Biology subjects

Kaur, G.

Publications and source records attributed to Kaur, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Overlooked signals: Highly stable quorum sensing molecule in phage lysates induces quorum sensing response

Phage-bacterial interaction studies routinely apply phage lysates at final concentrations of up to 10% of the culture. Consequently, bacterial metabolites such as quorum sensing (QS) signaling molecules are transferred along with the phage lysate to recipient bacterial cells. Here, we show that the Pseudomonas aeruginosa QS molecules 3OC12-HSL and C4-HSL are rapidly degraded in phage lysates. In contrast, the hydrophobic QS molecule PQS is remarkably stable, for at least one year, due to its binding within outer membrane vesicles. Strikingly, we find that PQS exceeds concentrations of 10 {micro}M in standard phage lysate preparations. We show that PQS carried over from phage lysates induces QS-controlled production of the virulence factor pyocyanin in P. aeruginosa. This PQS carryover does not oppose previous conclusions of phage infection-induced PQS production, as we show here, that this response is also triggered by PQS-free phage lysates. Since other bacterial species, including Paracoccus and Vibrio harveyi, also produce hydrophobic QS molecules that are bound within outer membrane vesicles, it is likely that phage lysates from these bacteria may similarly contain stable QS molecules. Collectively, we demonstrate that membrane-bound QS molecules may significantly confound QS-related physiological outcomes of phage-host interaction studies. This can be avoided by using QS synthase mutants for phage propagation or by purifying phage particles from lysates to eliminate QS molecule carryover.

microbiology↗

bioRxiv2025

Structural basis of substrate specificity of Helix pomatia AMP deaminase and a chimeric ADGF adenosine deaminase

HPAMPD, an enzyme enriched in the foot muscle of the mollusk Helix pomatia, exhibits deaminase activity on adenosine-5-monophosphate (AMP). HPAMPD is the first member of the adenosine deaminase-related growth factor (ADGF) family to prefer the nucleotide, AMP, over the nucleoside, adenosine. To investigate the substrate selectivity of HPAMPD, we determined its structure in the apo form and in complex with the adenosine analogs pentostatin (2- deoxycoformycin) and pentostatin-5-monophosphate. Structurally, HPAMPD adopts a fold similar to human ADA2, an ADGF family member. HPAMPD has acquired the ability to interact with the 5-monophosphate group of AMP through polar and charged residues located in three key structural elements: (1) the loop immediately following strand {beta}1, (2) the loop between helices H and I, and (3) the end of strand {beta}5 and its adjacent loop. We engineered a chimeric deaminase by integrating these elements from HPAMPD into another related mollusk nucleoside adenosine deaminase, the A. californica ADGF. The chimeric enzyme efficiently deaminates AMP, demonstrating a gained substrate specificity, while retaining the adenosine deamination activity of Aplysia ADGF. The phosphate-binding feature of HPAMPD is a hallmark of nucleotide deaminases, conserved among AMP and N6-methyl-AMP (6mAMP) deaminases. We discuss the human adenosine deaminases each with distinct substrate specificities for the nucleoside, the nucleotide (AMP), and the methylated form, 6mAMP. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/645602v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@be415forg.highwire.dtl.DTLVardef@b5f7a5org.highwire.dtl.DTLVardef@957514org.highwire.dtl.DTLVardef@397e9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Gut bacterial lactate stimulates lung epithelial mitochondria and exacerbates acute lung injury

Acute respiratory distress syndrome (ARDS) is an often fatal critical illness where lung epithelial injury leads to intrapulmonary fluid accumulation. ARDS became widespread during the COVID-19 pandemic, motivating a renewed effort to understand the complex etiology of this disease. Rigorous prior work has implicated lung endothelial and epithelial injury in response to an insult such as bacterial infection; however, the impact of microorganisms found in other organs on ARDS remains unclear. Here, we use a combination of gnotobiotic mice, cell culture experiments, and re-analyses of a large metabolomics dataset from ARDS patients to reveal that gut bacteria impact lung cellular respiration by releasing metabolites that alter mitochondrial activity in lung epithelium. Colonization of germ-free mice with a complex gut microbiota stimulated lung mitochondrial gene expression. A single human gut bacterial species, Bifidobacterium adolescentis, was sufficient to replicate this effect, leading to a significant increase in mitochondrial membrane potential in lung epithelial cells. We then used genome sequencing and mass spectrometry to confirm that B. adolescentis produces L-lactate, which was sufficient to increase mitochondrial activity in lung epithelial cells. Finally, we found that serum lactate was significantly associated with disease severity in patients with ARDS from the Early Assessment of Renal and Lung Injury (EARLI) cohort. Together, these results emphasize the importance of more broadly characterizing the microbial etiology of ARDS and other lung diseases given the ability of gut bacterial metabolites to remotely control lung cellular respiration. Our discovery of a single bacteria-metabolite pair provides a proof-of-concept for systematically testing other microbial metabolites and a mechanistic biomarker that could be pursued in future clinical studies. Furthermore, our work adds to the growing literature linking the microbiome to mitochondrial function, raising intriguing questions as to the bidirectional communication between our endo- and ecto-symbionts.

molecular biology↗

bioRxiv2025

Single-cell transcriptomics identifies a dampened neutrophil function and accentuated T-cell cytotoxicity in tobacco flavored e-cigarette exposed mouse lungs

E-cigarettes (e-cigs) are a public health concern for young adults due to their rising popularity despite evidence of harmful effects. Yet an extensive study defining the cell-specific immune changes upon exposure to flavored e-cigs remains elusive. To determine the immunological lung landscape upon acute nose-only exposure of C57BL/6J to flavored e-cig aerosols, we performed single-cell RNA sequencing (scRNA seq). Analyses of the levels of metals in the e-cig aerosol generated daily during exposure revealed a flavor-dependent variation in the day-to-day leaching of the levels of metals like Ni, Cu, K and Zn, among others. scRNA profiles of 71,725 cells generated from control and treatment groups (n=2/sex/group) found maximum dysregulation of (a) myeloid cell function in menthol (324 differentially enriched genes (DEG)) and tobacco (553 DEGs) -flavor exposed and (b) lymphoid cell function in fruit (112 DEGs)-flavored e-cig aerosol exposed mouse lungs as compared to air. Flow cytometry analyses identified marked increase in the neutrophil percentage and a decrease in the eosinophil count in menthol and tobacco-flavored e-cig aerosol exposed mouse lungs which corroborated with our scRNA seq data. We further found an: (a) increase in CD8+ T cell percentages, (b) upregulation of inflammatory genes like Stat4, Il1bos, Il1b, Il1ra and Cxcl3 and (c) enrichment of terms like T-helper cell 1cytokine function and NK cell degranulation in the lungs of e-cig aerosol exposed mouse when compared to control. Interestingly, the increase in the level of immature neutrophils characterized by Ly6G deficiency and reduction in the S100A8 (marker for neutrophil activation) using immunofluorescence in tobacco-flavored e-cig aerosol exposed mouse lung sections point towards a possible shift in the neutrophil dynamics upon exposure to e-cigs. Overall, this study identifies flavor dependent changes in myeloid and lymphoid-cell mediated responses in the mouse lungs exposed to acute nose-only e-cig aerosols and provides a resource to influence future research in select /specific cell types to understand the immunological implications of long-term use of e-cigs.

pharmacology and toxicology↗

bioRxiv2024

A modular gate system for autonomous control of rodent behavior

Rodent mazes have been used for decades to study the neural basis of behavior. Advancements in rapid prototyping techniques and access to affordable electronics allows laboratories with sufficient expertise in engineering and programming to customize and construct maze apparatuses and behavioral tasks, thereby increasing the ability of their studies to answer specific scientific questions. We designed and built a rodent gate system that lowers this bar of expertise even further. The NC4gate system is a robust mechanical design that can be built using low-cost hardware and execute thousands of cycles before maintenance. Up to 512 gates can be controlled using a single computer. Users can control the gates interactively using a Python-based graphical interface and programmatically using an extensible API. We hope that the open-source hardware / software and extensive documentation enables laboratories to build these affordable and robust gates and seamlessly incorporate automatic behavior control into their existing or new rodent tasks. Significance StatementRodent mazes are used by thousands of laboratories and research institutions across the world to study learning and memory, as well as the effects of pharmacological, genetic and environmental manipulations. Ideally, maze and task designs should be customized to the scientific questions at hand. It is challenging, however, for many laboratories to build, program, and operate custom mazes, requiring them instead to rely on expensive and proprietary commercial solutions. The most complex components of most mazes are moving gates that restrict and direct rodent behavior. Here we provide the open-source hardware and software for a gate system that is extensible, affordable and robust, removing this critical barrier to customized mazes.

animal behavior and cognition↗

bioRxiv2024

GENCODE: massively expanding the lncRNA catalog through capture long-read RNA sequencing

Accurate and complete gene annotations are indispensable for understanding how genome sequences encode biological functions. For more than twenty years, the GENCODE consortium has developed reference annotations for the human and mouse genomes, becoming a foundation for biomedical and genomics communities worldwide. Nevertheless, collections of important yet poorly-understood gene classes like long non-coding RNAs (lncRNAs) remain incomplete and scattered across multiple, uncoordinated catalogs. To address this, GENCODE has undertaken the most comprehensive lncRNA annotation effort to date. This is founded on the manually supervised computational annotation of full-length targeted long-read sequencing, on matched embryonic and adult tissues, of orthologous regions in human and mouse. Altogether 17,931 human genes (140,268 transcripts) and 22,784 mouse genes (136,169 transcripts) have been added to the GENCODE catalog representing a 2-fold and 6-fold growth in transcripts, respectively - the greatest increase in the number of annotated human genes since the sequencing of the human genome. Our targeted design assigned human-mouse orthologs at a rate beyond previous studies, tripling the number of human disease-associated lncRNAs that have mouse orthologs. Novel lncRNA genes consistently exhibit biological signals of functionality, and they greatly enhance the functional interpretability of the human genome. While poorly expressed in bulk RNA-Seq samples, many of them are highly expressed in specific cell populations, maybe even contributing to cell-type determination. The expanded GENCODE lncRNA annotations mark a critical step toward deciphering the human and mouse genomes.

genomics↗

bioRxiv2024

Atypical chemokine receptor 3 regulates synaptic removal in disease astrocytes

Astrocytes participate in the clearance of obsolete or unwanted neuronal synapses. However, the molecular machinery involved in recognizing these synapses remains unclear, particularly under pathological conditions. Here, we investigated the phagocytic process of astrocytes through individual gene silencing with a druggable gene library. Our study demonstrates that astrocyte- mediated synapse engulfment is regulated by the Atypical chemokine receptor 3 (Ackr3). Mechanistically, we showed that Ackr3 recognizes phosphatidylethanolamine (PE)-bound C-X-C motif chemokine 12 (CXCL12) at synaptic terminals both in vitro and in vivo, thus serving as a novel marker of synaptic dysfunction. Notably, both the receptor and its ligand are upregulated in post- mortem human Alzheimers disease (AD) brains, and AD mouse models. Downregulation of the Ackr3 in AD mice significantly diminishes astrocyte-mediated synaptic elimination, and rescues pathological phenotypes, including synapse loss and cognitive impairment. Overall, this work unveils a novel, possibly targetable mechanism of astrocyte-mediated synaptic engulfment implicated in neurodegenerative disease.

neuroscience↗

bioRxiv2024

Advancements in Human Norovirus Cultivation in Human Intestinal Enteroids

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically-modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights to this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells into intestinal organoids that were transplanted and matured in mice before making enteroids (H9tHIEs), genetically-engineered (J4FUT2 knock-in [KI], J2STAT1 knock-out [KO]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of GI and GII HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research. ImportanceHuNoVs cause global diarrheal illness and chronic infections in immunocompromised patients. This manuscript reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from i) different intestinal segments of single donors, ii) human embryonic stem cell-derived organoids transplanted into mice, iii) genetically-modified lines, and iv) a patient with chronic variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically-modified J4FUT2-KI HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

microbiology↗

bioRxiv2024

MicroRNA Tae-miR1130p targets wheat ferroportin1 (TaFPN1) in the absence of iron-responsive element/iron-regulatory protein1 module

Ferroportin (FPN) belongs to the Major Facilitator Superfamily of transporters and is a known iron (Fe) exporter in humans, with orthologues also present in plant species. Human FPN is subjected to multi-level regulation at transcriptional, post-transcriptional, and post-translational levels. How plant FPNs are regulated remains to be explored. In the current study, we have characterized wheat FPN1, a plasma-membrane localized protein, for its role in Fe homeostasis. A spatial-temporal expression analysis of wheat FPN1 suggested that its tissue-specific expression is differentially upregulated during Fe deficiency conditions. Unlike human FPN, plant FPN lacks the necessary sites for hepcidin binding, thereby emphasizing the need to explore the transcriptional/post-transcriptional mode of regulation. The lack of Iron Responsive Elements (IRE) in TaFPN1 promoter suggests no direct regulation through the Iron Regulatory Protein (IRP) mechanism like in humans. Further, to explore the miRNA-mediated regulation, we identified Fe-regulated tae-miR1130b-3p capable of targeting TaFPN1 under in-vivo conditions. Transcript expression of tae-miR1130b-3p negatively correlates with the TaFPN1. This alternative regulation pathway suggests a complex network of interactions governing the expression of genes involved in iron homeostasis, highlighting the intricacies of cellular regulatory mechanisms. Altogether, the work will unravel the cellular and physiological role of wheat FPN and contribute to a comprehensive understanding of plant iron homeostasis.

plant biology↗

bioRxiv2024

Modular, Vascularized Hypertrophic Cartilage Constructsfor Bone Tissue Engineering Applications

Insufficient vascularization is a main barrier to creating engineered bone grafts for treating large and ischemic defects. Modular tissue engineering approaches have promise in this application because of the ability to combine tissue types and to localize microenvironmental cues to drive desired cell function. In direct bone formation approaches, it is challenging to maintain sustained osteogenic activity, since vasculogenic cues can inhibit tissue mineralization. This study harnessed the physiological process of endochondral ossification to create multiphase tissues that allowed concomitant mineralization and vessel formation. Mesenchymal stromal cells in pellet culture were differentiated toward a cartilage phenotype, followed by induction to chondrocyte hypertrophy. Hypertrophic pellets exhibited increased alkaline phosphatase activity, calcium deposition, and osteogenic gene expression relative to chondrogenic pellets. In addition, hypertrophic pellets secreted and sequestered angiogenic factors, and supported new blood vessel formation by co-cultured endothelial cells and undifferentiated stromal cells. Multiphase constructs created by combining hypertrophic pellets and vascularizing microtissues and maintained in unsupplemented basal culture medium were shown to support robust vascularization and sustained tissue mineralization. These results demonstrate a new in vitro strategy to produce multiphase engineered constructs that concomitantly support the generation of mineralize and vascularized tissue in the absence of exogenous osteogenic or vasculogenic medium supplements. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/582166v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@12d3051org.highwire.dtl.DTLVardef@1bf53feorg.highwire.dtl.DTLVardef@234ba4org.highwire.dtl.DTLVardef@13b2723_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Intermolecular Energy Migration via HomoFRET Captures the Modulation in the Material Property of Phase-Separated Biomolecular Condensates

Biomolecular condensation via phase separation of proteins and nucleic acids has emerged as a crucial mechanism underlying the spatiotemporal organization of cellular components into functional membraneless organelles. However, aberrant maturation of these dynamic, liquid-like assemblies into irreversible gel-like or solid-like aggregates is associated with a wide range of fatal neurodegenerative diseases. New tools are essential to dissect the changes in the internal material properties of these biomolecular condensates that are often modulated by a wide range of factors involving the sequence composition, truncations, mutations, post-translational modifications, and the stoichiometry of nucleic acids and other biomolecules. Here, we employ homo-Forster Resonance Energy Transfer (homoFRET) as a proximity ruler to study intermolecular energy migration that illuminates the molecular packing in the nanometric length-scale within biomolecular condensates. We used the homoFRET efficiency, measured by a loss in the fluorescence anisotropy due to rapid depolarization, as a readout of the molecular packing giving rise to material properties of biomolecular condensates. Using single-droplet anisotropy imaging, we recorded spatially-resolved homoFRET efficiencies of condensates formed by fluorescent protein-tagged Fused in Sarcoma (FUS). By performing single-droplet picosecond time-resolved anisotropy measurements, we were able to discern various energy migration events within the dense network of polypeptide chains in FUS condensates. Our homoFRET studies also captured the modulation of material properties by RNA, ATP, and post-translational modification. Additionally, we utilized mammalian cell lines stably expressing FUS to study nuclear FUS and oxidative stress-induced stress granule formation in the cytoplasm. Our studies demonstrate that spatially-resolved homoFRET methodology offers a potent tool for studying intracellular phase transitions in cell physiology and disease.

biophysics↗

bioRxiv2024

Identifying the genetic and epigenetic basis for asymmetric bZIP expression in temperature-stressed bread wheat

Asymmetric expression in the bread wheat (Triticum aestivum) genome refers to the differential expression of genes from A, B, and D parental genomes. Bread wheat is a hexaploid crop with six copies of each chromosome. This complexity can result in unequal expression of genes from each parental genome, leading to asymmetry in gene expression. In other polyploid crops like cotton, transcription factors (TF) exhibit genome-biased expression; however, there are no comparable studies for bread wheat. One of the most prominent TFs families in plants is the basic Leucine Zippers (bZIP) which are eukaryote-specific proteins and regulate various biological processes, including stress-related responses. bZIP proteins are dimeric and several heptads long. They exhibit typical coiled-coil structures with strategically placed amino acids in each heptad, responsible for their stability and specificity. Here, we aim to decipher the structural basis of the asymmetric expression of the bZIP TFs in wheat under low and high-temperature conditions. Furthermore, 19 highly expressed stress-related TabZIP TFs were analysed for their asymmetric expression profiles as plants were exposed to temperature-stress conditions. Two benchmarks were used to analyse the asymmetric gene expression of bZIPs, i.e., a) the promoters occupancy by the epigenetic marker histones, namely, H3K4me3, H3k9ac (active) and H3K27me3 (repressive), b) density and diversity of cis-regulatory elements in the promoters. Notably, the genetic basis of the differences in protein sequences of bZIP triads was explored, which may impart structural stability to a specific homeolog enabling the plant to endure the stress conditions better.

plant biology↗

bioRxiv2023

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

The trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element.

biochemistry↗

bioRxiv2023

Investigation of microglial diversity in a mouse model of Parkinson's disease pathology

Microglia, the central nervous system resident immune cells, are now recognized to critically impact homeostasis maintenance and contribute to the outcomes of various pathological conditions including Parkinsons disease (PD). Microglia are heterogenous, with a variety of states recently identified in aging and neurodegenerative disease models, including the disease-associated microglia (DAM) which present a selective enrichment of CLEC7A encoding the CLEC7A or DECTIN1 protein, and the dark microglia (DM) displaying markers of cellular stress at the ultrastructural level. However, the roles of CLEC7A-positive microglia and DM in the pathology of PD have remained largely elusive. By applying immunofluorescence and scanning electron microscopy, we aimed to characterize 1) the CLEC7A -positive cell population, and 2) their possible relationships to DM in a mouse model harboring a G2019S pathogenic mutation of the LRRK2 gene, the most common mutation linked to PD. We examined 18-month-old mice, comparing between LRRK2 G2019S knock-in mice and wild-type controls. In the dorsal striatum, a region affected by PD pathology, extensive ultrastructural features of cellular stress (e.g., endoplasmic reticulum and Golgi apparatus dilation), as well as reduced direct cellular contacts, were observed for microglia from LRRK2 G2019S mice versus controls. CLEC7A-positive microglia exhibited extensive phagocytic ultrastructural characteristics in the LRRK2 G2019S mice. Additionally, the LRRK2 G2019S mice presented a higher proportion of DM. Lastly, immunofluorescence and biochemical analysis revealed higher number of CLEC7A-positive cells in Lrrk2 G2019S genotype versus controls both in tissues and in primary microglia cells. Of note, CLEC7A-positive cells present a selective enrichment of ameboid morphology and tend to cluster in the pathogenic animal. In summary, we provide novel insights into the involvement of recently-defined microglial states, CLEC7A-positive cells and DM, in the context of LRRK2 G2019S PD pathology.

neuroscience↗

bioRxiv2023

RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as platforms for viral replication

Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of non-enveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoV) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA. TeaserPolymerase of a positive-sense RNA virus forms LLPS to regulate replication as an elegant solution for an enigmatic question.

biochemistry↗

bioRxiv2023

A Standardized Antiviral Pipeline for Human Norovirus in Human Intestinal Enteroids Demonstrates No Antiviral Activity of Nitazoxanide

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis. In immunocompetent hosts, symptoms usually resolve within three days; however, in immunocompromised persons, HuNoV infection can become persistent, debilitating, and sometimes life-threatening. There are no licensed therapeutics for HuNoV due to a near half-century delay in its cultivation. Treatment for chronic HuNoV infection in immunosuppressed patients anecdotally includes nitazoxanide, a broad-spectrum antimicrobial licensed for treatment of parasite-induced gastroenteritis. Despite its off-label use for chronic HuNoV infection, nitazoxanide has not been clearly demonstrated to be an effective treatment. In this study, we established a standardized pipeline for antiviral testing using multiple human small intestinal enteroid (HIE) lines representing different intestinal segments and evaluated whether nitazoxanide inhibits replication of 5 HuNoV strains in vitro. Nitazoxanide did not exhibit high selective antiviral activity against any HuNoV strains tested, indicating it is not an effective antiviral for norovirus infection. HIEs are further demonstrated as a model to serve as a pre-clinical platform to test antivirals against human noroviruses to treat gastrointestinal disease.

microbiology↗

bioRxiv2023

Simultaneous enhancement of multiple functional properties using evolution-informed protein design

Designing optimized proteins is important for a range of practical applications. Protein design is a rapidly developing field that would benefit from approaches that enable many changes in the amino acid primary sequence, rather than a small number of mutations, while maintaining structure and enhancing function. Homologous protein sequences contain extensive information about various protein properties and activities that have emerged over billions of years of evolution. Evolutionary models of sequence co-variation, derived from a set of homologous sequences, have proven effective in a range of applications including structure determination and mutation effect prediction. In this work we apply one of these models (EVcouplings) to computationally design highly divergent variants of the model protein TEM-1 {beta}-lactamase, and characterize these designs experimentally using multiple biochemical and biophysical assays. Nearly all designed variants were functional, including one with 84 mutations from the nearest natural homolog. Surprisingly, all functional designs had large increases in thermostability and most had a broadening of available substrates. These property enhancements occurred while maintaining a nearly identical structure to the wild type enzyme. Collectively, this work demonstrates that evolutionary models of sequence co-variation (1) are able to capture complex epistatic interactions that successfully guide large sequence departures from natural contexts, and (2) can be applied to generate functional diversity useful for many applications in protein design.

synthetic biology↗

bioRxiv2023

Zika virus E protein alters blood-brain barrier by modulating brain microvascular endothelial cell and astrocyte functions.

Neurotropic viruses can cross the otherwise dynamically regulated blood-brain barrier (BBB) and affect the brain cells. Zika virus (ZIKV) is an enveloped neurotropic Flavivirus known to cause severe neurological complications, such as encephalitis and foetal microcephaly. In the present study, we used human brain microvascular endothelial cells (hBMECs) and human progenitor derived astrocytes to form a physiologically relevant BBB model. We used this model to investigate the effects of ZIKV envelope (E) protein on properties of cells comprising the BBB. E protein is the principal viral protein involved in interaction with host cell surface receptors, facilitating the viral entry. Our findings show that ZIKV E protein results in activation of both hBMECs and astrocytes. hBMECs showed reduced expression of endothelial junction proteins - ZO-1, Occludin and VE-Cadherin, which are crucial in establishing and maintaining the BBB. As a result, ZIKV E protein triggered alteration in BBB integrity and permeability. We also found upregulation of genes involved in leukocyte recruitment along with increased proinflammatory chemokines and cytokines upon exposure to E protein. Furthermore, E protein resulted in astrogliosis as seen by increased expression of GFAP and Vimentin. Both BBB cell types exhibited inflammatory response following exposure to E protein which may influence viral access into the central nervous system (CNS), resulting in infection of other CNS cells. Overall, our study provides valuable insights into the transient changes that occur at the site of BBB upon ZIKV infection.

neuroscience↗