Biology subjects

Mohanty, S.

Publications and source records attributed to Mohanty, S..

51 records · Page 3Linked to original sources

bioRxiv2021

Reversible brain edema in experimental cerebral malaria is associated with transcellular blood-brain barrier disruption and delayed microhemorrhages

Brain swelling occurs in cerebral malaria (CM) and may either reverse or result in fatal outcome. It is currently unknown how brain swelling in CM reverses, as investigations have been hampered by inadequate animal models. In this study, we show that reversible brain swelling in experimental murine cerebral malaria (ECM) can be induced reliably after single vaccination with radiation-attenuated sporozoites as revealed by in vivo high-field (9.4T) magnetic resonance imaging. Our results provide evidence that parenchymal fluid increase and consecutive brain swelling results from transcellular blood-brain barrier disruption (BBBD), as revealed by electron microscopy. This mechanism enables reversal of brain swelling but does not prevent persistent focal brain damage, evidenced by microhemorrhages, in areas of most severe BBBD. In a cohort of 27 pediatric and adult CM patients (n=4 fatal, n=23 non-fatal) two out of four fatal CM patients (50%) and 8 out of 23 non-fatal CM patients (35%) showed microhemorrhages on MRI at clinical field strength of 1.5T, emphasizing the translational potential of the experimental model. Our data suggest that targeting transcellular BBBD may represent a promising adjunct therapeutic approach in cerebral brain swelling to reduce edema and may ultimately lead to a reduced permanent brain damage and a better longtime neurological outcome. Author summaryBrain swelling, which occurs in diseases such as cerebral malaria, is not necessarily fatal, and may reverse. Even upon reversal of brain swelling, neurological sequelae can still occur. The factors contributing to the reversibility of brain edema are not known, and treatment options remain therefore limited. Identifying the mechanisms leading to such reversibility could inform clinical management aimed at decreasing brain swelling and consecutive brain injury. Here we introduce a reproducible and simple animal model that allows comprehensive in vivo studies of reversible brain swelling in cerebral malaria at the peak of disease and upon recovery. We identify a specific type of blood-brain barrier disruption (BBBD) as a mechanism that occurs in brain swelling. We show that BBBD can reverse, but also highlight remaining brain damage in areas of most severe BBBD. As the ECM model introduced here bares crucial similarities to the CM in humans, our findings open strategies to study new therapeutic avenues and point to compounds that specifically target transcellular BBBD to reduce brain edema, and increase survival rates.

neuroscience↗

bioRxiv2021

MINK1 modulates 5FU resistance in OSCC through AKT/MDM2 mediated regulation of p53

Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responder as compared to chemotherapy responders. The nude mice and zebrafish xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. The patient derived TPF resistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC.

cancer biology↗

bioRxiv2020

Genoprotective effects of Chaga mushroom (Inonotus obliquus) polysaccharides in UVB-exposed embryonic zebrafish (Danio rerio) through coordinated expression of DNA repair genes

BackgroundChaga mushroom (Inonotus obliquus) is one of the most promising antioxidants with incredible health-promoting effects. Chaga polysaccharides (IOP) have been reported to enhance immune response and alleviate oxidative stress during development. However, the effects of IOP on the genotoxicity in model organisms are yet to be clarified. MethodsZebrafish embryos (12 hours post fertilization, hpf) were exposed to transient UVB (12 J/m2/s, 310 nm) for 10 secs using a UV hybridisation chamber, followed by IOP treatment (2.5 mg/mL) at 24 hpf for up to 7 days post fertilization (dpf). The genotoxic effects were assessed using acridine orange staining, alkaline comet assay, and qRT-PCR for screening DNA repair genes. ResultsWe found significant reduction in DNA damage and amelioration of the deformed structures in the IOP-treated zebrafish exposed to UVB (p < 0.05) at 5 dpf and thereafter. In addition, the relative mRNA expressions of XRCC-5, XRCC-6, RAD51, P53, and GADD45 were significantly upregulated in the IOP-treated UVB-exposed zebrafish. Pathway analysis demonstrated coordinated regulation of DNA repair genes, suggesting collective response during UVB exposure. ConclusionsOverall, IOP treatment ameliorated the genotoxic effects in UVB-exposed zebrafish embryos, which eventually assisted in normal development. The study suggested the efficacy of Chaga mushroom polysaccharides in mitigating UV-induced DNA damage.

molecular biology↗

bioRxiv2020

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-kB activation

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-{kappa}B pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-{kappa}B; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify novel Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-{kappa}B activation, whereas knockdown of UBE4B impaired Tax-induced NF-{kappa}B activation and the induction of NF-{kappa}B target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-{kappa}B activation. Author SummaryInfection with the retrovirus HTLV-1 leads to the development of either CD4+CD25+ leukemia/lymphoma (ATLL) or a demyelinating neuroinflammatory disease (HAM/TSP) in a subset of infected individuals. The HTLV-1 Tax protein is a regulatory protein which regulates viral gene expression and persistently activates cellular signaling pathways such as NF-{kappa}B to drive the clonal expansion and longevity of HTLV-1 infected CD4+ T cells. Polyubiquitination of Tax is a key mechanism of NF-{kappa}B activation by assembling and activating I{kappa}B kinase (IKK) signaling complexes; however, the host factors regulating Tax ubiquitination have remained elusive. Here, we have identified the E3/E4 ubiquitin conjugation factor UBE4B as a novel Tax binding protein that promotes both K48- and K63-linked polyubiquitination of Tax. Knockdown or knockout of UBE4B impairs Tax-induced NF-{kappa}B activation and triggers apoptosis of HTLV-1 transformed cells. Therefore, UBE4B is an integral host factor that supports HTLV-1 Tax polyubiquitination, NF-{kappa}B activation and cell survival.

microbiology↗

bioRxiv2020

CMTM6 drives cisplatin resistance in OSCC by regulating AKT mediated Wnt signaling

Chemoresistance is one of the important factors for treatment failure in OSCC, which can culminate in progressive tumor growth and metastatic spread. Rewiring tumor cells to undergo drug-induced apoptosis is a promising way to overcome chemoresistance, which can be achieved by identifying the causative factors for acquired chemoresistance. In this study, to explore the key cisplatin resistance triggering factors, we performed global proteomic profiling of OSCC lines representing with sensitive, early and late cisplatin-resistant patterns. The top ranked up-regulated protein appeared to be CMTM6. We found CMTM6 to be elevated in both early and late cisplatin-resistant cells with respect to the sensitive counterpart. Analyses of OSCC patient samples indicate that CMTM6 expression is upregulated in chemotherapy-non-responder tumors as compared to chemotherapy-naive tumors. Stable knockdown of CMTM6 restores cisplatin-mediated cell death in chemoresistant OSCC lines. Similarly, upon CMTM6 overexpression in CMTM6KD lines, the cisplatin resistant phenotype was efficiently rescued. Mechanistically, it was found that CMTM6 interacts with membrane bound Enolase-1 and stabilized its expression, which in turn activates the AKT-GSK3{beta} mediated Wnt signaling. CMTM6 triggers the translocation of {beta}-catenin into the nucleus, which elevates the Wnt target pro-survival genes like Cyclin D, c-Myc and CD44. Moreover, incubation with lithium chloride, a Wnt signaling activator, efficiently rescued the chemoresistant phenotype in CMTM6KD OSCC lines. In a patient-derived cell xenograft model of chemoresistant OSCC, knock-down of CMTM6 restores cisplatin induced cell death and results in significant reduction of tumor burden. CMTM6 has recently been identified as a stabilizer of PD-L1 and henceforth it facilitates immune evasion by tumor cells. Herewith for the first time, we uncovered another novel role of CMTM6 as one of the major driver of cisplatin resistance.

cancer biology↗

bioRxiv2020

RRBP1 rewires cisplatin resistance in Oral Squamous Cell Carcinoma by regulating YAP-1

Cisplatin-based chemotherapy still remains as one of the primary treatment modalities for OSCC. Several OSCC patients experience relapse owing to development of chemoresistance. To identify key resistance triggering molecules, we performed global proteomic profiling of human OSCC lines presenting with sensitive, early and late cisplatin resistance patterns. From the proteomic profiling study, human RRBP1 was identified to be upregulated in both early and late cisplatin-resistant cells with respect to the sensitive counterpart. Analysis of OSCC patient sample indicates that RRBP1 expression is elevated in chemotherapy-non-responder tumors as compared to chemotherapy-naive tumors. Knocking out RRBP1 resulted in restoring cisplatin mediated cell death in chemoresistant lines and patient derived cells (PDC). Mechanistically, RRBP1 regulates YAP-1 to induce chemoresistance in OSCC. The chemoresistant PDC xenograft data suggests that knock out of RRBP1 induces cisplatin mediated cell death and facilitates a significant reduction of tumor burden. We also found Radezolid, a novel oxazolidinone antibiotic represses the expression of RRBP1 and restores cisplatin-induced cell death in chemoresistant OSCC. This unique combinatorial approach needs further clinical investigation to target advanced OSCC. Here with for the first time, we uncover the novel role of RRBP1 as potential modulator of cisplatin resistance in advanced OSCC.

cancer biology↗

bioRxiv2019

Allosteric feed-forward activation of AP-2 via FCHO- BMP2K axis promotes clathrin-mediated endocytosis

Spatio-temporal regulation of central adaptor complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). We recently discovered that FCHO proteins trigger clathrin-coated pit (CCP) formation by allosterically activating AP-2 on plasma membrane (Umasankar et al., 2014). Here, we demonstrate that this activation promotes AP-2 phosphorylation via recruitment and stabilization of BMP-2 inducible kinase (BMP2K), a bona fide AP-2 kinase leading to CCP maturation. Accordingly, BMP2K mislocalizes and degrades in FCHO knockout/ AP-2 depleted cells. Functional inactivation of kinase impairs AP-2 phosphorylation leading to altered lattice morphology and CME phenotypes reminiscent of CCP maturation defects. Reexpression of FCHO rescues AP-2 phosphorylation defects in FCHO knockout cells implying membrane activation of AP-2 is a prerequisite for kinase function. Furthermore, gain- and loss-of function phenotypes of FCHO and BMP2K are analogous and mirror altered AP-2 functions during zebrafish embryogenesis. Together, our findings reveal an in vivo allosteric feed-forward axis for operation of CME.

cell biology↗

bioRxiv2019

Seasonal Variability and Shared Molecular Signatures of Inactivated Influenza Vaccination in Young and Older Adults

The seasonal influenza vaccine is an important public health tool but is only effective in a subset of individuals. The identification of molecular signatures provides a mechanism to understand the drivers of vaccine-induced immunity. Most previously reported molecular signatures of influenza vaccination were derived from a single age group or season, ignoring the effects of immunosenescence or vaccine composition. Thus, it remains unclear how immune signatures of vaccine response change with age across multiple seasons. Here we profile the transcriptional landscape of young and older adults over five consecutive vaccination seasons to identify shared signatures of vaccine response as well as marked seasonal differences. Along with substantial variability in vaccine-induced signatures across seasons, we uncovered a common transcriptional signature 28 days post-vaccination in both young and older adults. However, gene expression patterns associated with vaccine-induced antibody responses were distinct in young and older adults; for example, increased expression of Killer Cell Lectin Like Receptor B1 (KLRB1; CD161) 28 days post-vaccination positively and negatively predicted vaccine-induced antibody responses in young and older adults, respectively. These findings contribute new insights for developing more effective influenza vaccines, particularly in older adults.

immunology↗

bioRxiv2019

Nano-enhanced optical gene delivery to retinal degenerated mice

The efficient and targeted delivery of genes and other impermeable therapeutic molecules into retinal cells is of immense importance for therapy of various visual disorders. Traditional methods for gene delivery require viral transfection, or chemical methods that suffer from one or many drawbacks such as invasiveness, low efficiency, lack of spatially targeted delivery, and can generally have deleterious effects such as unexpected inflammatory responses and immunological reactions. Here, we introduce a continuous wave near-infrared laser-based Nano-enhanced Optical Delivery (NOD) method for spatially controlled delivery of opsin-encoding genes into retina in-vivo. In this method, the optical field enhancement by gold nanorods is utilized to transiently permeabilize cell membrane enabling delivery of exogenous impermeable molecules to nanorod-binding cells in laser-irradiated regions. The successful delivery and expression of opsin in targeted retina after in-vivo NOD in the mice models of retinal degeneration opens new vista for re-photosensitizing retina with geographic atrophies as in dry age-related macular degeneration (AMD).

bioengineering↗

bioRxiv2018

Bioluminescent Multi-Characteristic Opsin for monitoring visual cortical activity upon optical stimulation

Non-invasive detection of neural activity is important for the diagnosis of neurological diseases, evaluation of therapeutic outcomes, and collection of real-time feedback for stimulation based therapeutic approaches. In the case of vision loss, due to retinal degeneration, optic neuropathy, or enucleation of the eye, there is a need to map changes in visual cortical activity during disease progression and subsequent vision restoration by retinal, optic nerve or cortical stimulation. Existing technologies allow interrogation of neuronal circuits by both read and write activity, albeit with inherent limitations. Here, we present the development of bioluminescent multi-characteristic opsin (bMCO-11), which is comprised of a highly photosensitive ambient light-activatable domain and a Ca2+-sensitive bioluminescence reporter. The high quantum efficiency of bMCO-11 enables light activation and recording of cellular activity upon local as well as wide area optical stimulation. Furthermore, persistent Ca2+ influx was achieved by bioluminescence based cyclic activation of the opsin-domain of bMCO-11 by transporting the ions only into bMCO expressing cells. This allowed us to continuously monitor visual cortical activity in wild type and retinal degenerated mice, without requiring any additional external excitation source.

neuroscience↗

bioRxiv2018

Macrophage migration inhibitory factor (MIF) of Syrian golden hamster (Mesocricetus auratus) has similar structure and function as human MIF and promotes pancreatic tumor growth in vivo

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize structure and function of hamster MIF, and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant hamster MIF (rha-MIF) was cloned, expressed and purified in bacterial expression system. The rha-MIF primary sequence, biochemical properties and crystal structure analysis showed a greater similarity with human MIF. The crystal structure of hamster MIF illustrates that it forms a homotrimer as known in human and mouse. However, hamster MIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that rha-MIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of rha-MIF into HapT1 pancreatic tumor bearing hamsters significantly enhanced the tumor growth and tumor associated angiogenesis. Together, the current study shows a structural and functional similarity between hamster and human MIF. Moreover, it has demonstrated that a high-level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo.

biochemistry↗

bioRxiv2018

Taking a Dive: Experiments in Deep Learning for Automatic Ontology-based Annotation of Scientific Literature

I.Text mining approaches for automated ontology-based curation of biological and biomedical literature have largely focused on syntactic and lexical analysis along with machine learning. Recent advances in deep learning have shown increased accuracy for textual data annotation. However, the application of deep learning for ontology-based curation is a relatively new area and prior work has focused on a limited set of models.\n\nHere, we introduce a new deep learning model/architecture based on combining multiple Gated Recurrent Units (GRU) with a character+word based input. We use data from five ontologies in the CRAFT corpus as a Gold Standard to evaluate our models performance. We also compare our model to seven models from prior work. We use four metrics - Precision, Recall, F1 score, and a semantic similarity metric (Jaccard similarity) to compare our models output to the Gold Standard. Our model resulted in a 84% Precision, 84% Recall, 83% F1, and a 84% Jaccard similarity. Results show that our GRU-based model outperforms prior models across all five ontologies. We also observed that character+word inputs result in a higher performance across models as compared to word only inputs.\n\nThese findings indicate that deep learning algorithms are a promising avenue to be explored for automated ontology-based curation of data. This study also serves as a formal comparison and guideline for building and selecting deep learning models and architectures for ontology-based curation.

bioinformatics↗

bioRxiv2018

An Atlas of Human and Murine Genetic Influences on Osteoporosis

Osteoporosis is a common debilitating chronic disease diagnosed primarily using bone mineral density (BMD). We undertook a comprehensive assessment of human genetic determinants of bone density in 426,824 individuals, identifying a total of 518 genome-wide significant loci, (301 novel), explaining 20% of the total variance in BMD--as estimated by heel quantitative ultrasound (eBMD). Next, meta-analysis identified 13 bone fracture loci in ~1.2M individuals, which were also associated with BMD. We then identified target genes from cell-specific genomic landscape features, including chromatin conformation and accessible chromatin sites, that were strongly enriched for genes known to influence bone density and strength (maximum odds ratio = 58, P = 10-75). We next performed rapid throughput skeletal phenotyping of 126 knockout mice lacking eBMD Target Genes and showed that these mice had an increased frequency of abnormal skeletal phenotypes compared to 526 unselected lines (P < 0.0001). In-depth analysis of one such Target Gene, DAAM2, showed a disproportionate decrease in bone strength relative to mineralization. This comprehensive human and murine genetic atlas provides empirical evidence testing how to link associated SNPs to causal genes, offers new insights into osteoporosis pathophysiology and highlights opportunities for drug development.

genomics↗

bioRxiv2018

22-hydroxycholesterol leads to efficient dopaminergic specification of human Mesenchymal Stem Cells

In the current study, we aim to investigate the neurogenic effect of 22 (R)- hydroxycholesterol (22-HC), on hMSCs obtained from bone marrow, adipose tissue and dental pulp. The effect was evaluated on the basis of detailed morphological and morphometric analysis, expression of genes and proteins associated with maturation of neurons (NF, MAP2, TUJ1, TH), channel ion proteins (Kv4.2 & SCN5A), chemical functionality (DAT), synapse forming tendency of neurons (Synaptophysin) and transcription factors (ngn2 & Pitx3), efficiency of generation of dopaminergic neurons and functional assessment. The percentage of non- DA cells generation (Ach, TPH2, S100 and GFAP positive cells) was also evaluated to confirm selective neurogenic potential of 22-HC. Post analysis, it was observed that 22-HC yields higher percentage of functional DA neurons and has differential effect on various tissue- specific primary human MSCs. This study, which is one of its kinds, may help in improvising the approach of cell based treatment regimes and drug testing in pharmaceutical industry for Parkinsons disease.

neuroscience↗

bioRxiv2017

A 23bp Indel Polymorphism in TLR2 Gene Enhances Inflammation and Disease Severity in Dengue

BackgroundDengue is the most rapidly spreading viral disease transmitted by the bite of infected Aedes mosquitos. Pathogenesis of dengue is still unclear; although host genetic factors, immune responses and virus serotypes have been proposed to contribute to disease severity. The development of high-throughput methods have allowed to scale up capabilities of identifying the key markers of inflammation. Since NS1 protein of dengue virus has been reported to activate immune cells towards enhanced inflammation through TLR2, we examined the role of a polymorphism, a 23bp deletion in 5UTR region of TLR2 gene in patients with dengue (with and without warning signs) and correlated with plasma levels of inflammatory mediators with disease severity and viral serotypes.\n\nMethodsEighty nine patients classified as per WHO 2009 criteria during dengue outbreak in Odisha, India in 2016 were included in the current study. Presence of dengue virus (DENV) was demonstrated by detecting NS1 antigen, IgM capture ELISA and serotypes in circulation were discriminated by type-specific RT-PCR and/or sequencing. Sixty-one confirmed dengue cases were typed for TLR2 indel polymorphism and compared with 485 disease free controls. Plasma samples were assayed for 41-plex cytokine/ chemokines using Luminex bead based immunoassay.\n\nResultsPresence of 23bp deletion allele of TLR2 gene was significantly more in patients with severe dengue in comparison to dengue fever cases (p= 0.03; Odds ratio 4.05) although the frequency of insertion (Ins) allele of TLR2 was comparable in healthy controls and dengue cases (82.4 and 87.9 % respectively). Seventy-three (82%) samples were found to be positive by NS1/IgM capture ELISA/ RT-PCR. DENV-2 was predominant (58%) during the outbreak. Among the host inflammatory biomarkers 9 molecules were significantly altered in dengue patients when compared to healthy controls. The increased levels of IFN-{gamma}, GM-CSF, IL-10, IL-1R and MIP-1{beta} correlated significantly with severe dengue.\n\nConclusionsThe frequency of 23bp Indel mutation of TLR2 was comparable between healthy controls and dengue fever (with and without warning signs), suggesting that this indel mutation does not contribute significantly to susceptibility/ resistance to dengue; however, del allele of TLR2 gene was significantly more associated in patients with severe dengue symptoms when compared to dengue fever cases.

microbiology↗