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Kumar, N.

Publications and source records attributed to Kumar, N..

At least 109 records · Page 6Linked to original sources
bioRxiv2023

Widespread gene regulator Psu inhibits transcription termination factor rho by forced hyper-oligomerization

Many bacteriophages modulate the host transcription machinery for efficient expression of their own genomes. Phage P4 polarity suppression protein, Psu, is a building block of the viral capsid and inhibits the hexameric transcription termination factor, {rho}, by presently unknown mechanisms. We elucidated cryogenic electron microscopy structures of {rho}-Psu complexes, showing that Psu dimers laterally clamp two inactive, open {rho} rings and promote their expansion to higher-oligomeric states. Systematic ATPase, nucleotide binding and nucleic acid binding studies revealed that Psu hinders {rho} ring closure and traps nucleotides in their binding pockets on {rho}. Structure-guided mutagenesis in combination with growth, pull-down and termination assays further delineated the functional {rho}-Psu interfaces. Bioinformatic analyses suggested that, in addition to guarding its own genome against {rho}, Psu enables expression of diverse phage-defense systems commonly found in P4-like mobile genetic elements across bacteria. Thus, Psu is a widespread gene regulator that inhibits {rho} via forced hyper-oligomerization.

molecular biology↗
bioRxiv2023

Faulty Metabolism: A Potential Instigator of an Aggressive Phenotype in Cdk5-dependent Medullary Thyroid Carcinoma

Mechanistic modeling of cancers such as Medullary Thyroid Carcinoma (MTC) to emulate patient-specific phenotypes is challenging. The discovery of potential diagnostic markers and druggable targets in MTC urgently requires clinically relevant animal models. Here we established orthotopic mouse models of MTC driven by aberrantly active Cdk5 using cell-specific promoters. Each of the two models elicits distinct growth differences that recapitulate the less or more aggressive forms of human tumors. The comparative mutational and transcriptomic landscape of tumors revealed significant alterations in mitotic cell cycle processes coupled with the slow-growing tumor phenotype. Conversely, perturbation in metabolic pathways emerged as critical for aggressive tumor growth. Moreover, an overlapping mutational profile was identified between mouse and human tumors. Gene prioritization revealed putative downstream effectors of Cdk5 which may contribute to the slow and aggressive growth in the mouse MTC models. In addition, Cdk5/p25 phosphorylation sites identified as biomarkers for Cdk5-driven neuroendocrine tumors (NETs) were detected in both slow and rapid onset models and were also histologically present in human MTC. Thus, this study directly relates mouse and human MTC models and uncovers vulnerable pathways potentially responsible for differential tumor growth rates. Functional validation of our findings may lead to better prediction of patient-specific personalized combinational therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC="FIGDIR/small/544755v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@196ed2dorg.highwire.dtl.DTLVardef@1ad2d59org.highwire.dtl.DTLVardef@7c6e84org.highwire.dtl.DTLVardef@13bd7db_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICGRP driven aberrant Cdk5 activation develops early onset aggressive MTC C_LIO_LIGenetic alterations in mouse and human tumors disrupt common pathways C_LIO_LIAggressive tumor model characterized by alterations in metabolic pathways C_LIO_LISlow growing tumor model elicits disruption of mitotic spindle assembly C_LI

cancer biology↗
bioRxiv2023

Aeroplane wing, a new recessive autosomal phenotypic marker in malaria vector, Anopheles stephensi Liston

A novel and distinct mutant, with an aeroplane wing (ae) phenotype, is reported for the first time in the urban malaria vector, Anopheles stephensi. The mutant mosquitoes exhibit extended wings that are easily visible to naked eyes in both sexes. This mutant was first observed in a nutritionally stressed experimental isofemale line and characterized for its genetic inheritance and other related parameters. Meticulous and strategic genetic crosses revealed that the ae gene is an autosomal, recessive, non-sex-linked and monogenic trait with full penetrance and uniform expression in the adult stage. Cytogenetic study of the ovarian polytene chromosome revealed an inversion on the 3L chromosome (3Li) in both the ae mutant and its parent line. No significant differences in wing venation and other parameters were observed in ae mutants compared to their normal parental lines. This ae mutant would be an excellent marker that can be used by researchers to study the function of related genes within the genome. Author summaryIn the present study, we have established and describe the inheritance of an unusual novel aeroplane (ae) winged mutant in Anopheles stephensi, an urban malaria vector in India. The ae mutant lines exhibit three open-wing orientations in both the sexes of adults viz. left (LW), right (RW) and double wing (DW) during its resting phase. Through various inbreeding crosses we demonstrated the mode of inheritance of the ae gene to be autosomal, recessive and monogenic in nature. The morphometric studies of eggs and wings revealed that mutant lines are on par with their parental lines. Cytogenetic study of polytene chromosome of ae mutant revealed the presence of heterogenic inversion on the 3L chromosome arm, which might help in adaptation mechanism. We strongly believe that ae phenotypic markers have great applications bridging both basic and applied genetic research such as constructing linkage maps, identifying loci of quantitative and/or qualitative traits and as guides for insect transformation studies.

genetics↗
bioRxiv2023

Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii

Sorghum (Sorghum bicolor) is a major food and bioenergy grass species cultivated worldwide. To promote more robust and sustainable growth of this important crop, we need a deeper understanding of the plant-microbe interactions between sorghum and soil microbial communities that benefit plant host resiliency and enhance nutrient acquisition. The release of specific metabolites from plant roots, or root exudation, drives these plant-microbe interactions, but the molecular pathways by which root exudates shape the sorghum rhizosphere microbiome require further elucidation. To investigate these complex interkingdom interactions in the sorghum rhizosphere, we developed a photoaffinity probe based on sorgoleone, a hydrophobic secondary metabolite and allelochemical produced in sorghum seedling root exudates. Here, we apply a new synthetic sorgoleone diazirine alkyne photoaffinity probe (SoDA-PAL) to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth promoting microbe derived from Sorghum bicolor rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins that were complementary to a previously identified gene cluster involved in sorgoleone catabolism. Proteins identified by SoDA-PAL included a select set of putative transporters, transcription regulators, and a subset of proteins with lipid and secondary metabolic activities. We confirm binding of SoDA-PAL to a putative hydrolase in the /{beta} fold family (OH685_09420) through structural bioinformatics and in-vitro recombinant protein analysis. This photoaffinity labeling approach using metabolite-based probes can be extended in the future to proteomic profiling of complex rhizosphere microbiomes to discover genes that can be leveraged to promote beneficial plant-microbe interactions. ImportanceHere we demonstrate a photoaffinity-based chemical probe modeled after sorgoleone, a known secondary metabolite released from the roots of sorghum, can be used to dissect complicated plant-microbe interactions. Applying this probe to the sorghum-associated bacterium Acinetobacter pittii identified diverse proteins that directly interact with sorgoleone. We show that probe labeling is dose-dependent and is sensitive to competition with purified sorgoleone, demonstrating the probe is selective for protein targets that directly interact with sorgoleone. By using the probe to broadly profile proteins that interact with sorgoleone, we identified bacterial catabolic pathways, unintuitive transcriptional regulation pathways, and vital exchange mechanisms involving transporters that may be involved in sorgoleone utilization and cellular response toward this plant metabolite. We envision that this workflow will expand our understanding of the sorghum root exudate interactome and elucidate the molecular mechanisms by which specific metabolites shape the sorghum rhizosphere microbiome.

biochemistry↗
bioRxiv2023

Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH, reveals the unique mode of mycobacterial ribosome hibernation

Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome and slows down its overall protein synthesis. The mechanism is well studied in enteric bacteria, which mainly hibernate its ribosome in 100S disome form through a dual domain, long HPF (HPFlong) or a single domain, short HPF (HPFshort) in concert with another ribosome modulation factor. Mycobacteria under hypoxia (low oxygen) stress overexpresses RafH protein regulated under DosR regulon, a critical factor for its survival. The RafH, a dual domain HPF, an orthologue of bacterial HPFlong, hibernates ribosome in 70S monosome form only. Here we report the cryo-EM structure of Mycobacterium smegmatis, a close homologue of M. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 [A] resolution. The RafH N-terminus domain (NTD) is conserved and binds to the decoding center of the ribosomal small subunit, a similar binding site of HPFlong NTD, but additionally it also interacts with the inter subunit bridge, B2a. Contrary to the HPFlong CTD, the RafH CTD, which is larger, binds to a unique site at the platform binding center of the ribosomal small subunit and sandwiches between bS1 and uS11 ribosomal proteins. The two domain connecting linker regions, which remain mostly disordered in earlier reported HPFlong structures, interacts mainly with the anti-Shine Dalgarno sequence of the 16S rRNA. The helix H54a of 23S rRNA, unique to the mycobacterial ribosome, adopts a different conformation and come close to RafH CTD, suggesting its role in ribosome hibernation. RafH inhibits in-vitro protein synthesis in a concentration dependent manner. Further, the modeling studies provided the structural basis for the incompatibility of mycobacterial ribosomes forming 100S like hibernating ribosomes.

biochemistry↗
bioRxiv2023

Efficient self-organization of blastoids solely from mouse ESCs is facilitated by transient reactivation of 2C gene network

Human pluripotent stem cells (hPSCs) can self-organize into a blastocyst-like structure (blastoid) by virtue of their full developmental potential. The pluripotent mouse embryonic stem cells (mESC) are considered to lack this potential and hence can form blastoids only when combined with trophoblast stem cells. We performed a small molecule and cytokine screen to demonstrate that mESC have full potential to efficiently self-organize themselves into E-blastoids (ESC-blastoids). The morphology, cell lineages and the transcriptome of these blastoids resemble the mouse blastocyst. The E-blastoids undergo implantation and in utero development in mice. The transient reactivation of the 2C-gene network by retinoid signaling is essential for E-blastoid generation. GSK3{beta} activity is critical for retinoid signaling and consequent 2C gene network activation. Collectively, the mESC possess full developmental potential to generate blastoids similar to hPSCs and other mammals. The plasticity of PSCs to self-organize into blastoids is not exclusive to humans or larger mammals; rather, it could be a general feature shared by most mammals, including rodents.

developmental biology↗
bioRxiv2023

Dysregulated miR-3959-3p in response to Lumpy skin disease virus

Lumpy skin disease virus (LSDV), a member of the Capripoxvirus genus, causes substantial economic losses in the livestock industry and is rapidly spreading among various LSDV-free countries across the globe. Viral infections are known to alter the cellular miRNA expression profile of the host significantly.Besides being important biomarker candidates, circulating miRNAs have a significant role in controlling viral infection and antiviral immune responses, including several molecular mechanisms. miR-3959-3p, a significantly downregulated miRNA revealed in RNA-sequencing results of LSDV-infected LT cells, was selected to bedetected in the serum of LSDV-infected and uninfected cattle sera (40 LSDV-infected and 40 LSDV-uninfected). We optimized quantitative real-time PCR (qRT-PCR) for quantitative determination of miR-3959-3p in the bovine sera samples. The level of miR-3959-3p appears to be towards lower side in the LSDV-infected as compared to the uninfected animals. However, no significant correlation could be established between the two groups due to fluctuations in the miRNA levels in both groups. This is the first report on the detection of circulating miRNA in LSDV-infected cattle sera.The dysregulation pattern of miR-3959-3p appears to suggest that several other miRNAs need to be explored and may serve as biomarkers for LSDV infection. However, this needs further investigation by screening several other miRNAs and on large number of LSDV positive and negative animals. Author summaryMicroRNAs are the key regulators of viral infections. However, in LSDVinfection the miRNA response is greatly unknown. In this study, miRNA expression in Vero cell linepost LSDV infection was studied for the first time. One of the miRNAs identified in the RNA-sequencing results i.emiR-3959-3p, was shown to be downregulated LSDV infection. We detected the levels of mir-3959-3p in sera of LSDV-infected and uninfected cattle to explore its potential as a biomarker.

molecular biology↗
bioRxiv2023

Effective networks mediate right hemispheric dominance of human 40 Hz auditory steady-state response

Auditory steady-state responses (ASSR) are induced from the brainstem to the neocortex when humans hear periodic amplitude-modulated tonal signals. ASSRs have been argued to be a key marker of auditory temporal processing and pathological reorganization of ASSR - a biomarker of neurodegenerative disorders. However, most of the earlier studies reporting the neural basis of ASSRs were focused on looking at individual brain regions. Here, we seek to characterize the large-scale directed information flow among cortical sources of ASSR entrained by 40 Hz external signals. Entrained brain rhythms with power peaking at 40 Hz were generated using both monaural and binaural tonal stimulation. First, we confirm the presence of ASSRs and their well-known right hemispheric dominance during binaural and both monaural conditions. Thereafter, reconstruction of source activity employing individual anatomy of the participant and subsequent network analysis revealed that while the sources are common among different stimulation conditions, differential levels of source activation and differential patterns of directed information flow using Granger causality among sources underlie processing of binaurally and monaurally presented tones. Particularly, we show bidirectional interactions involving the right superior temporal gyrus and inferior frontal gyrus underlie right hemispheric dominance of 40 Hz ASSR during both monaural and binaural conditions. On the other hand, for monaural conditions, the strength of inter-hemispheric flow from left primary auditory areas to right superior temporal areas followed a pattern that comply with the generally observed contralateral dominance of sensory signal processing.

neuroscience↗
bioRxiv2023

A method for predicting linear and conformational B-cell epitopes in an antigen from its primary sequence

B-cell is an essential component of the immune system that plays a vital role in providing the immune response against any pathogenic infection by producing antibodies. Existing methods either predict linear or conformational B-cell epitopes in an antigen. In this study, a single method was developed for predicting both types (linear/conformational) of B-cell epitopes. The dataset used in this study contains 3875 B-cell epitopes and 3996 non-B-cell epitopes, where B-cell epitopes consist of both linear and conformational B-cell epitopes. Our primary analysis indicates that certain residues (like Asp, Glu, Lys, Asn) are more prominent in B-cell epitopes. We developed machine-learning based methods using different types of sequence composition and achieved the highest AUC of 0.80 using dipeptide composition. In addition, models were developed on selected features, but no further improvement was observed. Our similarity-based method implemented using BLAST shows a high probability of correct prediction with poor sensitivity. Finally, we came up with a hybrid model that combine alignment free (dipeptide based random forest model) and alignment-based (BLAST based similarity) model. Our hybrid model attained maximum AUC 0.83 with MCC 0.49 on the independent dataset. Our hybrid model performs better than existing methods on an independent dataset used in this study. All models trained and tested on 80% data using cross-validation technique and final model was evaluated on 20% data called independent or validation dataset. A webserver and standalone package named "CLBTope" has been developed for predicting, designing, and scanning B-cell epitopes in an antigen sequence (https://webs.iiitd.edu.in/raghava/clbtope/).

bioinformatics↗
bioRxiv2023

The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc

BackgroundG proteins mediate cell responses to various ligands and play key roles in organ development. Dysregulation of G-proteins or Ca2+ signaling impacts many human diseases and results in birth defects. However, the downstream effectors of specific G proteins in developmental regulatory networks are still poorly understood. MethodsWe employed the Gal4/UAS binary system to inhibit or overexpress Gq in the wing disc, followed by phenotypic analysis. Immunohistochemistry and next-gen RNA sequencing identified the downstream effectors and the signaling cascades affected by the disruption of Gq homeostasis. ResultsHere, we characterized how the G protein subunit Gq tunes the size and shape of the wing in the larval and adult stages of development. Downregulation of Gq in the wing disc reduced wing growth and delayed larval development. Gq overexpression is sufficient to promote global Ca2+ waves in the wing disc with a concomitant reduction in the Drosophila final wing size and a delay in pupariation. The reduced wing size phenotype is further enhanced when downregulating downstream components of the core Ca2+ signaling toolkit, suggesting that downstream Ca2+ signaling partially ameliorates the reduction in wing size. In contrast, Gq-mediated pupariation delay is rescued by inhibition of IP3R, a key regulator of Ca2+ signaling. This suggests that Gq regulates developmental phenotypes through both Ca2+-dependent and Ca2+-independent mechanisms. RNA seq analysis shows that disruption of Gq homeostasis affects nuclear hormone receptors, JAK/STAT pathway, and immune response genes. Notably, disruption of Gq homeostasis increases expression levels of Dilp8, a key regulator of growth and pupariation timing. ConclusionGq activity contributes to cell size regulation and wing metamorphosis. Disruption to Gq homeostasis in the peripheral wing disc organ delays larval development through ecdysone signaling inhibition. Overall, Gq signaling mediates key modules of organ size regulation and epithelial homeostasis through the dual action of Ca2+-dependent and independent mechanisms.

developmental biology↗
bioRxiv2022

m6A modification of TERRA RNA is required for telomere maintenance and is a therapeutic target for ALT positive Neuroblastoma

Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.

cancer biology↗
bioRxiv2022

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed using an Indian field strain (LSDV/India/2019/Ranchi). The virus was attenuated by continuous passage (P=50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n=10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune response and exhibited complete protection upon virulent LSDV challenge. We observed a minimum Neethling response (0.018% animals; 5 out of 26940 animals) of the vaccine in field trials among 26940 animals. There was no significant reduction in the milk yield in lactating animals (n=10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n=2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

microbiology↗
bioRxiv2022

Balancing competing effects of tissue growth and cytoskeletal regulation during Drosophila wing disc development

Cytoskeletal structure and force generation within cells must be carefully regulated as the developing organ grows to reach a final size and shape. However, how the complex regulation of multiple features of tissue architecture is simultaneously coordinated remains poorly understood. Through iterations between experiments and novel computational multi-scale model simulations, we investigate the combined regulation of cytoskeletal regulation and proliferation in the growing wing imaginal disc. First, we found through experiments and calibrated model simulations that the local curvature and nuclear positioning of cells in the growing wing disc are defined by patterning of nested spatial domains of peaks in apical and basal contractility. Additionally, predictions from model simulations that incorporate a mechanistic description of interkinetic nuclear migration demonstrate that cell proliferation increases the local basal curvature of the wing disc. This is confirmed experimentally as basal curvature increases when growth and proliferation are increased through insulin signaling. In surprising contrast, we experimentally found that Decapentaplegic (Dpp), the key morphogen involved in both growth control and patterning of the anterior-posterior axis, counteracts increases in tissue bending due to cell proliferation via a combined mechanism that balances the competing impacts of both proliferation and patterning of cell contractility. Overall, the high conservation of these regulatory interactions suggests an important balancing mechanism through dual regulation of proliferation and cytoskeleton to meet the multiple criteria defining tissue morphogenesis.

developmental biology↗
bioRxiv2022

Enrichment of phenotype among biological forms of Anopheles stephensi Liston through establishment of isofemale lines

The success of vector management programs relies on knowledge of the biology and genetic make-up of mosquitoes so that they can be interlaced with modern tools for developing suitable intervention strategies. There are many reports available for rearing varied species of mosquito vectors. However, there are limited studies addressing the development of isofemale lines among mosquitoes to homogenize the population to obtain both high-quality genome assemblies and enrichment of phenotype. Anopheles stephensi, an urban malaria vector, is one of the major invasive vectors of malaria distributed throughout the Indian subcontinent, Middle East, and has recently been expanding its range in Africa. With the existence of three biological forms, distinctly identifiable based on the number of ridges on eggs with varying vectorial competence, An. stephensi is a perfect species for developing a method for the successful establishment of isofemale lines, which can be tested for retention of the expected vectorial competence for the various forms. We describe the key steps in the establishment and validation of isofemale lines, which include monitoring the transgenerational fitness traits, morphometrics of eggs, and adult wing size during every generation. After the initial inbreeding depression, as proof of the tedious selection process, no significant morphometric differences were observed in the wings and egg size between the parental and their respective isofemale lines. We observed a significant change in the vectorial competence between the respective isofemale and parental lines enriching expected differential susceptibility towards malaria parasites by the type and intermediate forms. Interestingly, IndCh and IndInt strains showed variations in resistance to different insecticides belonging to all the four major classes. These variant lines have been characterized for their levels of homozygosity both at the phenotype and genotype levels and can be used as a standard reference or as a biological resource for other studies related to urban malaria research. Author summaryIsofemale lines can be a valuable resource for characterizing and enhancing several genotypic and phenotypic traits. This is the first detailed report of the establishment of two isofemale lines of type and intermediate biological forms in Anopheles stephensi. The work encompasses the characterization of fitness traits among the two lines through a transgenerational study. Further, isofemale colonies were established and used to characterize insecticide susceptibility and vector competence. The study provides valuable insights into the differential susceptibility status of the parental and isofemale to different insecticides belonging to the same class. Corroborating with the earlier hypothesis we exemplify the high vector competence in type form than the intermediate from using homozygous isofemale lines. Using these isofemale lines it is now possible to study host-parasite interactions and identify factors that might be responsible for altered susceptibility and increased vector competence in An. stephensi biological forms that would also pave way for developing better vector management strategies.

genetics↗
bioRxiv2022

G protein-coupled Receptor Contributions to Wing Growth and Morphogenesis in Drosophila melanogaster

The development of multicellular organisms relies on a symphony of spatiotemporally coordinated signals that regulate gene expression. G protein-coupled receptors (GPCRs) are the largest group of transmembrane receptors that play a pivotal role in transducing extracellular signals into physiological outcomes. Emerging research has implicated neurotransmitter GPCRs, classically associated with communication in neuronal tissues, as regulators of pattern formation and morphogenesis. However, how these receptors interact amongst themselves and signaling pathways to regulate organogenesis is still poorly understood. To address this gap, we performed a systematic RNA interference (RNAi)-based screening of 111 GPCRs along with 8 G, 3 G{beta}, and 2 G{gamma} protein subunits in Drosophila melanogaster. We performed a coupled, machine learning-based quantitative and qualitative analysis to identify both severe and more subtle phenotypes. Of the genes screened, 25 demonstrated at least 60% penetrance of severe phenotypes with several of the most severe phenotypes resulting from the knockdown of neuropeptide and neurotransmitter GPCRs that were not known previously to regulate epithelial morphogenesis. Phenotypes observed in positive hits mimic phenotypic manifestations of diseases caused by dysregulation of orthologous human genes. Quantitative reverse transcription polymerase chain reaction and meta-analysis of RNA expression validated positive hits. Overall, the combined qualitative and quantitative characterization of GPCRs and G proteins identifies an extensive set of GPCRs involved in regulating epithelial morphogenesis and relevant to the study of a broad range of human diseases.

developmental biology↗
bioRxiv2022

Genomic and ecologic characteristics of the airway microbial-mucosal complex

SO_SCPLOWUMMARYC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWPARAGRAPHC_SCPLOWLung diseases due to infection and dysbiosis affect hundreds of millions of people world-wide1-4. Microbial communities at the airway mucosal barrier are conserved and highly ordered5, reflecting symbiosis and co-evolution with human host factors6. Freed of selection to digest nutrients for the host, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the results of the first systematic culture and whole-genome sequencing of the principal airway bacterial species, identifying abundant novel organisms within the genera Streptococcus, Pauljensenia, Neisseria and Gemella. Bacterial genomes were enriched for genes encoding antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. RNA-targeting CRISPR elements in some taxa suggest the potential to prevent or treat specific viral infections. Homologues of human RO60 present in Neisseria spp. provide a possible respiratory primer for autoimmunity in systemic lupus erythematosus (SLE) and Sjogren syndrome. We interpret the structure and biogeography of airway microbial communities from clinical surveys in the context of whole-genome content, identifying features of airway dysbiosis that may presage breakdown of homeostasis during acute attacks of asthma and chronic obstructive pulmonary disease (COPD). We match the gene content of isolates to human transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways that can sustain host interactions with the microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosal immunity in lung diseases of global significance.

microbiology↗
bioRxiv2022

A novel fluorobenzothiazole RBx 10080758 as a dual inhibitor against bacterial GyraseB (GyrB) and Topoisomerase IV (parE) of Gram-positive pathogens causing skin and respiratory infections

Development of a novel inhibitor targeting Gyrase B and Topoisomerase IV offers a potential opportunity to combat the drug resistance. In the present study, we extensively investigated the efficacy of RBx 10080758, a novel fluorobenzothiazole, against skin and respiratory infections caused by Staphylococci and Streptococci in in vitro and in vivo models. RBx 10080758 showed a potent IC50 of 0.06 M against Gyrase and Topoisomerase IV and also exhibited a strong whole cell in vitro activity with MIC ranges of 0.015-0.06, 0.015-0.03, 0.008-0.03, 0.008-0.03, 0.008-0.0.06, 0.015-0.06 g/ml against Staphylococcus. aureus, Streptococcus pneumoniae, Coagulase negative Staphylococci, Streptococcus viridans, Streptococcus pyogenes and Enterococcus, respectively. As expected from the novel class of molecule, it retains potent even against linezolid and vancomycin resistant strains. Interesting, in mouse systemic infection 10 mg/kg, IV dose protected 100% mice from lethal infections. In rat thigh infection model with MRSA WCUH29 at 45 mg/kg exhibited >3-log10 cfu reduction in thigh muscles. RBx 10080758 displayed potent in vitro and in vivo activity against a panel of MDR Gram-positive bacteria. As a novel chemical class, the fluorobenzothiazoles have the potential to become clinically viable antibiotics, to address the drug resistance problem by its unique dual targeting mechanism of action.

microbiology↗