical role in maintaining CNS homeostasis and has been implicated in Multiple Sclerosis (MS) pathology. Thus, warranting further studies in experimental animal models to understand how modulation of Cx43 expression can influence MS pathology. Intracranial infection with murine-{beta}-coronavirus Mouse Hepatitis Virus (MHV-A59) in mice results in acute pathology characterized by high viral titers, glial activation in the brain and chronic neuroinflammatory demyelination, effectively mimicking key pathological hallmarks of MS and serving as a robust model to investigate its viral etiology. MHV-A59 infection leads to a pronounced downregulation of Cx43 during the acute phase, emphasizing its critical role in virus-induced CNS pathology. In this study, we investigated the potential of in vivo 4-phenylbutyric acid (4-PBA) administration in modulating Cx43 expression in this MHV-induced model and its consequence on chronic virus-induced demyelination. Our results reveal that 4-PBA treatment reduced acute MHV-A59 infectivity and viral spread in the brain while modulating the glial cell response, mounting host immunity. Treatment with 4-PBA effectively preserved the expression of both Cx43 and Cx47 in infected CNS cells, counteracting their infection-induced downregulation. Furthermore, MHV-A59 infection downregulated the expression of ER-resident thioredoxin family protein (ERp29), a well-known molecular chaperone of Cx43, which was rescued by 4-PBA treatment. We further validated if such downregulation of ERp29 is also evident in MS demyelinating plaques. In human MS patient-derived brain tissue, reduced Cx43 and ERp29 staining was observed in demyelinating plaques. Our studies revealed that 4-PBA treatment not only limits viral replication and spread throughout the brain but also protects the mice against severe chronic neuroinflammatory demyelination. These findings suggest that targeting Cx43 with 4-PBA holds significant therapeutic potential for addressing virus-induced neuroinflammatory demyelination and MS by preserving gap junction intercellular communication.

neuroscience↗

bioRxiv2024

Learning from and improving upon high-throughputscreens for protein fitness with Generative AI - Application to BBB-crossing AAV design

Deep Science is enabling high-throughput experimentation (HTE) to design novel biological entities with desired properties. E.g., blood-brain-barrier (BBB) crossing adeno-associated virus (AAV) vectors, needed for systemic delivery of gene therapies to brain cells, have been identified through innovative directed evolution assays such as M-CRE-ATE and TRACER. But, even these high-throughput experiments are only able to explore a miniscule portion of the large design space of biological entities. In this paper, we introduce autograd based maximization of protein fitness (AutoMaxProFit) to learn from and improve upon protein designs generated with high-throughput screens. Using a transformer based generative AI network and protein language models, we improve upon the design of a variant previously discovered through HTE, to yield 2x better enrichment in brain endothelial cells, as estimated by molecular dynamics (MD) simulations. This shows that Deep Tech models can learn from the observations generated by Deep Science experiments and go on to find more optimal design candidates for application in Biopharma.

bioinformatics↗