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Mishra, S. K.

Publications and source records attributed to Mishra, S. K..

At least 19 recordsLinked to original sources

Protein disorder controls allostery in DNA

Metabolism, gene expression, and signaling all require the adaptation of protein activity to the mixture of reactants and products in a cell. This trait to adapt, called allostery, is hardwired in the structure of proteins. Binding a ligand at one location in a protein can change distant locations, thus tuning protein activity. How allostery works has been subject of intense research since its discovery sixty years ago. The challenge is to understand the order of events that follow ligand-binding in the three-dimensional architecture of proteins. Here we simplify this task by studying allostery in DNA, a nearly one-dimensional system. DNA can transmit allosteric signals over many nanometers to generate cooperativity in the binding of transcription factors, an archetype of the long-range action of allostery. We found that binding of the transcription factor ComK amplifies intrinsic microsecond structural fluctuations in DNA many nanometers distant from the binding site. Yet, it is not protein binding per se, but the intrinsically disordered region (IDR) of the protein that amplifies these fluctuations. IDR removal does not only rigidify DNA, but it also abolishes allostery. The result is a structurally distorted protein-DNA complex that lost its function. These findings have important implications for our understanding of transcription activation and suggest a new functional role for IDRs in transcription factors.

biophysics↗

CD4+ T cells promote fibrosis during metabolic dysfunction-associated steatohepatitis

Unresolved inflammation and fibrosis are the two key features of metabolic dysfunction-associated steatohepatitis (MASH), a progressive form of steatotic liver disease that can evolve into cirrhosis and liver cancer. Although innate immunity has been well studied in MASH, the role of CD4 T cells remains underexplored despite their potential to coordinate immune responses by providing help to other immune cells, promoting inflammation, or regulating immune activity through effector and regulatory subsets. To better understand the role of CD4+ T cells in the pathogenesis of MASH, we comprehensively characterized hepatic CD4+ T cells in murine and human MASH at a single-cell protein, transcriptional, and functional level. Mass cytometry and CITE-sequencing revealed a marked shift in intrahepatic CD4 T-cell composition in MASH, with enrichment of Th1, regulatory, and cytotoxic CD4 T cells. Similar phenotypic changes were mirrored in the peripheral blood and validated in human MASH samples. Functional assays demonstrated increased production of IFN{gamma} and TNF by hepatic CD4 T cells, highlighting their proinflammatory effector activity. Transcriptomic profiling identified Tnfrsf4 (OX40) upregulation in hepatic CD4 T cells during MASH. Therapeutic blockade of the OX40L-OX40 axis reversed hepatic fibrosis and improved histologic disease scores in mice with established MASH, and also decreased inflammatory markers in a human ex vivo liver model. Together, these studies provide a proteogenomic single-cell atlas for hepatic CD4 T cells and uncover a CD4 T cell-dependent immunopathogenic circuit as a promising immunotherapeutic target to alleviate MASH and liver fibrosis.

immunology↗

Competing 5' splice sites of SRC1/HEH1 are marked for alternative splicing by U5 and U6 snRNAs during the B to Bact transition of the spliceosome

Alternative splicing of precursor-messenger RNA (pre-mRNA) bearing introns with competing and overlapping 5 splice sites (5SS) produces more mRNAs. We investigated the mechanism of this form of alternative splicing by monitoring RNA and protein products of the yeast SRC1/HEH1 gene. Its alternative splicing requires a sixteen-nucleotide pre-mRNA segment spanning the two 5SS. The nucleotides are decoded by U5 and U6 small nuclear RNAs (snRNA), further supported by proteins of the spliceosomal B and Bact complexes. Specific variants of the pre-mRNA segment with recalibrated binding to U5 and U6 snRNAs allowed alternative splicing independently of the trans-acting proteins. Conversely, snRNA variants specified for modified 5SS selection suppressed the splicing defects in the B complex mutants. Further data suggest that alternative splicing from HEH1-type introns containing overlapping and competing 5SS relies on pre-mRNA-snRNA interactions during B to Bact transitions of the spliceosome, supported by proteins that stabilize low-fidelity, high-efficiency conformations.

molecular biology↗

Selective targeting of Plasmodium falciparum hexose transporter by phytochemical Ginsenoside Rg1 disrupts glucose metabolism and block development of parasite

The emergence of resistance to first-line antimalarial therapies highlights the critical need for next-generation drugs that target distinct molecular pathways and employ novel mechanisms of action. Notably, the intra-erythrocytic parasite development is highly dependent on a sustained glucose supply as their fundamental energy source. Therefore, exploiting a "selective starvation" strategy, by targeting the parasites reliance on glucose metabolism, particularly through the Plasmodium falciparum hexose transporter (PfHT1), which is critical for parasite survival can serve as a promising therapeutic approach to combat multidrug-resistant Plasmodium parasites. Through molecular docking and structure-based drug design approach, we identified a natural compound, Ginsenoside Rg1 (G-Rg1) from drug bank database library, as a potential PfHT1 inhibitor. The PfHT1 specificity of G-Rg1 was validated using yeast complementation model. Subsequently, to investigate the role of PfHT1 in drug resistant Pf parasites we investigated the stage-specific expression of PfHT1 in both artemisinin (ART)-sensitive and resistant Pf parasites and reported its elevated expression in resistant parasites, predicting its role in their survival. Notably, in vitro growth inhibition studies demonstrated that G-Rg1 effectively suppressed the growth of both ART-sensitive and resistant Pf parasites. Additionally, G-Rg1 potentiated the efficacy of dihydroartemisinin in combination and ring survival assays, indicating its potential to circumvent resistance mechanisms. G-Rg1 administration alone and in combination with ART, in P. berghei ANKA-infected mice reduced parasite multiplication and increased mean survival time. Our findings support G-Rg1 as a promising candidate for drug development against malaria, highlighting the potential of targeting PfHT1 to combat drug-resistant malaria.

pharmacology and toxicology↗

Metabolic Reprogramming of Pathogenic CD4+ T Helper Cells Attenuates Inflammatory Bowel Disease Pathogenesis

BACKGROUND & AIMSCD4+ T helper 1 (Th1) cells are involved in human inflammatory bowel disease (IBD) pathogenesis; however, mechanisms governing the persistent inflammatory function of these cells are unclear, leading us to examine how metabolism governs Th1 cell-induced IBD. METHODSTh1 cells supplemented with methyl pyruvate (MePyr) were analyzed to define how enforced mitochondrial pyruvate metabolism and subsequent glycogen synthase kinase 3{beta} (GSK3{beta}) deactivation reprogram cellular state. Re-analysis of the inflamed ileal single-cell RNA sequencing dataset from Crohns disease patients was performed to assess non-Treg CD4+ T cell metabolic gene signature. We assessed the capacity of a repurposed GSK3{beta} inhibitor to restrain pathogenic CD4+ T cell-driven murine colitis. RESULTSEffector Th1 cells exhibit a distinct metabolic program exemplified by glucose-driven glycolysis but low mitochondrial respiration. MePyr deactivates GSK3{beta}, glycolysis, and histone H3 acetylation on cytokine promoter region, resulting in reduced interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor- (TNF-) expression in Th1 cells with concomitant gain of regulatory T cell-like program. GSK3{beta} inhibition with LY2090314 mirrored the anti-inflammatory effect of MePyr in a manner reversible by acetate supplementation, implying that GSK3{beta} potentially sustains glycolysis-derived acetyl-coenzyme A needed for histone acetylation and Th1 cell inflammatory response. Interleukin-21 exacerbates Th1 cell inflammatory response by maintaining a GSK3{beta}-driven glycolytic program. The Th1 cell metabolic gene signature downregulated by MePyr or GSK3{beta} inhibition in vitro is enriched in refractory Crohns disease patients. GSK3{beta} inhibition with LY2090314 retrains T cell-induced colitis in mice. CONCLUSIONSMePyr impairs GSK3{beta}-mediated glycolysis and Th1 cell immune response. GSK3{beta} inhibition may mitigate Th1 cell-induced human IBD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/649047v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@5b4acdorg.highwire.dtl.DTLVardef@18c03fforg.highwire.dtl.DTLVardef@15a75cforg.highwire.dtl.DTLVardef@1eca84d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

1,2,4-Triazole-based first-in-class non-nucleoside inhibitors of bacterial enzyme MraY

MraY, a bacterial enzyme crucial for the synthesis of peptidoglycans, represents a promising yet underexplored target for the development of effective antibacterial agents. Nature has provided several classes of nucleoside inhibitors of MraY and scientists have modified these structures further to obtain natural product-like inhibitors of MraY. The natural products and their synthetic analogs suffer from non-optimal in vivo efficacy, and the synthetic complexity of the structures renders the synthesis and structure-activity relationship (SAR) studies of these molecules particularly challenging. In this study, we present our findings on the discovery of first-in-class 1,2,4-triazole-based MraY inhibitors that are not nucleoside-derived. A series of 1,2,4-triazole analogous were identified by a structure-activity-relationship (SAR) study using a structure-based drug design strategy. Compound 1, with an IC50 of 171 {micro}M against MraY from Staphylococcus aureus (MraYSA), was optimized to compound 12a, exhibiting an IC50 of 25 {micro}M. Molecular docking studies against MraYSA provided insights into these compounds binding interactions and activity. Furthermore, screening against the ESKAPE bacterial panel was also conducted, through which we discovered compounds demonstrating broad-spectrum antibacterial activity against E. faecium, methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococci (VRE) strains and Mycobacterium tuberculosis. The novel, first-in-class non-nucleoside inhibitors of MraY highlighted in this work provide a strong proof-of-concept of how to leverage structural information of the protein to develop future antibacterial agents targeting MraY. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/635793v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@14b1f84org.highwire.dtl.DTLVardef@1024f45org.highwire.dtl.DTLVardef@a246d0org.highwire.dtl.DTLVardef@1d70e52_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Association of oropharyngeal cancer recurrence with tumor-intrinsic and immune-mediated sequelae of reduced genomic instability

BackgroundLimited understanding of the biology predisposing certain human papillomavirus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) to relapse impedes therapeutic personalization. We aimed to identify molecular traits that distinguish recurrence-prone tumors. Methods50 HPV+ OPSCCs that later recurred (cases) and 50 non-recurrent controls matched for stage, therapy, and smoking history were RNA-sequenced. Groups were compared by gene set enrichment analysis, and select differences were validated by immunohistochemistry. Features discriminating groups were scored in each tumor using gene set variation analysis, and scores were evaluated for recurrence prediction ability. ResultsCases downregulated pathways linked to anti-tumor immunity (FDR-adjusted p<.05) and contained fewer tumor-infiltrating lymphocytes (p<.001), including cytotoxic T-cells (p=.005). Cases also upregulated pathways related to cell division and other aspects of tumor progression. Upregulated and downregulated pathways were respectively used to define a tumor progression score (TPS) and immune suppression score (ISS) for each tumor. Correlation between TPS and ISS (r=.603, p<.001) was potentially explained by observed upregulation of DNA repair pathways in cases, which might enhance their progression directly and by limiting cytosolic DNA-induced inflammation. Accordingly, cases contained fewer double-strand breaks based on staining for phospho-RPA32 (p=.006) and {gamma}-H2AX (p=.005) and downregulated pro-inflammatory components of the cytoplasmic DNA sensing pathway. A combined score derived from TPS and ISS optimized recurrence prediction and stratified survival in a manner generalizable to three external cohorts. ConclusionsWe provide novel evidence that limiting genomic instability makes tumor-intrinsic and immune-mediated contributions to HPV+ OPSCC recurrence risk, opening opportunities to detect and target this treatment-resistant biology.

cancer biology↗

Analysis of the progression of cervical cancer in Guatemala- from pre-malignancy to invasive disease

To better understand cervical cancer progression, we analyzed RNA from 262 biopsies from women referred for colposcopy We determined HPV type and analyzed the expression of 51 genes. HPV31 was significantly more prevalent in precancer than stage 1 cancer and invasive cancer (p < 0.0001) and HPV16 increased in invasive disease (p < 0.0001). CCNE1, MELTF, and ULBP2 were significantly increased in HPV16-positive compared to HPV31 precancers while NECTIN2 and HLA-E expression decreased. Markers of the innate immune system, DNA repair genes, and cell cycle genes are significantly increased during cancer progression (p = 0.0001). In contrast, the TP53 and RB1 tumor suppressor gene expression is significantly decreased in cancer cells. TheT cell markers CD28 and FLT3LG expression decreased in cancer while FOXP3, IDO1, and ULBP2 expression increased. There is a significantly higher survival rate in individuals with increased expression of CD28 (p = 0.0005), FOXP3 (p = 0.0002), IDO1 (p = 0.038), FLT3LG (p = 0.026), APOBEC3B (p = 0.0011), and RUNX3 (p = 0.019), and a significantly lower survival rate in individuals with increased expression of ULBP2 (p = 0.035). These results will help us understand the molecular factors influencing the progression of cervical precancer to cancer.

cancer biology↗

Overexpression of barley heat stress transcription factor HvHsfA6a provide thermotolerance by thermopriming

Adverse impacts of climate change, including high temperature on cereal crop production, have been evidenced globally. In plants, heat shock factors (HSFs) are crucial components of heat stress associated rescue mechanisms and are also required for normal biological processes. Here, we functionally characterized a highly heat stress responsive HvHSFA6a in barley by developing constitutively overexpressing transgenic lines. These transgenic lines showed heat tolerant phenotype via improved photosynthesis, antioxidants and upregulation of HSPs and metabolites involved in stress amelioration and keeping thermomemory as compared to wild type plants. Global transcriptomics and ChIP sequencing revealed that HvHSFA6a orchestrates the expression of several genes through direct binding with other HSFs containing consensus HSE in their promoter regions. A GC-MS based metabolomics analysis also revealed the alterations in key metabolic processes such as carbohydrate metabolism, citric acid cycle, amino acids and secondary metabolism. Higher accumulation of key metabolites such as sucrose, galactinol, shikimate and ascorbate has been observed under both control and heat stress in transgenic lines as compared to wild type plants. Taken together, the results suggest that overexpression of HvHsfA6a prime the plants for heat stress conditions by alteration in gene expression and metabolic status. HighlightPriming is a mechanism by which plants respond to various abiotic and biotic stresses. Through multi omics approach we found that barley HsfA6a provide thermotolernce in transgenic plants through priming effect on transcriptome and metabolome.

molecular biology↗

Interleukin-21 Drives a Hypermetabolic State and CD4+ T Cell-associated Pathogenicity in Chronic Intestinal Inflammation

BACKGROUND & AIMSIncapacitated regulatory T cells (Tregs) contribute to immune-mediated diseases. Inflammatory Tregs are evident during human inflammatory bowel disease (IBD); however, mechanisms driving the development of these cells and their function are not well understood. Therefore, we investigated the role of cellular metabolism in Tregs relevant to gut homeostasis. METHODSUsing human Tregs, we performed mitochondrial ultrastructural studies via electron microscopy and confocal imaging, biochemical and protein analyses using proximity ligation assay, immunoblotting, mass cytometry and fluorescence-activated cell sorting, metabolomics, gene expression analysis, and real-time metabolic profiling utilizing Seahorse XF analyzer. We utilized Crohns disease single-cell RNA sequencing dataset to infer therapeutic relevance of targeting metabolic pathways in inflammatory Tregs. We examined the superior functionality of genetically-modified Tregs in CD4+ T cell-induced murine colitis models. RESULTSMitochondria-endoplasmic reticulum (ER) appositions, known to mediate pyruvate entry into mitochondria via VDAC1, are abundant in Tregs. VDAC1 inhibition perturbed pyruvate metabolism, eliciting sensitization to other inflammatory signals reversible by membrane-permeable methyl pyruvate (MePyr) supplementation. Notably, IL-21 diminished mitochondria-ER appositions, resulting in enhanced enzymatic function of glycogen synthase kinase 3 {beta} (GSK3{beta}), a putative negative regulator of VDAC1, and a hypermetabolic state that amplified Treg inflammatory response. MePyr and GSK3{beta} pharmacologic inhibitor (LY2090314) reversed IL-21-induced metabolic rewiring and inflammatory state. Moreover, IL-21-induced metabolic genes in Tregs in vitro were enriched in human Crohns disease intestinal Tregs. Adoptively transferred Il21r-/- Tregs efficiently rescued murine colitis in contrast to wild-type Tregs. CONCLUSIONSIL-21 triggers metabolic dysfunction associated with Treg inflammatory response. Inhibiting IL-21-induced metabolism in Tregs may mitigate CD4+ T cell-driven chronic intestinal inflammation.

immunology↗

Quercetin selectively reduces expanded repeat RNA levels in models of myotonic dystrophy

Myotonic dystrophy is a multisystemic neuromuscular disease caused by either a CTG repeat expansion in DMPK (DM1) or a CCTG repeat expansion in CNBP (DM2). Transcription of the expanded alleles produces toxic gain-of-function RNA that sequester the MBNL family of alternative splicing regulators into ribonuclear foci, leading to pathogenic mis-splicing. There are currently no approved treatments that target the root cause of disease which is the production of the toxic expansion RNA molecules. In this study, using our previously established HeLa DM1 repeat selective screening platform, we identified the natural product quercetin as a selective modulator of toxic RNA levels. Quercetin treatment selectively reduced toxic RNA levels and rescued MBNL dependent mis-splicing in DM1 and DM2 patient derived cell lines and in the HSALR transgenic DM1 mouse model where rescue of myotonia was also observed. Based on our data and its safety profile for use in humans, we have identified quercetin as a priority disease-targeting therapeutic lead for clinical evaluation for the treatment of DM1 and DM2. One Sentence SummaryThe natural product quercetin reduces toxic RNA in myotonic dystrophy.

molecular biology↗

Human Dectin-1 is O-glycosylated and serves as a ligand for C-type lectin receptor CLEC-2

C-type lectin receptors (CLRs) elicit immune responses upon recognition of glycoconjugates present on pathogens and self-components. While Dectin-1 is the best-characterized CLR recognizing {beta}-glucan on pathogens, the endogenous targets of Dectin-1 are not fully understood. Herein, we report that human Dectin-1 is a ligand for CLEC-2, another CLR expressed on platelets. Biochemical analyses revealed that Dectin-1 is a mucin-like protein as its stalk region is highly O-glycosylated. A sialylated core 1 glycan attached to the EDxxT motif of human Dectin-1, which is absent in mouse Dectin-1, provides a ligand moiety for CLEC-2. Strikingly, the expression of human Dectin-1 in mice rescued the lethality and lymphatic defect resulting from a deficiency of Podoplanin, a known CLEC-2 ligand. This finding is the first example of an innate immune receptor also functioning as a physiological ligand to regulate ontogeny upon glycosylation.

immunology↗

Chloroplast activity provides in vitro regeneration capability in contrasting cultivars

Existence of potent in vitro regeneration system is a prerequisite for efficient genetic transformation and functional genomics of crop plants. We know little about why only some cultivars in crop plants are tissue culture friendly. In this study, tissue culture friendly cultivar Golden Promise (GP) and tissue culture resistant DWRB91(D91) were selected as contrasting cultivars to investigate the molecular basis of regeneration efficiency. Multiomics studies involving transcriptomics, proteomics, metabolomics, and biochemical analysis were performed using GP and D91 callus to unravel the regulatory mechanisms. Transcriptomics analysis revealed 1487 differentially expressed genes (DEGs), in which 795 DEGs were upregulated and 692 DEGs were downregulated in the GP-D91 transcriptome. Genes encoding proteins localized in chloroplast and involved in ROS generation were upregulated in the embryogenic calli of GP. Moreover, proteome analysis by LC-MSMS revealed 3062 protein groups and 16989 peptide groups, out of these 1586 protein groups were differentially expressed proteins (DEPs). Eventually, GC-MS based metabolomics analysis also revealed the higher activity of plastids and alterations in key metabolic processes such as sugar metabolism, fatty acid biosynthesis, and secondary metabolism. Higher accumulation of sugars, amino acids and metabolites corresponding to lignin biosynthesis were observed in GP as compared to D91. HighlightsMulti omics analysis revealed chloroplast play crucial role in providing in vitro regeneration capability in contrasting genotypes

plant biology↗

Protein Stability Changes upon Point Mutations Identified with a Gaussian Network Model Simulating Protein Unfolding Behavior

Understanding the effects of missense mutations on protein stability is a widely acknowledged significant biological problem. Genomic missense mutations may alter one or more amino acids leading to increased or decreased stability of the encoded proteins. In this study, we propose a novel approach - Protein Stability Prediction with a Gaussian Network Model (PSP-GNM) to study the effect of single amino acid substitutions on protein stability. Specifically, PSP-GNM employs a coarse-grained Gaussian Network Model (GNM) that has interactions between amino acids weighted by the Miyazawa-Jernigan (MJ) statistical potential. We use PSP-GNM to simulate partial unfolding of the wildtype and mutant structures and then, use the difference in energies of the unfolded wildtype and mutant protein structures to estimate the experimentally obtained unfolding free energy change ({Delta}{Delta}G). We verify the extent of correspondence between the {Delta}{Delta}G calculated by PSP-GNM and the {Delta}{Delta}G obtained experimentally using three datasets: 350 forward mutations from 66 proteins, 2298 forward mutations from 126 proteins and 611 forward and reverse mutations from 66 proteins and observe Pearson correlation coefficient (PCC) as high as 0.58 and root mean-squared error (RMSE) as low as 1.24 kcal/mol. The performance is comparable to the existing state of the art methods. Importantly, we do observe an increase in the correlation to 0.73 and decrease in RMSE to 1.07 when considering only those measurements made close to 25{degrees}C and neutral pH, suggesting a strong dependence on temperature and pH. PSP-GNM is written in Python and is available as a free downloadable package at https://github.com/sambitmishra0628/PSP-GNM. Author SummaryUnderstanding how genomic missense mutations impact the thermodynamic stability of encoded proteins is important to understand disease etiology. Specifically, mutant proteins are often functionally inactive and underlie numerous genetic and neurodegenerative diseases. A classic example is sickle cell anemia - a single amino acid change significantly affects hemoglobins binding affinity for oxygen. To be able to identify mutations that would likely impact the protein function and stability is therefore essential and is the focus of our study. We present an approach that relies on utilizing the intrinsic dynamics of protein structures to predict the effect of single amino acid mutations (point mutations) on protein stability. In our approach, we model proteins as coarse-grained beads (amino acids) and springs (interactions), simulate protein unfolding and identify putative residue-residue contacts that are broken during the unfolding process. We demonstrate that the knowledge of broken contacts and their order is essential in describing the thermodynamic differences between wildtype and mutant proteins. We also highlight the importance of residue-residue interactions at the mutation site in the context of protein stability prediction. Our findings present novel avenues to interpret how genomic mutations may manifest in the encoded proteins.

bioinformatics↗

Validated Determination of NRG1 Ig-like Domain Structure by Mass Spectrometry Coupled with Computational Modeling

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally-informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure leaving questions of unintentional bias and applicability to unknown structures unresolved. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 [A]. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

biophysics↗

Artemin and its cognate receptor, GFRα3, play a function role in osteoarthritis pain

Osteoarthritis associated pain (OA-pain) is a significant global problem. OA-pain limits limb use and mobility, and is associated with widespread sensitivity. Therapeutic options are limited, and the ones that are available are often associated with side or adverse effects. The lack of therapeutic options is partly due to a lack of understanding of clinically relevant underlying neural mechanisms of OA-pain. In previous work in naturally occurring OA-pain in dogs, we identified potential signaling molecules (artemin/GFR3) that were upregulated. Here, we use multiple approaches including knockout mice, immunological suppression in a mouse model of OA, and clinically relevant measures of sensitivity and limb use to explore the functional role of artemin/GFR3 signaling in OA-pain. We found the monoiodoacetate (MIA)-induced OA model in mice is associated with decreased limb use and hypersensitivity. GFR3 expression is increased in sensory neurons. Exogenous artemin induces heat, cold and mechanical hypersensitivity, and anti-artemin monoclonal antibody administration reverses this hypersensitivity and restores limb use in mice with MIA-induced OA pain. Our results provide a molecular basis of arthritis pain linked with artemin/GFR3 signaling and indicate that further work is warranted to investigate the neuronal plasticity and the pathways that drive pain in OA.

neuroscience↗

Imaging the Transmembrane and Transendothelial Sodium Gradients in Gliomas

High sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane ({Delta}Na+mem) and weak transendothelial ({Delta}Na+end) gradients respectively, which reflect cell membrane potential (Vm) and blood-brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic contrast agent to measure {Delta}Na+mem and {Delta}Na+end. In vitro 23Na-MRSI established that the 23Na signal is strongly shifted by the agent compared to biological factors. In vivo 23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together created weakened {Delta}Na+mem and enhanced {Delta}Na+end in rat gliomas. Specifically, RG2 and U87 tumors maintained weakened {Delta}Na+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, and RG2 tumors displayed elevated {Delta}Na+end suggesting altered BBB integrity. 23Na-MRSI will allow explorations of perturbed Na+ homeostasis in vivo for the tumor neurovascular unit.

cancer biology↗

Characterization of Bacterial Isolates in Vermicompost Produced from a Mixture of Cow dung, Straw, Neem leaf and Vegetable Wastes

Vermicomposting is a non-thermophilic biological oxidation process of composting where certain species of earthworm are used to enhance the process of conversion of organic waste to compost. Earthworm helps in influencing the growth of certain microbial species, and also improves the physical and chemical properties of the soil. The microbial population present in vermicompost play an important role in increasing the productivity of crop as well as maintain the structural stability of the soil. Different types of bacteria found in vermicompost and is depends on the environmental condition and the raw materials used in vermicomposting. Owing to this, a study was carried out to identify the bacteria exist in vermicompost produced from cow dungs, straw, neem leafs and vegetable wastes. The phenotypic studies such as colony morphology, microscopic studies, and biochemical characterization have identified eight bacterial species namely Actinomyces israelli, Azotobacter, Micrococus luteus, Bacillus cereus, Bacillus subtillis, Pseudomonas aeruginosa, Enterobacter in the vermicompost. All these bacteria were present in the gut of Eesenia Foetida and found beneficial for the soil and crop plants.

microbiology↗