Biology subjects

Mishra, A.

Publications and source records attributed to Mishra, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Metabolomics of Mouse Embryonic CSF Following Maternal Immune Activation

The cerebrospinal fluid (CSF) serves various roles in the developing central nervous system (CNS), from neurogenesis to lifelong cognitive functions. Changes in CSF composition due to inflammation can impact brain function. We recently identified an abnormal cytokine signature in embryonic CSF (eCSF) following maternal immune activation (MIA), a mouse model of autism spectrum disorder (ASD). We hypothesized that MIA leads to other alterations in eCSF composition and employed untargeted metabolomics to profile changes in the eCSF metabolome in mice after inducing MIA with polyI:C. We report these data here as a resource, include a comprehensive MS1 and MS2 reference dataset, and present additional datasets comparing two mouse strains (CD-1 and C57Bl/6) and two developmental time points (E12.5 and E14.5). Targeted metabolomics further validated changes upon MIA. We show a significant elevation of glucocorticoids and kynurenine pathway related metabolites. Both pathways are relevant for suppressing inflammation or could be informative as disease biomarkers. Our resource should inform future mechanistic studies regarding the etiology of MIA neuropathology and roles and contributions of eCSF metabolites to brain development.

neuroscience↗

bioRxiv2023

Remyelination protects neurons from DLK-mediated neurodegeneration

Chronic demyelination and oligodendrocyte loss deprive neurons of crucial support. It is the degeneration of neurons and their connections that drives progressive disability in demyelinating disease. However, whether chronic demyelination triggers neurodegeneration and how it may do so remain unclear. We characterize two genetic mouse models of inducible demyelination, one distinguished by effective remyelination and the other by remyelination failure and chronic demyelination. While both demyelinating lines feature axonal damage, mice with blocked remyelination have elevated neuronal apoptosis and altered microglial inflammation, whereas mice with efficient remyelination do not feature neuronal apoptosis and have improved functional recovery. Remyelination incapable mice show increased activation of kinases downstream of dual leucine zipper kinase (DLK) and phosphorylation of c-Jun in neuronal nuclei. Pharmacological inhibition or genetic disruption of DLK block c-Jun phosphorylation and the apoptosis of demyelinated neurons. Together, we demonstrate that remyelination is associated with neuroprotection and identify DLK inhibition as protective strategy for chronically demyelinated neurons. HighlightsO_LICharacterization of a transgenic mouse model of demyelination without subsequent remyelination C_LIO_LIRemyelination protects neurons from axon loss and neuronal apoptosis C_LIO_LIMAPK and c-Jun phosphorylation are increased in mice featuring remyelination failure C_LIO_LIDLK is necessary for the apoptosis of chronically demyelinated neurons C_LI

neuroscience↗

bioRxiv2023

Arrested Agonist Paradigm For Selective Radiosensitization of Prostate Cancer

As a prototypical nuclear hormone receptor, the androgen receptor (AR) signals via a sequential cascade triggered by binding to androgenic ligands such as testosterone and dihydrotestosterone (DHT). This cascade includes dimerization of the ligand-receptor complex, nuclear translocation, chromatin binding to response elements, recruitment of TOP2B and co-activator complexes, and induction of an effector transcriptional program. In prostate cancers, this AR signaling cascade is an essential driver of growth and survival, yet its activity confers potential vulnerabilities through transient TOP2B-mediated DNA double strand breaks. We investigated the ability of non-steroidal AR ligands to activate initial steps of the AR signaling cascade up to the point of AR- and TOP2B-mediated double strand breaks, with subsequent arrest of the signaling cascade to prevent induction of pro-growth/survival transcriptional programs in prostate cancer cells. We identified hydroxyflutamide (FLU) as such an androgen receptor arrested agonist; in androgen-deprived conditions, FLU induced AR nuclear translocation, chromatin binding, and TOP2B-mediated double strand breaks, but failed to induce AR target gene expression and prostate cancer cell growth. The FLU-mediated arrest in the signaling cascade could be attributed to the inability of FLU to allow association of AR with SMARCD2, a critical component of the BAF chromatin remodeling complex required for androgen induced AR co-activation. Interestingly, the FLU-induced, AR- and TOP2B-mediated double strand breaks could be used to selectively sensitize AR-positive prostate cancer cells to ionizing radiation in vitro and in vivo. These findings support a novel arrested agonist paradigm for selective radiosensitization of prostate cancer cells without inducing AR-mediated pro-growth and survival transcriptional programs.

cancer biology↗

bioRxiv2023

Autophagy protein Atg7 is essential and druggable for maintaining malaria parasite cellular homeostasis and organelle biogenesis

Plasmodium parasites have a complex life cycle that transitions between mosquito and mammalian hosts and undergoes continuous cellular remodeling to adapt to various drastic environments. Following hepatocyte invasion, the parasite discards superfluous organelles for intracellular replication, and the remnant organelles undergo extensive branching and mature into hepatic merozoites. Autophagy is a ubiquitous eukaryotic process that permits the recycling of intracellular components. Here, we show that the P. berghei autophagy-related E1-like enzyme Atg7 is expressed in the blood and liver stages, localized to the parasite cytosol and is essential for the localization of Atg8 on the membrane and the development of parasite blood and liver forms. We found that depleting Atg7 abolishes exocytosis of micronemes, organelle biogenesis and the formation of merozoites during liver stage development. Furthermore, we identified the compounds from the Maybridge library with a high docking score against PfAtg7. We show that these compounds inhibit apicoplast biogenesis and parasite development in both blood and liver stages. Overall, this study establishes the essential functions of autophagy in Plasmodium blood and liver stages and highlights the potential of using Atg7 as a drug target against malaria.

microbiology↗

bioRxiv2023

SwrA extends DegU over an UP element to activate flagellar gene expression in Bacillus subtilis

SwrA activates flagellar gene expression in Bacillus subtilis to increase the frequency of motile cells in liquid and elevate flagellar density to enable swarming over solid surfaces. Here we use ChIP-seq to show that SwrA interacts with many sites on the chromosome in a manner that depends on the response regulator DegU. We identify a DegU-specific inverted repeat DNA sequence and show that SwrA synergizes with phosphorylation to increase DegU DNA binding affinity. We further show that SwrA increases the size of the DegU footprint expanding the region bound by DegU towards the promoter. The location of the DegU inverted repeat was critical and moving the binding site closer to the promoter impaired transcription more that could be explained by deactivation. We conclude that SwrA/DegU forms a heteromeric complex that enables both remote binding and interaction between the activator and RNA polymerase in the context of an interceding UP element. We speculate that multimeric activators that resolve cis-element spatial conflicts are common in bacteria and likely act on flagellar biosynthesis loci and other long operons of other multi-subunit complexes. IMPORTANCEIn Bacteria, the sigma subunit of RNA polymerase recognizes specific DNA sequences called promoters that determine where gene transcription begins. Some promoters also have sequences immediately upstream called an UP element that is bound by the alpha subunit of RNA polymerase and is often necessary for transcription. Finally, promoters may be activated by transcription factors that bind DNA specific sequences and help recruit RNA polymerase to weak promoter elements. Here we show that the promoter for the 32 gene long flagellar operon in Bacillus subtilis requires an UP element and is activated by a heteromeric transcription factor of DegU and SwrA. Our evidence suggests that SwrA oligomerizes DegU over the DNA to allow RNA polymerase to interact with DegU and the UP element simultaneously. Heteromeric activator complexes are known but poorly-understood in bacteria and we speculate they may be needed to resolve spatial conflicts in the DNA sequence.

microbiology↗

bioRxiv2023

Altered GM1 catabolism affects NMDAR-mediated Ca2+ signaling at ER-PM junctions and increases synaptic spineformation

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to {beta}-galactosidase deficiency drastically alters neuronal Ca2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated NMDAR Ca2+ channel, thereby increasing Ca2+ flux, activating ERK signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and exacerbates the generalized neuronal cell death characteristic of GM1-gangliosidosis.

neuroscience↗

bioRxiv2023

A Microfluidic Transistor for Liquid Signal Processing

Abstract/SummaryMicrofluidics have enabled significant advances in molecular biology1-3, synthetic chemistry4,5, diagnostics6,7, and tissue engineering8. However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity, and scalability of electronic circuits9-11. Just as the electronic transistor enabled unprecedented advances in the control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the complex, scalable control of reagents, droplets, and single cells on an autonomous microfluidic chip. Prior works on creating a microfluidic analogue to the electronic transistor12-14 could not replicate the transistors saturation behavior, which is crucial to perform analog amplification15 and is fundamental to modern circuit design16. Here we exploit the fluidic phenomenon of flow-limitation17 to develop a microfluidic element with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. As this microfluidic transistor successfully replicates all of the key operating regimes of the electronic transistor (linear, cut-off and saturation), we are able to directly translate a variety of fundamental electronic circuit designs into the fluidic domain, including the amplifier, regulator, level shifter, logic gate, and latch. Finally, we demonstrate a "smart" particle dispenser that senses single suspended particles, performs liquid signal processing, and accordingly controls the movement of said particles in a purely fluidic system without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic transistor-based circuits are easy to integrate at scale, eliminate the need for external flow control, and enable uniquely complex liquid signal processing and single-particle manipulation for the next generation of chemical, biological, and clinical platforms.

bioengineering↗

bioRxiv2023

A GALLIUM-68-LABELED PEPTIDE RADIOTRACER FOR CD38-TARGETED IMAGING IN MULTIPLE MYELOMA WITH PET

PurposeThe limited availability of molecularly targeted low-molecular-weight imaging agents for monitoring multiple myeloma (MM)-targeted therapies has been a significant challenge in the field. In response, we developed [68Ga]Ga-AJ206, a peptide-based radiotracer that can be seamlessly integrated into the standard clinical workflow and is specifically designed to non-invasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). Experimental designWe synthesized a high-affinity binder for quantification of CD38 levels. Affinity was tested using surface plasmon resonance, and In vitro specificity was evaluated using a gallium-68-labeled analog. Distribution, pharmacokinetics, and CD38 specificity of the radiotracer were assessed in MM cell lines and in primary patient-derived myeloma cells and xenografts (PDX) with cross-validation by flow cytometry and immunohistochemistry. Furthermore, we investigated the radiotracers potential to quantify CD38 pharmacodynamics induced by all-trans retinoic acid therapy (ATRA). Results[68Ga]Ga-AJ206 exhibited high CD38 binding specificity (KD: 19.1{+/-}0.99 nM) and CD38-dependent In vitro binding. [68Ga]Ga-AJ206-PET showed high contrast within 60 minutes and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga-AJ206 detected CD38 expression in xenografts, PDXs and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga-AJ206-PET successfully quantified CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following ATRA therapy. Conclusions[68Ga]Ga-AJ206 exhibited the salient features required for clinical translation, providing CD38-specific high contrast images in multiple models of MM. [68Ga]Ga-AJ206-PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement. STATEMENT OF TRANSLATIONAL RELEVANCEThere is an unmet need for functional imaging agents to monitor the pharmacodynamic effects of new therapeutics targeting multiple myeloma (MM). MM is a challenging bone marrow plasma cell cancer that is associated with heterogenous responses and universal recurrence. While minimal residual disease monitoring by blood and invasive bone marrow samples have improved prognostication of disease recurrence, molecularly targeted, non-invasive imaging options that can assess therapy response early remain limited. To address this gap, we report the development of a high affinity, first-in-class gallium-68 labeled peptide radiotracer, [68Ga]Ga-AJ206, for CD38 protein, which is highly expressed on MM cells. [68Ga]Ga-AJ206 provides high-contrast CD38-specific images by PET within the standard clinical workflow of 60 minutes. Furthermore, the potential of [68Ga]Ga-AJ206 PET to measure pharmacodynamics of CD38 was demonstrated. Further development of this new radiotracer may complement existing technologies and improve prognostication and monitoring of therapy response in patients with MM.

cancer biology↗

bioRxiv2023

Multiplexed fluorescence and scatter detection with single cell resolution using on-chip fiber optics for droplet microfluidic applications

Droplet microfluidics has emerged as a critical component of several high-throughput single cell analysis techniques in biomedical research and diagnostics. However, while there has been significant progress in the individual assays being developed, multi-parametric optical sensing of droplets and their encapsulated contents remains challenging. The current common approach of microscopy based high-speed imaging of droplets is technically complex and requires expensive instrumentation limiting their large-scale adoption. Here we have adapted the principles of the widely established technique of flow cytometry to a novel optofluidic setup - the OptiDrop platform, with on-chip detection of scatter and multiple fluorescence signals from microfluidic droplets and their contents using optical fibers. The highly customizable on-chip optical fiber-based signal detection system enables simplified, miniaturized, low-cost, multi-parametric sensing of optical signals with high sensitivity and single cell resolution within each droplet. Our work on differential expression analysis of the Major Histocompatibility Complex (MHC) protein in response to IFN{gamma} stimulation further demonstrates the OptiDrop platforms capabilities in sensitively detecting cell surface biomarkers using fluorescently labeled antibodies. The OptiDrop platform thus combines the versatility of flow cytometry with the power of droplet microfluidics to provide wide-ranging optical sensing solutions for research and diagnostics.

bioengineering↗

bioRxiv2023

Charting spatial ligand-target activity using Renoir

The advancement of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics has enabled the inference of cellular interactions in a tissue microenvironment. Despite the development of cell-cell interaction inference methods, there is a lack of methods capable of mapping the influence of ligands on downstream target genes across a spatial topology with specific cell type composition, with the potential to shed light on niche-specific relationship between ligands and their downstream targets. Here we present Renoir for charting the ligand-target activities across a spatial topology and delineating spatial communication niches harboring specific ligand-target activities. Renoir also spatially maps pathway-level activity of ligand-target genesets and identifies domain-specific ligand-target activities. Across spatial datasets with varying resolution (spot to single-cell) ranging from development to disease, Renoir inferred cellular niches with distinct ligand-target interactions, spatially mapped pathway activities, and identified context-specific novel cell-cell interactions. Renoir uncovers biological insights and therapeutically-relevant cellular crosstalk from spatial transcriptomics data.

bioinformatics↗

bioRxiv2023

Higher-order co-mutation interactions in mitochondrial genomes

Pair-wise co-mutation networks of the mitochondrial genome have already provided ample evidences about the roles of genetic interactions in the manifestation of phenotype under altered environmental conditions. Here, we present a method to construct and analyze higher-order interactions, namely, 3-uniform hypergraphs of the mitochondrial genome for different altitude populations to decipher the role of co-mutating variable sites beyond pair-wise interactions. While the weights distribution of such gene hyperedges manifested power-law for all the altitudes, we identified altitude-specific genes based on gene hyperedge weight. This framework of hypergraphs serves a promising avenue for future investigation of nuclear genomes in context of phenotypic association and genetic disorders.

systems biology↗

bioRxiv2022

Evolution of mate harm resistance in females from Drosophila melanogaster populations selection for faster development and early reproduction

Interlocus sexual conflict is predicted to result in sexually antagonistic coevolution between male competitive traits, which are also female-detrimental, and mate harm resistance (MHR) in females. Little is known about connection life-history evolution and sexually antagonistic coevolution. Here, we investigated the evolution of MHR in a set of experimentally evolved populations, where mate-harming ability has been shown to have evolved in males as a correlated response to the selection for faster development and early reproduction. We measured mortality and fecundity of females of these populations and those of their matched controls, under different male exposure conditions. We observed that the evolved females were more susceptible to mate harm - suffering from significantly higher mortality under continuous exposure to control males within the twenty-day assay period. Though these evolved females are known to have shorter lifespan, such higher mortality was not observed under virgin and single-mating conditions. We used fecundity data to show that this higher mortality in evolved females is unlikely due to cost of egg production. Further analysis indicated that this decreased MHR is unlikely to be due purely to the smaller size of these females. Instead, it is more likely to be an indirect experimentally evolved response attributable to the changed breeding ecology, and/or male trait evolution. Our results underline the implications of changes in life history traits, including lifespan, to the evolution of MHR in females.

evolutionary biology↗

bioRxiv2022

Functional mRNA delivery to hematopoietic stem and progenitor cells in vivo.

Gene correction of hematopoietic stem cells (HSC) is a promising therapeutic approach for multiple disorders. Current methods, however, require HSC collection from patients, gene correction during ex vivo culture, and re-infusion of corrected HSC into patients conditioned with chemotherapeutic agents. These approaches are complex, and the conditioning creates toxicities. We show that a lipid nanoparticle (LNP) can deliver mRNA encoding a reporter or a gene editing protein to HSC, with one injection transfecting [~]25% of mouse HSC, and repeated doses resulting in higher editing efficiencies. We also demonstrate LNP-driven in vivo mRNA delivery to HSC in non-human primates and humanized mice. These results demonstrate a translatable approach to deliver mRNA encoding therapeutic proteins, or gene correcting tools, to HSC that do not require cell culture or toxic conditioning. One-Sentence SummaryLNP can deliver functional mRNA to mouse, non-human primate, and human HSC.

immunology↗

bioRxiv2022

Genetic variants of human platelet antigens in the Indian population from 1029 whole genomes

BackgroundGenetic variants in human platelet antigens (HPAs) considered as allo- or auto antigens are associated with various disorders including neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and post-transfusion purpura. While global differences in genotype frequencies were observed, the distribution of HPA variants in the Indian population are largely unknown. This study aims to explore the landscape of HPA variants in India to provide a basis for risk assessment and management of related complications. Materials and methodsPopulation specific frequencies of genetic variants associated with the 35 classes of HPAs (HPA-1 to HPA-35) were estimated by systematically analyzing genomic variations of 1029 healthy Indian individuals as well as from global population genome datasets.. ResultsAllele frequencies of the most clinically relevant HPA systems in the Indian population were found as follows, HPA-1a - 0.89, HPA-1b - 0.15, HPA-2a - 0.94, HPA-2b - 0.05, HPA-3a - 0.66, HPA-3b - 0.36, HPA-4a - 1.00, HPA-4b - 0, HPA-5a - 0.92, HPA-5b - 0.08, HPA-6a - 1.00, HPA-6b - 0, HPA-15a - 0.58 and HPA-15b - 0.42. In addition, HPA-4b allele frequencies were found to be significantly higher in India in comparison to global populations. ConclusionThis study provides the first comprehensive analysis of HPA allele and genotype frequencies using large scale representative whole genome sequencing data of the Indian population.

genomics↗

bioRxiv2022

Glucuronoxylomannan intranasal challenge prior to Cryptococcus neoformans pulmonary infection enhances cerebral cryptococcosis in rodents

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a "Trojan horse" mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening the human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB), and promotes C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated [~]200,000 deaths worldwide each year. AUTHOR SUMMARYCryptococcus neoformans infection of the central nervous system (CNS) typically begins by inhalation of fungal spores and results in devastating mortality rates worldwide. Over 200,000 deaths have been reported annually, with cryptococcal meningoencephalitis being the most severe form of the disease. This study investigates the ability of the fungus to invade, colonize, and cause damage to the host through properties of the fungal polysaccharide capsule, which allows the microbe a variety of both protective and offensive abilities. This capsule, made primarily of the polysaccharide glucuronoxylomannan (GXM), has been implicated in the progression and severity of cryptococcal infection in the CNS. We determined that GXM increases the fungal burden in the lungs of mice and enhances fungal migration to the brain. Interaction of GXM with the blood-brain barrier, which is a protective structure that regulates movement of particles into the CNS, demonstrated that GXM can disrupt the integrity of this barrier, compromising the delicate balances of fluids, immune cells, and other factors vital to the maintenance of the CNS. The findings of this study reveal the substantial role of GXM in establishing C. neoformans infection in the brain and necessitate future studies to further understand these interactions.

microbiology↗

bioRxiv2022

A meta-analysis of genome-wide association studies identifies new genetic loci associated with all-cause and vascular dementia

Dementia is multifactorial with Alzheimer (AD) and vascular (VaD) pathologies making the largest contributions. Genome-wide association studies (GWAS) have identified over 70 genetic risk loci for AD but the genomic determinants of other dementias, including VaD remain understudied. We hypothesize that common forms of dementia will share genetic risk factors and conducted the largest GWAS to date of "all-cause dementia" (ACD) and examined the genetic overlap with VaD. Our dataset includes 809,299 individuals from European, African, Asian, and Hispanic ancestries with 46,902 and 8,702 cases of ACD and VaD, respectively. We replicated known AD loci at genome-wide significance for both ACD and VaD and conducted bioinformatic analyses to prioritize genes that are likely functionally relevant, and shared with closely related traits and risk factors. For ACD, novel loci identified were associated with energy transport (SEMA4D), neuronal excitability (ANO3), amyloid deposition in the brain (RBFOX1), and MRI markers of small vessel disease (HBEGF). Novel VaD loci were associated with hypertension, diabetes, and neuron maintenance (SPRY2, FOXA2, AJAP1, and PSMA3). Our study identified genetic risks underlying all-cause dementia, demonstrating overlap with neurodegenerative processes, vascular risk factors (Type-II diabetes, blood pressure, lipid) and cerebral small vessel disease. These novel insights could lead to new prevention and treatment strategies for all dementias.

genetics↗

bioRxiv2022

Novel triplex nucleic acid lateral flow immuno-assay (NALFIA) for rapid detection of Nipah virus, Middle East respiratory syndrome coronavirus and Reston ebolavirus

We report the development of the first triplex Nucleic Acid Lateral Flow Assay (NALFIA) for detection of genomes of Nipah virus (NiV), Middle East respiratory syndrome coronavirus (MERS-CoV) and Reston ebolavirus (REBOV), intended for screening of bats as well as other hosts and reservoirs of these three viruses. Our triplex NALFIA is a two-step assay format wherein the target nucleic acid in sample is first amplified using tagged primers, and the tagged ds DNA amplicons are captured by immobilized antibodies on NALFIA device resulting to signal development from binding of streptavidin-colloidal gold conjugate to biotin tag on the captured amplicons. Triplex amplification of N gene of NiV, UpE gene of MERS-CoV, and Vp40 gene of REBOV was optimized using primers adapted from validated real-time RT-PCR assays of previous studies and the compatible combinations of hapten-labels and antibodies for triplex NALFIA device were identified. Digoxigenin, rhodamine red and alexa fluor 488 were identified as suitable 5 labels on forward primers. The lowest copy number detected by the triplex NALFIA with 2 l of triplex RT-PCR product were up to 8.21e4 for NiV N target, 7.09e1 for MERS-CoV UpE target, and 1.83e4 for REBOV Vp40 target. Using simulated samples and Taqman real-time RT-PCR as standard, the sensitivity and positive predictive values were found to be 100% for MERS-CoV UpE and REBOV Vp40 targets and 91% for NiV N target while the specificity and negative predictive values were 100% for MERS-CoV UpE targets and REBOV Vp40, and 93.3% for NiV N target.

molecular biology↗

bioRxiv2022

Identification of prognostic biomarkers for suppressing tumorigenesis and metastasis of Hepatocellular carcinoma through transcriptome analysis

Cancer is one of the deadliest diseases developed through tumorigenesis and could be fatal if reached at metastatic phase. The novality of present investigation is to explore the prognostic biomarkers in hepatocellular carcinoma (HCC) that could develop glioblastoma multiforme (GBM) due to metastasis. The analysis was done using RNA-Seq datasets for both HCC (PRJNA494560 and PRJNA347513) and GBM (PRJNA494560 and PRJNA414787) from Gene Expression Omnibus (GEO). This study identified 13 hub genes found to be overexpressed in both GBM and HCC. Promoter methylation study showed these genes to be hypomethylated. Validation through genetic alteration and missense mutations resulted in chromosomal instability leading to improper chromosome segregation causing aneuploidy. A 13-gene predictive model was obtained and validated using KM plot. These hub genes could be prognostic biomarkers and potential therapeutic targets, inhibition of which could suppress tumorigenesis and metastasis.

bioinformatics↗