Biology subjects

Kumar, H.

Publications and source records attributed to Kumar, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

The intracellular vacuolar pathogen Coxiella burnetii promotes exocytosis in a type IVB secretion system dependent manner

Coxiella burnetii is an obligate intracellular bacterial pathogen that causes a zoonotic disease known as Q fever. Upon internalization into host cells, Coxiella extensively remodels host vesicle trafficking and replicates within acidic, lysosome-derived, spacious vacuoles by secreting a suite of effectors through the type IVB secretion system (T4BSS), and hijacking the host cell machinery. However, how fundamental lysosomal functions, such as exocytosis, are subverted during Coxiella infection has not been well understood. In this study, we aimed to investigate the regulation and role of exocytosis in the context of Coxiella infection. Biochemical and fluorescence-based imaging approaches indicated that C. burnetii infection promotes release of extracellular vesicles (EVs). Increase in extracellular levels of endolysosomal proteins (LAMP1 and cathepsin D) and surface LAMP1 expression were identified in both phagocytic and non-phagocytic cells, during later stages of infection. Interestingly, infection-induced exocytosis was dependent on the activity of the bacterial T4BSS. Modulating the activity of TRPML1, a host calcium channel that induces exocytosis by regulating the release of calcium from lysosomes into the cytosol, using synthetic agonist/antagonist, led to a decrease in intracellular C. burnetii replication, suggesting a complex, tightly regulated role of TRPML1 during infection. Together, this study demonstrates that infection by the lysosome-adapted pathogen Coxiella burnetii activates exocytosis in a temporal, host and bacterial factors-dependent manner.

cell biology↗

bioRxiv2025

circEPSTI1 Enables Dengue Innate Immune Evasion by Sponging miR-942-5p, a Repressor of AKT1 and the Viral Genome

Circular RNAs (circRNAs) have recently been identified as crucial regulators of gene expression in various diseases. Their role in dengue remains unexplored. In this study, circEPSTI1 induction was observed in peripheral blood mononuclear cells (PBMCs) and plasma of dengue patients. The induction of circEPSTI1 is interferon-dependent and enhances DENV infection by sponging the expression of miR-942-5p. The antiviral role of miR-942-5p is mediated by bidirectional inhibition through targeting conserved genomic sequences of the DENV genome of different subtypes across NS1, NS3, and NS5 and the host AKT1 signaling pathway. RNA-seq analysis of DENV-infected circEPSTI1 knockdown A549 cells identified SERPINE1 and AKT1 signaling pathways to be dysregulated significantly. circEPSTI1 relieves the inhibition of miR-942-5p over DENV genomic RNA and the host SERPINE1 to activate AKT1 signaling. The activation of AKT1 signaling facilitates cell survival and enhances DENV replication. The expression of SERPINE1 and circEPSTI1 was upregulated in Dengue patients, and pharmaceutical inhibition of SERPINE1 through Tiplaxtinin inhibits the DENV replication by reducing the expression of circEPSTI1. Overall, our result demonstrates the therapeutic potential of Tiplaxtinin by targeting the circ-EPSTI1-miR-942-5p-SERPINE1-AKT axis in dengue treatment.

immunology↗

bioRxiv2025

Bioprinting Taurine-Incorporated Gelatin Methacrylate Hydrogels for Enhanced Muscle Tissue Regeneration

Skeletal muscle diseases like myopathies and muscular dystrophies present significant clinical challenges with few effective treatments. To better understand disease mechanisms and accelerate therapy development, robust in vitro muscle models are needed. Extrusion- and light-based bioprinting offer precise fabrication of tissue-like constructs, but creating bioinks that support muscle cell function remains difficult. Here, we report a novel bioink in which taurine is first methacrylated--substituting its -NH2 groups to afford taurine methacrylate (TMA)--enabling covalent integration into gelatin methacryloyl (GelMA) networks. We systematically compared GelMA-Taur (physical blend) versus GelMA-TMA (covalent) hydrogels, assessing mechanical stiffness, swelling behavior, and photocrosslinking kinetics. Incorporating TMA yielded tighter crosslinking control, minimized overcure in DLP-printed features, and improved shape fidelity. SEM revealed finer pore structures and homogeneous TMA distribution, and release assays confirmed prolonged TMA retention compared to rapidly leaching taurine. Photopatterning and 3D printing of complex geometries demonstrated excellent printability of the GelMA-TMA bioink. Finally, C2C12 myoblasts encapsulated in GelMA-TMA scaffolds exhibited accelerated early differentiation, higher myosin heavy chain expression, and more extensive myotube formation than controls. Together, these results establish GelMA-TMA as a printable, mechanically tunable, and biologically active platform for engineering skeletal muscle tissue in disease modeling and regenerative applications.

bioengineering↗

bioRxiv2025

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

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

immunology↗

bioRxiv2024

A Novel GelMA-OrnMA Electrically Conductive Bioink for Developing Engineered Neural Tissues

Electrical conductivity is a crucial requirement of matrices for developing engineered neural tissues. A conductive matrix not only supports cell growth but also provides potential to stimulate the cells. However, electrically conductive matrices often require inclusion of synthetic polymers, nanomaterials and large number of ionic species. While enhancing electrical conductivity, often properties like transparency, mechanical stiffness and biocompatibility are compromised which can render the resulting matrices partially suitable for neural tissue engineering. Further, the byproducts of matrix degradation can have unforeseen influences. Therefore, electrically active matrices are required which provide a suitable combination of electrical conductivity, mechanical properties and biocompatibility. In this work, a novel biomaterial is described which results in optically transparent, electrically conductive and highly biocompatible matrices along with ability to match the native neural tissue stiffness. Using gelatin methacryloyl (GelMA) as base hydrogel, we covalently incorporated zwitterionic functional groups to obtain a composite matrix. The zwitterion moieties were derived from Ornithine by synthesizing ornithine methacryloyl (OrnMA) and blending with GelMA inks. Through systematic characterization we demonstrated the suitability of GelMA-OrnMA hydrogels in providing mechanical stiffness matching the native neural tissues, supporting proliferation of human astrocytes in 3D culture and electrical conductivity in the range required for electrically active cell types like astrocytes. Owing to their electrical conductivity, these matrices also influenced the growth of astrocytes which manifested as significant changes in their organization and morphology. These findings suggest that GelMA-OrnMA has immense potential as a bioink for developing engineered neural tissues.

bioengineering↗

bioRxiv2024

Machine Learning Driven Optimization for High Precision Cellular Droplet Bioprinting

Controlled volume microliter cell-laden droplet bioprinting is important for precise biologics deposition, reliably replicating 3D microtissue environments for building cell aggregates or organoids. To achieve this, we propose an innovative machine-learning approach to predict cell-laden droplet volumes according to input parameters. We developed a novel bioprinting platform capable of collecting high-throughput droplet images and generating an extensive dataset for training machine learning and deep learning algorithms. Our research compared the performance of three machine learning and two deep learning algorithms that predict droplet volume based on numerous bioprinting parameters. By adjusting bioink viscosity, nozzle size, printing time, printing pressure, and cell concentration as input parameters, we precisely could control droplet sizes, ranging from 0.1 {micro}L to 50 {micro}L in volume. We utilized a hydrogel precursor composed of 5% gelatin methacrylate and a mixture of 0.5% and 1% alginate, respectively. Additionally, we optimized the cell bioprinting process using green fluorescent protein-tagged 3T3 fibroblast cells. These models demonstrated superior predictive accuracy and revealed the interrelationships among parameters while taking minimal time for training and testing. This method promises to advance the mass production of organoids and microtissues with precise volume control for various biomedical applications.

bioengineering↗

bioRxiv2024

MicroRNA hsa-miR-203a-3p promotes H1N1 and NDV Virus Replication by suppressing the IFNA signaling.

MicroRNAs (miRNAs) are small non-coding molecules that act as essential post-transcriptional regulators in various biological processes. Many studies suggest that miRNAs may modulate the hosts immune response during virus infections. We analyzed publicly available transcriptomics data involving infection with different RNA viruses and selected the most prominent candidate, miR-203a-3p. miR-203a-3p is upregulated during H7N1, HCV, and HIV+HPV infections. Interestingly, pathway analysis of microRNA-targeted genes shows that miR-203a-3p targets the type-I interferon pathway. In this study, we report that the expression of miR-203a-3p is elevated in response to polyinosinic-polycytidylic acid [poly(I:C)] transfection and infection with RNA viruses like Newcastle Disease Virus (NDV) and H1N1 influenza virus. We found that overexpression of miR-203-3p promotes the replication of H1N1 virus by suppressing the hosts type-I interferons and interferon-stimulated genes. In addition, miR-203a-3p overexpression reduced the expression of ISGs, and is attributed to the binding of miR-203a-3p to 3 UTRs of Janus-activated kinase 1 (JAK1), STAT1, SOCS3, and multiple IFNA transcripts, as shown by luciferase and Ago-2 pulldown assays. Altogether, these findings strongly suggest that miR-203a-3p acts as a pro-viral molecule during H1N1 and NDV infection by targeting the hosts IFN signaling pathways.

immunology↗

bioRxiv2024

3D bioprinting of Liver Microenvironment Model Using Photocrosslinkable Decellularized Extracellular Matrix based Hydrogel

The liver, as one of the vital organs in the body, plays a crucial role in various bodily functions. Numerous factors can cause liver damage, that the sole remedy for severe liver conditions is the transplantation of healthy liver tissue. In response to the transplantation challenges, innovative approaches involving hydrogel-based technologies have emerged, leading to the creation of highly functionalized tissues. The development of three-dimensional printing and patterning of cell-laden biomaterial matrices offers promising advances for creating tissue-specific structures in tissue engineering and bioprinting. However, the matrix materials currently employed in bioprinting liver microtissue often fail to capture the complexity of the natural extracellular matrix (ECM), hindering their ability to restore innate cellular shapes and functions. Liver ECM-based hydrogels are increasingly recognized for their potential as biomimetic 3D cell culture systems that facilitate the exploration of liver disease, metabolism, and toxicity mechanisms. Yet, the conventional production of these hydrogels relies on slow thermal gelation processes, which restrict the manipulation of their mechanical characteristics. In this research, we introduce a novel approach with a functionalized photocrosslinkable liver decellularized extracellular matrix (dECM). By combining liver dECM methacrylate (LdECMMA) with gelatin methacrylate (GelMA), we achieved accelerated crosslinking under visible light irradiation and the ability to tune the mechanical, rheological, and physiological properties of the material. We encapsulated human hepatocellular carcinoma cells within an optimal concentration of the GelMA-LdECMMA hybrid hydrogel and examined cell proliferation and function over an extended period. The findings revealed that the GelMA-LdECMMA hybrid hydrogel enhances liver cell proliferation and function, holding significant promise for applications in drug screening and liver cancer metastasis research.

bioengineering↗

bioRxiv2024

Direct Ink Writing of Graphene Oxide Reinforced 13-93B1 Bioactive Glass Scaffolds for Bone Tissue Engineering Applications

Graphene-reinforced bioactive glass scaffolds have gained significant attention in the field of bone tissue engineering due to their unique combination of mechanical strength, bioactivity, and electrical conductivity. Additive manufacturing techniques, such as 3D printing, provide a versatile platform for fabricating these scaffolds with precise control over their architecture and composition. Consequently, in this work, we have fabricated graphene oxide (GO)-reinforced 13-93B1 bioactive glass scaffold using the direct ink writing technique. A Pluronic F-127- based ink was prepared for scaffold fabrication, and its rheological properties were assessed for shear thinning behaviour, structural support, and recovery. Further, the fabricated scaffolds were characterized using micro-computed tomography, scanning electron microscopy, and energy dispersive x-ray spectroscopy. Additionally, computational fluid dynamics simulations with Dulbeccos modified eagle medium and blood were performed to evaluate the perfusion kinetics of the scaffolds. The inclusion of GO enhanced the compressive strength of the fabricated scaffolds by [~]225%. The morphological characterization based on micro-computed tomography showed that additively manufactured scaffolds have appropriate porosity, pore size, pore throat size, and interconnectivity. The live-dead assay results showed no cytotoxicity towards C2C12 mouse myoblast cells. Also, cell adhesion and cell viability results show better cell growth on the nanocomposite scaffolds. Overall, the fabricated scaffold is found suitable for bone tissue engineering applications.

bioengineering↗

bioRxiv2023

Polymer Model Unveils Quantitative Association of Chromatin Conformation and Gene Regulation

The spatial organization of chromatin in the nucleus is of essence for regulating gene transcription. However, the mechanisms governing the intricate interplay of chromatin structure and gene transcription remain poorly understood. Hi-C experiments have unveiled a multiscale chromatin organization, significantly enriching our understanding of the structural control of gene expression. We introduce a computational framework to link chromatin structural modifications to gene regulation. This framework generates an ensemble of three-dimensional conformations of a given genomic locus using a bead-spring polymer model where the cHiC contact map is used as an input. We then correlate such chromatin conformations to the transcription level of the encoded genes using a Markov chain-based model, where binding/unbinding rates extracted from the molecular dynamics trajectories are used. By considering a specific example, we have demonstrated that the deletion of the CTCF binding domain between two consecutive TADs leads to a significant change in the enhancer-promoter interaction and gene transcription of the encoded genes, namely sox9 and kcnj2, responsible for limb development. Such a change in gene expression level is quantitatively consistent with experiments. Further insight from the polymer-based 3D conformation reveals that the higher gene expression level of the kcnj2 gene is caused by the specific enhancers of kcnj2, present in the sox9 TAD, which become accessible after boundary deletion. By quantifying the impact of these enhancers, our model can also be used to identify the functional enhancers. Together, the present computational framework not only advances our understanding of the relationship between the spatial architecture of the chromosome and the function of the cell but also provides invaluable insights into potential therapeutic interventions targeting aberrant gene regulation in pathological contexts.

biophysics↗

bioRxiv2023

Vascularized Liver Tissue Embedded Bioprinting Utilizing GelMA/Nanoclay-based Composite hydrogels

As the aging population grows, the need to regenerate non-self-repairing tissues becomes increasingly crucial for enhancing our quality of life. Tissue engineering offers a promising solution, particularly in recreating the intricate networks of blood vessels crucial for tissue vitality. These tissues rely on effective nutrient and oxygen circulation, with an optimal oxygen diffusion range of 100-200 {micro}m. Yet, crafting vascularized in vitro tissues remains a significant challenge. This study addresses the challenge by using GelMA-based hydrogels as a photocrosslinkable support bath, a biocompatible and versatile choice for biological applications. To enhance the rheological properties for in vitro tissue engineering, Laponite (LPN) is introduced as a rheology modifier. The study optimizes the GelMA-LPN nanocomposite hydrogel composition, ensuring the desired physical, mechanical, and rheological properties, including recovery. The research also explores the biological implications, encapsulating liver cells within the nanocomposite hydrogel, and studying their behavior under perfusion conditions. This research presents a promising avenue for creating vascularized in vitro tissues, potentially advancing tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2023

Computational design of DNA binding domain-retained fusion proteins and virtual screening against FDA-approved drugs

Even though the transcription factors (TFs) are not regarded as good drug targets, mutated or dysregulated TFs can be a unique class of drug targets. Specifically, the TF fusion protein, which is the translated structural variants including TFs may affect downstream to promote tumorigenesis. To date, we lack the fusion protein sequence information and 3D structure information in identifying the potential drugs of fusion proteins. In this study, we predicted the 3D structures of 732 transcription factor fusion proteins (TFFPs). For the top five most frequent TFFPs, we performed the virtual screening across the FDA-approved drugs. Our study will provide an initial platform to develop novel therapeutic targets in the transcription factor fusion proteins.

bioinformatics↗

bioRxiv2023

A core Nrf2 gene set defined through comprehensive transcriptomic analysis predicts drug resistance and poor multi-cancer prognosis

The NRF2-KEAP1 pathway plays an important role in the cellular response to oxidative stress but may also contribute to metabolic changes and drug resistance in cancer. We investigated the activation of NRF2 in human cancers and fibroblast cells through KEAP1 inhibition and cancer associated KEAP1/NRF2 mutations. We define a core set of 14 upregulated NRF2 target genes from seven RNA-Sequencing databases that we generated and analyzed, which we validated this gene set through analyses of published databases and gene sets. An NRF2 activity score based on expression of these core target genes correlates with resistance to drugs such as PX-12 and necrosulfonamide but not to paclitaxel or bardoxolone methyl. We validated these findings and also found NRF2 activation led to radioresistance in cancer cell lines. Finally, our NRF2 score is prognostic for cancer survival and validated in additional independent cohorts for novel cancers types not associated with NRF2-KEAP1 mutations. These analyses define a core NRF2 gene set that is robust, versatile, and useful as a NRF2 biomarker and for predicting drug resistance and cancer prognosis.

cancer biology↗

bioRxiv2022

Latent representation of single-cell transcriptomes enables algebraic operations on cellular phenotypes

Single-cell RNA-sequencing (scRNA-seq) coupled with robust computational analysis facilitates the characterization of phenotypic heterogeneity within tumors. Current scRNA-seq analysis pipelines are capable of identifying a myriad of malignant and non-malignant cell subtypes from single-cell profiling of tumors. However, given the extent of intra-tumoral heterogeneity, it is challenging to assess the risk associated with individual cell subpopulations, primarily due to the complexity of the cancer phenotype space and the lack of clinical annotations associated with tumor scRNA-seq studies. To this end, we introduce SCellBOW, a scRNA-seq analysis framework inspired by document embedding techniques from the domain of Natural Language Processing (NLP). SCellBOW is a novel computational approach that facilitates effective identification and high-quality visualization of single-cell subpopulations. We compared SCellBOW with existing best practice methods for its ability to precisely represent phenotypically divergent cell types across multiple scRNA-seq datasets, including our in-house generated human splenocyte and matched peripheral blood mononuclear cell (PBMC) dataset. For tumor cells, SCellBOW estimates the relative risk associated with each cluster and stratifies them based on their aggressiveness. This is achieved by simulating how the presence or absence of a specific cell subpopulation influences disease prognosis. Using SCellBOW, we identified a hitherto unknown and pervasive AR-/NElow (androgen-receptor-negative, neuroendocrine-low) malignant subpopulation in metastatic prostate cancer with conspicuously high aggressiveness. Overall, the risk-stratification capabilities of SCellBOW hold promise for formulating tailored therapeutic interventions by identifying clinically relevant tumor subpopulations and their impact on prognosis.

bioinformatics↗

bioRxiv2022

Intragastric administration of low-dose rotenone post-colitis exacerbates damage to the nigrostriatal dopaminergic system in Parkinson's disease: The pace accelerates even more

BackgroundThe contribution of gastrointestinal (GI) inflammation and local exposure to neurotoxins in the gut offers the most in-depth explanation of Parkinsons disease (PD) etiopathogenesis through abnormal accumulation and spreading of alpha-synuclein (-syn) aggregates from the gut to the brain. ObjectivesThis study was designed to investigate whether dextran sodium sulfate (DSS)-mediated colitis may have lasting effects on dopaminergic pathways in the brain and whether or not colitis exacerbated susceptibility to later exposure to the neurotoxin rotenone. MethodsTo induce chronic colitis, 10 months old C57BL/6 mice were pre-exposed to 3 cycles of 7 days of 1% (w/v) DSS administration in drinking water followed by 14 days of regular drinking water. After colitis-induction, animals received a low dose of intragastric rotenone for the next 8 weeks, followed by testing for Parkinsonian behavior and GI phenotypes of inflammation. At the end of the 8th week after colitis, colon, brain stem, and midbrain tissue were isolated and analyzed for -syn, inflammatory markers, and dopaminergic neuronal loss. Gut microbial composition was assessed by 16S rRNA sequencing analysis. ResultsWe found that local rotenone exposure for 8 weeks did not affect colitis severity and colonic tight junction(TJ) protein expression (ZO-1, Occludin, and Claudin-1). On the other hand, we found that while eight weeks of chronic rotenone administration led to an increase in inflammatory markers, the presence of pre-existing colitis resulted in a considerable change in gut microbiota composition and a decrease in TJs protein expression. In addition, the administration of rotenone in mice post-colitis caused gastrointestinal function impairment and poor behavioral performances. Itworsened rotenone-induced -syn pathology in the colon, which extended upward and resulted in severe dopaminergic neuron loss and significant astroglia activation in the dorsal motor nucleus of the vagus (DMV), locus coeruleus, substantia nigra as well as in striatum. Interestingly, in the case of rotenone alone, we found that -syn induced ChAT+ neuronal death is restricted to the DMV. These findings indicate that long-term rotenone exposure in conjunction with early inflammatory intestinal milieu exacerbates the progression of -syn pathology and aggravates neurodegeneration in the intragastric mouse PD model. ConclusionsThis work provides detailed insight into the involvement of GI inflammation triggered after a neurotoxic insult in the colon and explores their potential to impact central dopaminergic degeneration in PD. This way, we can identify potential therapeutic targets that stop the enteric inflammatory processes involved in progressing PD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/521569v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@e0b5a4org.highwire.dtl.DTLVardef@b556c0org.highwire.dtl.DTLVardef@1526d27org.highwire.dtl.DTLVardef@7c506f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILow-dose intragastric rotenone post-colitis aggravates gastrointestinal dysfunction and accelerates the onset of motor impairment. C_LIO_LILow-dose intragastric rotenone did not alter colitis clinical and histological aspects. C_LIO_LILow-dose intragastric rotenone post-colitis exacerbated the progression of -syn pathology from the GI tract to the brain, leading in central dopaminergic neuronal degeneration. C_LI

neuroscience↗

bioRxiv2022

De novo discovery of traits co-occurring with chronic obstructive pulmonary disease

Epidemiological research indicates that chronic obstructive pulmonary disease (COPD) is a heterogeneous group of chronic lung conditions that are typically accompanied by cardiovascular disease, depression, lung cancer and other conditions. Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with COPD and the co-occuring conditions, suggesting common biological mechanisms underlying COPD and these co-occuring conditions. To identify them, we have integrated information across different biological levels (i.e. genetic variants, lung-specific 3D genome structure, gene expression and protein-protein interactions) to build lung-specific gene regulatory and protein-protein interaction networks. We have queried these networks using disease-associated SNPs for COPD, unipolar depression and coronary artery disease. Our results show that COPD-associated SNPs can control genes involved in the regulation of lung or pulmonary function, asthma, brain region volumes, cortical surface area, depressed affect, neuroticism, Parkinsons disease, white matter microstructure and smoking behaviour. We describe the regulatory connections, genes and biochemical pathways that underly these co-occuring trait-SNP-gene associations. Collectively, our findings provide new avenues for the investigation of the underlying biology and diverse clinical presentations of COPD. In so doing, we identify a collection of genetic variants and genes that may aid COPD patient stratification and treatment.

genomics↗

bioRxiv2022

Conformational flexibility, lipid binding, and regulatory domains in CelTOS

CelTOS is an essential Plasmodium traversal protein and conserved in apicomplexan parasites. We showed that CelTOS forms pores in cell membranes to enable traversal of parasites through cells (Jimah et al., 2016). Here, we establish roles for the distinct regions of CelTOS, examine the mechanism of pore formation and evaluate the immunogenicity of engineered CelTOS variants. CelTOS dimer dissociation is required for pore formation as disulfide bridging between monomers inhibits pore formation and this inhibition is rescued by disulfide-bridge reduction. A helix destabilizing Pro127 allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C-terminus of CelTOS is a negative regulator that limits pore formation. Lipid binding is a pre-requisite for pore assembly as mutation of a phospholipid binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. The disulfide-locked and N-terminal deletion mutants showed improved immunogenicity relative to wild-type CelTOS in mice. These findings have implications for pore-forming proteins that are essential for diverse functions, identify critical regions in CelTOS, and will guide the design of effective CelTOS-targeting vaccines to combat infection and transmission of malaria and apicomplexan parasites.

microbiology↗

bioRxiv2021

Innate Immune sensing of Influenza A viral RNA through IFI16 promotes pyroptotic cell death

Programmed cell death pathways are triggered by various stresses or stimuli, including viral infections. The mechanism underlying the regulation of these pathways upon Influenza A virus IAV infection is not well characterized. We report that a cytosolic DNA sensor IFI16 is essential for the activation of programmed cell death pathways in IAV infected cells. We have identified that IFI16 functions as an RNA sensor for influenza A virus by binding to genomic RNA. The activation of IFI16 triggers the production of type I, III interferons, and also other pro-inflammatory cytokines via the STING-TBK1 and Pro-caspase-1 signaling axis, thereby promoting cell death (apoptosis and pyroptosis in IAV infected cells). Whereas, IFI16 knockdown cells showed reduced inflammatory responses and also prevented cell mortality during IAV infection. These results demonstrate the pivotal role of IFI16-mediated IAV sensing and its essential role in activating programmed cell death pathways.

immunology↗