Biology subjects

Sharma, S.

Publications and source records attributed to Sharma, S..

At least 379 records · Page 21Linked to original sources

bioRxiv2020

HSPs, Ubiquitins and Antioxidants aid in Heat Tolerance in Tharparkar Indicine cattle

Transcriptome profiling of Vrindavani and Tharparkar cattle revealed that more numbers of genes dysregulated in Vrindavani than in Tharparkar. A contrast in gene expression was observed with 18.5 % of upregulated genes in Vrindavani were downregulated in Tharparkar and 17.5% upregulated genes in Tharparkar were downregulated in Vrindavani. Functional annotation of genes differentially expressed in Tharparkar and Vrindavani revealed that the systems biology in Tharparkar is moving towards counteracting the effects due to heat stress. Unlike Vrindavani, Tharparkar is not only endowed with higher expression of the scavengers (UBE2G1, UBE2S, and UBE2H) of misfolded proteins but also with protectors (VCP, Serp1, and CALR) of naive unfolded proteins. Further, higher expression of the antioxidants in Tharparkar enables it to cope up with higher levels of free radicals generated as a result of heat stress. In this study we found relevant genes dysregulated in Tharparkar in the direction that can counter heat stress.

physiology↗

bioRxiv2020

Changes to the resistome of Pseudomonas aeruginosa clone ST308 associated with corneal infection over time

ObjectivesThis study compared the resistomes of isolates of Pseudomonas aeruginosa clone ST308 from 2018 and 1997 from India. MethodsTwo ocular clonal type ST308 isolates of Pseudomonas aeruginosa (198 and 219) isolated in 2018 and five historical isolates (31, 32, 33, 35 and 37) isolated in 1997 at the LV Prasad Eye Institute in India were analysed for their susceptibilities to ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. DNA was extracted using the DNeasy(R) Blood and Tissue. Paired-end library was prepared using Nextera XT DNA library preparation kit. Libraries were sequenced on Illumina(R) MiSeq bench top sequencer generating 300 bp paired-end reads. Spades v3.12.0 was used for assembly, Resfinder v3.1. for acquired resistance genes and Snippy V2 for variants calling. Integron finder v1.5.1 was used to identify the integrons present in the genomes. ResultsThe recent isolate 219 was resistant to all tested antibiotics except polymyxin while isolate 198 was resistant to ciprofloxacin, levofloxacin, gentamicin and tobramycin. Among historical isolates five were resistant to gentamicin, tobramycin and ciprofloxacin, four were resistant to levofloxacin while two were resistant to polymyxin. Twenty-four acquired resistance genes were present in the 2018 isolates compared to 11 in the historical isolates. All isolates contained the following genes encoding for aminoglycoside aph(6)-Id, aph(3')-lIb, aph(3'')-Ib), beta-lactam (blaPAO), tetracycline (tet(G)), fosfomycin (fosA), chloramphenicol (catB7), sulphonamide (sul1), quaternary ammonium (qacEdelta1) and fluoroquinolone (crpP) resistance. Isolate 198 possessed aph(3')-VI, rmtD2, qnrVC1, blaOXA-488, blaPME-1, while 219 possessed aadA1, rmtB, aac(6')-Ib-cr, blaTEM-1B, blaVIM-2, mph(E), mph(A), msr(E). In the isolate 219 genes blaTEM-1b, blaVIM-2, sul1, qnrvc1, rmtB and aadA1 were carried on class 1 integron. While an incomplete class 1 integron was also found in isolate 198 which was located on the genome where gene rmtB, blaPME-1, qnrVC1 and sul1 genes were positioned. There were no notable differences in the number of single nucleotide polymorphisms, but recent isolates carried more insertions and deletions in their genes. ConclusionP. aeruginosa ocular clonal isolates have changed over time, with strains acquiring genes and having more insertions and deletions in their chromosomal genes that confirm resistance to antibiotics. HighlightsO_LIRecent clonal ocular isolates of Pseudomonas aeruginosa from India have acquired a number of resistance genes compared to historical clones C_LIO_LIConsequently, resistance to antibiotics particularly fluoroquinolones in recent clones of P. aeruginosa appears to have increased. C_LIO_LIThe acquired resistance genes found in the recent P. aeruginosa isolates were related to mobile genetic elements. C_LI

microbiology↗

bioRxiv2020

Rapid, field-deployable nucleobase detection and identification using FnCas9

Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids has provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19.

molecular biology↗

bioRxiv2020

A versatile high throughput strategy for cloning the env gene of HIV-1

The trimeric envelope glycoprotein (gp120/gp41)3 of human immunodeficiency virus-1 (HIV-1) mediates viral and host cell membrane fusion, initiated by binding of viral envelope gp120 protein to the CD4 receptor on host immune cells. Functional env genes from infected individuals have been widely used as templates for vaccine design, for setting up viral neutralization assays and to study the viral evolution and pathogenesis. Traditional topoisomerase or T4 DNA polymerase mediated approaches for cloning single genome amplified (SGA) env genes are labor-intensive, cost-ineffective with low-throughput, thereby enabling functional analysis of only a limited number of env genes from the diverse circulating quasispecies in infected individuals. Herein, we report an efficient, easy to optimize and high-throughput approach for cloning diverse HIV-1 env genes. Multiple env/rev gene cassettes, derived from infected infants, were subjected to SGA using Phusion polymerase and utilized as megaprimers in overlap extension PCR mediated cloning (OEC), circumventing the requirement for novel enzymes. Furthermore, utilization of Phusion polymerase for both the amplification of env/rev cassettes and OEC allows convenient monitoring and optimization, thereby providing much greater flexibility and versatility for analysis of env genes from HIV-1 infected individuals.

synthetic biology↗

bioRxiv2020

Circulating immune cell phenotype dynamics reflect the strength of tumor-immune cell interactions in patients during immunotherapy

The extent that immune cell phenotypes in the peripheral blood reflect within-tumor immune activity prior to and early in cancer therapy is unclear. To address this question, we studied the population dynamics of tumor and immune cells, and immune phenotypic changes, using clinical tumor and immune cell measurements and single cell genomic analyses. These samples were serially obtained from a cohort of advanced gastrointestinal cancer patients enrolled on a trial with chemotherapy and immunotherapy. Using an ecological population model, fitted to clinical tumor burden and immune cell abundance data from each patient, we find evidence of a strong tumor-circulating immune cell interaction in responder patients, but not those patients that progress on treatment. Upon initiation of therapy, immune cell abundance increased rapidly in responsive patients, and once the peak level is reached, tumor burden decreases, similar to models of predator-prey interactions; these dynamic patterns were absent in non-responder patients. To interrogate phenotype dynamics of circulating immune cells, we performed single cell RNA sequencing at serial time points during treatment. These data show that peripheral immune cell phenotypes were linked to the increased strength of patients tumor-immune cell interaction, including increased cytotoxic differentiation and strong activation of interferon signaling in peripheral T-cells in responder patients. Joint modeling of clinical and genomic data highlights the interactions between tumor and immune cell populations and reveals how variation in patient responsiveness can be explained by differences in peripheral immune cell signaling and differentiation soon after the initiation of immunotherapy. One sentence summaryPeripheral immune cell differentiation and signaling, upon initiation of immunotherapy, reflects tumor attacking ability and patient response. Significance statementThe evolution of peripheral immune cell abundance and signaling over time, as well as how these immune cells interact with the tumor, may impact a cancer patients response to therapy. By developing an ecological population model, we provide evidence of a dynamic predator-prey like relationship between circulating immune cell abundance and tumor size in patients that respond to immunotherapy. This relationship is not found either in patients that are non-responsive to immunotherapy or during chemotherapy. Single cell RNA-sequencing (scRNAseq) of serial peripheral blood samples from patients show that the strength of tumor-immune cell interactions is reflected in T-cells interferon activation and differentiation early in treatment. Thus, circulating immune cell dynamics reflect a tumors response to immunotherapy.

cancer biology↗

bioRxiv2020

RNA editing enzyme APOBEC3A promotes pro-inflammatory (M1) macrophage polarization

Pro-inflammatory (M1) macrophage polarization is associated with microbicidal and antitumor responses. We recently described APOBEC3A-mediated cytosine-to-uracil (C>U) RNA editing during M1 polarization. However, the functional significance of this editing is unknown. Here, we find that APOBEC3A-mediated cellular RNA editing can also be induced by influenza or Maraba virus infections of normal human macrophages, and by interferons in tumor-associated macrophages. Gene knockdown and RNA_Seq analyses show that APOBEC3A mediates C>U RNA editing of 209 exonic/UTR sites in 203 genes during M1 polarization. The highest level of deleterious C>U RNA editing occurred in THOC5, encoding a nuclear mRNA export protein implicated in M-CSF-driven macrophage differentiation. Knockdown of APOBEC3A reduces pro-inflammatory M1 markers including IL6, IL23A and IL12B gene expression, CD86 surface protein expression, and TNF-, IL-1{beta} and IL-6 cytokine secretion, and increases glycolysis and glycolytic capacity. Thus, APOBEC3A cytidine deaminase plays an important role in transcriptomic and functional polarization of M1 macrophages.

immunology↗

bioRxiv2020

Proofreading deficiency in mitochondrial DNA polymerase does not affect total dNTP pools in mouse embryos

Mitochondrial DNA (mtDNA) mutator mice express proofreading-deficient mtDNA polymerase gamma (PolgD257A) and age prematurely1,2, whereas mice with other mitochondrial defects do not show global signs of early aging. The reason for this discrepancy is not completely understood. Hamalainen et al. recently reported that both induced pluripotent stem cells (iPSCs) and primary mutator embryonic fibroblasts harboring PolgD257A demonstrate increased mtDNA replication frequency leading to depletion of the total cellular dNTP pool3. The authors proposed that the decreased availability of cellular dNTPs for nuclear genome replication leads to compromised nuclear genome maintenance and premature aging. Here we report that total cellular dNTP pools are normal in mtDNA mutator mouse embryos (genotype: PolgD257A/D257A), which shows that a living organism exclusively expressing PolgD257A has normal total dNTP pools despite ubiquitous rapid cell division.

cell biology↗

bioRxiv2020

SAMHD1 limits the efficacy of forodesine in leukaemia by protecting cells against cytotoxicity of dGTP

The anti-leukaemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphates (dNTPs), protected cells against the effects of dNTP imbalances. SAMHD1-deficient cells induced intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine acted synergistically to kill cells lacking SAMHD1. Using mass cytometry, we found that these compounds killed SAMHD1-deficient malignant cells from patients with chronic lymphocytic leukaemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation were unaffected. We therefore propose to use forodesine as a precision medicine for leukaemia, stratifying patients by SAMHD1 genotype or expression. O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/951517v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@69c367org.highwire.dtl.DTLVardef@19be007org.highwire.dtl.DTLVardef@91156corg.highwire.dtl.DTLVardef@1158ec6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOSupplementary Figure 5.C_FLOATNO Graphical Abstract C_FIG Highlights O_LISAMHD1-deficient cells die upon exposure to deoxyribonucleosides (dNs) C_LIO_LIDeoxyguanosine (dG) is the most toxic dN, inducing apoptosis in cells lacking SAMHD1 C_LIO_LISAMHD1-mutated leukaemic cells can be killed by dG and the PNP-inhibitor forodesine C_LI In BriefSAMHD1 degrades deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Davenne et al. found that SAMHD1 protects cells against dNTP imbalances. Exposure of SAMHD1-deficient cells to deoxyguanosine (dG) results in increased intracellular dGTP levels and subsequent apoptosis. This can be exploited to selectively kill cancer cells that acquired SAMHD1 mutations.

cancer biology↗

bioRxiv2020

Deep phylo-taxono-genomics (DEEPT genomics) reveals misclassification of Xanthomonas species complexes into Xylella, Stenotrophomonas and Pseudoxanthomonas

Genus Xanthomonas encompasses specialized group of phytopathogenic bacteria with genera Xylella, Stenotrophomonas and Pseudoxanthomonas being its closest relatives. While species of genera Xanthomonas and Xylella are known as serious phytopathogens, members of other two genera are found in diverse habitats with metabolic versatility of biotechnological importance. Few species of Stenotrophomonas are multidrug resistant opportunistic nosocomial pathogens. In the present study, we report genomic resource of genus Pseudoxanthomonas and further in-depth comparative studies with publically available genome resources of other three genera. Surprisingly, based on deep phylo-taxono-genomic or DEEPT criteria, all the four genera were found to be one genus and hence synonyms of Xanthomonas. The members of Pseudoxanthomonas are more diverse and ancestral and rest forming two mega species groups (MSGs) i.e. Xanthomonas Xylella (XX-MSG) and Stenotrophomonas (S-MSG). Within XX-MSG, there are 3 species complexes i.e., X. citri complex (Xcc) member species are primarily pathogenic to dicots, X. hyacinthi complex (Xhc) member species are primarily pathogenic to monocots and Xanthomonas (Xylella) fastidosa complex (Xfc) with diverse phytopathogenic potential. Interestingly, Xylella seems to be a variant Xanthomonas lineage or species complex that is sandwiched between Xcc and Xhc. Like Xylella, within S-MSG, we find a species complex of clinical origin Xanthomonas (Stenotrophomonas) maltophilia complex (Xmc). Comparative studies revealed selection and role of xanthomonadin pigment and xanthan gum in emergence of plant pathogenic XX-MSG. Pan genome analysis also revealed large set of unique genes with particular functions suited for plant/animal lifestyle responsible for emergence of variant Xfc and Xmc species complexes. Overall, our systematic and large scale genera based study has allowed us to understand the origin and to clarify the taxonomic breadth of genus of high importance in agriculture, medicine and industry. Such DEEPT genomics studies are also way forward to identify right markers or functions for diagnosis and drug development of any pathogenic bacteria. RepositoriesGenome Submission Accession Number: MWIP00000000, PDWO00000000, PDWN00000000, PDWT00000000, PDWS00000000, PDWW00000000, PDWU00000000, PDWR00000000, PDWL00000000, PDWQ00000000, PDWM00000000, PDWP00000000, PDWV00000000, PDWK00000000 and QOVG00000000

genomics↗

bioRxiv2020

Design, optimization, and analysis of large DNA and RNA nanostructures through interactive visualization, editing, and molecular simulation

This work seeks to remedy two deficiencies in the current nucleic acid nanotechnology software environment: the lack of both a fast and user-friendly visualization tool and a standard for common structural analyses of simulated systems. We introduce here oxView, a web browser-based visualizer that can load structures with over 1 million nucleotides, create videos from simulation trajectories, and allow users to perform basic edits to DNA and RNA designs. We additionally introduce open-source software tools for extracting common structural parameters to characterize large DNA/RNA nanostructures simulated using the coarse-grained modeling tool, oxDNA, which has grown in popularity in recent years and is frequently used to prototype new nucleic acid nanostructural designs, model biophysics of DNA/RNA processes, and rationalize experimental results. The newly introduced software tools facilitate the computational characterization of DNA/RNA designs by providing multiple analysis scripts, including mean structures and structure flexibility characterization, hydrogen bond fraying, and interduplex angles. The output of these tools can be loaded into oxView, allowing users to interact with the simulated structure in a 3D graphical environment and modify the structures to achieve the required properties. We demonstrate these newly developed tools by applying them to in silico design, optimization and analysis of a range of DNA and RNA nanostructures.

biophysics↗

bioRxiv2020

Ketamine blocks the correlation between morphine-induced conditioned place preference and anxiety-like behaviors in mice.

Patients suffering from opioid use disorder often relapse during periods of abstinence, which is posited to be caused by negative affective states that drive motivated behaviors. Here, we explored whether conditioning mice with morphine in a CPP training paradigm evoked anxietylike behavior during morphine abstinence. To do this, mice were conditioned with morphine (10 mg/kg, i.p.) for five days. 24 h following conditioning, anxiety levels were tested by measuring time in the open arms of the elevated plus maze. The next day, mice were placed in the three compartment chamber to measure morphine-induced conditioned place preference (CPP). Our results show that following morphine conditioning, mice spent significantly less time in the open arm of the elevated plus maze and expressed robust morphine CPP on CPP test day. Furthermore, we found that an acute treatment with (R,S)-ketamine (10 mg/kg, i.p.), a medication demonstrating promise for preventing anxiety-related phenotypes, 30 min. prior to testing on post conditioning day 1, increased time spent in the open arm of the elevated plus maze in saline- and morphine-conditioned mice. Additionally, we found that a second injection of ketamine 30 min. prior to CPP tests on post conditioning day 2 prevented morphine-induced CPP, which lasted for up to 28 d post conditioning. Furthermore, we found that conditioning mice with 10% (w/v) sucrose using an oral self-administration procedure did not evoke anxietylike behavior, but elicited robust CPP, which was attenuated by ketamine treatment 30 min. prior to CPP tests. Overall, our results suggest that the ketamine-induced block of morphine CPP may not be attributed solely to alleviating negative affective states, but potentially through impaired memory of morphine-context associations.

neuroscience↗

bioRxiv2020

Human Telomerase Expression is under Direct Transcriptional Control of the Telomere-binding-factor TRF2

Tight regulatory mechanisms to maintain repression of human Telomerase (hTERT), the sole protein that synthesizes telomeres, is crucial for normal adult somatic cells. In contrast, enhanced telomerase activity and resulting pathological maintenance of telomeres, is widely understood as causal in >90% of human cancers. These implicate underlying mechanisms connecting hTERT regulation and telomeres, possibly through telomeric proteins, that remain unclear. In light of of recent work by us and others showing non-telomeric function of the telomere-binding protein TRF2, here we examined whether and how TRF2 affected hTERT regulation. Direct binding of TRF2 - spanning [~]450 bp of the hTERT promoter from the Transcriptional Start Site (TSS) - led to TRF2-dependent recruitment of the polycomb repressor complex PRC2 in both normal and cancer cells. This induced repressor histone modifications resulting in TRF2-dependent hTERT repression. Mutations in the hTERT promoter, found frequently in aggressive glioblastoma and reported to destabilize the G-quadruplex structure, resulted in loss of TRF2 binding and consequent hTERT over-expression. Conversely, using G-quadruplex-stabilizing ligands we regained TRF2 binding, hTERT re-suppression, in highly proliferating glioblastoma cells with telomerase hyperactivation due to hTERT promoter mutations. Together, results herein demonstrate direct control of hTERT through TRF2 in a G-quadruplex-dependent manner - implicating mechanisms of how telomerase regulation might be linked to telomeres in normal and cancer cells.

cancer biology↗

bioRxiv2020

The novel reversible LSD1 inhibitor SP-2577 promotes anti-tumor immunity in SWItch/Sucrose-NonFermentable (SWI/SNF) complex mutated ovarian cancer

Chromatin remodeling SWItch/Sucrose-NonFermentable (SWI/SNF) complexes, initially identified in yeast 20 years ago, are evolutionarily conserved multi-subunit protein complexes that use the energy from hydrolysis of adenosine triphosphate (ATP) to remodel nucleosome structure and modulate transcription. Mutations in proteins of SWI/SNF complexes occur in 20% of human cancers including ovarian cancer (OC). Approximately 50% of ovarian clear cell carcinoma (OCCC) carries mutations in the SWI/SNF subunit ARID1A while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is driven primarily by genetic inactivation of the SWI/SNF ATPase SMARCA4 (BRG1) alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Dual loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A) which regulates the chromatin landscape and gene expression by demethylating proteins, including histone H3. LSD1 associates with epigenetic complexes such as the nucleosome remodeling deacetylase complex (NuRD) and SWI/SNF to inhibit the transcription of genes involved in tumor suppression and cell differentiation. TGCA analysis of human cancers shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. Further, SCCOHT and OCCC cell lines show low nM IC50s for the reversible LSD1 inhibitor SP-2577 (Seclidemstat, currently in clinical phase I trials), supporting that these SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Recently, it has been also shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of Endogenous Retroviruses Elements (ERVs) and may thereby overcome resistance to checkpoint blockade. Additionally, SCCOHTs have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T cell infiltration. Thus, in this study we investigated the ability of SP-2577 to promote anti-tumor immunity and T cell infiltration in SWI/SNF-mutant SCCOHT and OCCC models. Our data shows that the reversible LSD1 inhibitor SP-2577 stimulates IFN-dependent anti-tumor immunity in SCCOHT cells in vitro in a 3D immune-organoid platform. Additionally, SP-2577 promoted the expression of PD-L1 in both SCCOHT and OCCC models. Together our findings suggest that SP-2577 and checkpoint inhibitors as a therapeutic combination may induce or augment immunogenic responses in these tumors.

cancer biology↗

bioRxiv2020

Opportunities for prioritizing and expanding conservation enterprise in India using carnivores as flagships

Conservation interventions in developing countries are frequently thwarted by socio-economic agendas, severely limiting the scope and rigor of biodiversity and habitat conservation. Very few ecological assessments incorporate human interests in conservation prioritization, creating asynchrony between planning and implementation. For conservation actions to be logistically feasible, multiple criteria including ecological, social, economic and administrative aspects must be considered. Understanding how these different dimensions interact spatially is also important for gauging the potential for conservation success. Here, we use a guild of select mammalian carnivores (wild canids and hyenas) in India to (i) generate distribution maps at the spatial scale of administrative sub-districts, that is relevant to management, (ii) examine ecological, social and biogeographic factors associated with their distribution, quantify key threats, and identify areas important for their conservation, (iii) use prioritization tools for balancing habitat conservation, human needs and economic growth, and (iv) evaluate the spatial congruence between areas with high conservation potential, and areas currently in focus for protection efforts, conservation investments, and infrastructure development. We find that the current Protected Area system does not adequately cover or represent diverse habitats, that there is immense potential for States to increase financial investments towards alternative conservation strategies, and, most infrastructure projects may be jeopardizing important carnivore habitats. Our framework allowed for identifying locations where conservation investments would lead to the highest dividends for flagship carnivores and associated species across habitats. We make a case for re-evaluating how large-scale prioritization assessments are made, and for broadening the purview of conservation policies in India and other developing countries.

ecology↗

bioRxiv2020

KDM6A mediated expression of the long noncoding RNA DINO causes TP53 tumor suppressor stabilization in Human Papillomavirus type 16 E7 expressing cells

HPV16 E7 has long been noted to stabilize the TP53 tumor suppressor. However, the molecular mechanism of TP53 stabilization by HPV16 E7 has remained obscure and can occur independent of E2F regulated MDM2 inhibitor, p14ARF. Here, we report that the Damage Induced Noncoding (DINO) lncRNA (DINOL) is the missing link between HPV16 E7 and increased TP53 levels. DINO levels are decreased in cells where TP53 is inactivated, either by HPV16 E6, expression of a dominant negative TP53 minigene or by TP53 depletion. DINO levels are increased in HPV16 E7 expressing cells. HPV16 E7 causes increased DINO expression independent of RB1 degradation and E2F1 activation. Similar to the adjacent CDKN1A locus, DINO expression is regulated by the histone demethylase, KDM6A. DINO stabilizes TP53 in HPV16 E7 expressing cells and as a TP53 transcriptional target, DINO levels further increase. Similar to other oncogenes such as adenovirus E1A or MYC, HPV16 E7 expressing cells are sensitized to cell death under conditions of metabolic stress and in the case of E7, this has been linked to TP53 activation. Consistent with earlier studies, we show that HPV16 E7 expressing keratinocytes are highly sensitive to metabolic stress induced by the antidiabetic drug, metformin. Metformin sensitivity of HPV16 E7 expressing cells is rescued by DINO depletion. This work identifies DINO as a critical mediator TP53 stabilization and activation in HPV16 E7 expressing cells. IMPORTANCEViral oncoproteins, including HPV16 E6 and E7 have been instrumental in elucidating the activities of cellular signaling networks including those governed by the TP53 tumor suppressor. Our study demonstrates that the long noncoding RNA DINO is the long sought missing link between HPV16 E7 and elevated TP53 levels. Importantly, the TP53 stabilizing DINO plays a critical role in the predisposition of HPV16 E7 expressing cells to cell death under metabolic stress conditions from metformin treatment.

cancer biology↗

bioRxiv2019

Discovery of dual inhibitors of KPC-3 and KPC-15 of Klebsiella pneumoniae - a ligand and structure based virtual screening study

BackgroundThe development of carbapenem resistance against Klebsiella pneumoniae is a situation of grave concern and requires urgent attention. Among the KPC produced by K.pneumoniae, KPC-3, and KPC-15, play a significant role in the development of resistance to carbapenem. Materials and methodsThe binding sites of KPC-3 and KPC-15 were predicted by the COACH server. Drug-like ligands from ZINC were then screened by ligand-based drug screening (LBVS) by keeping Relebactam as a template. The top 50,000 selected ligands were then screened by structure-based virtual screening using idock. For keeping an account of the dual inhibitors stability in complex with KPC-3 and KPC-15, MDS were carried out for each complex. ResultsBased on consensus weighted ranks, the top 3 ligands with the dual inhibitory property are ZINC76060350 (consensus weighted rank - 1.5), ZINC05528590 (2), ZINC72290395 (3.5). All the top 3 dual inhibitors have a reasonable probability of passing through the blood-brain barrier. The RDKit and Morgan fingerprint scores between Relebactam and the top three ligands were 0.24, 0.22, 0.23, and 0.26, 0.19, 0.25, respectively (showing only 20% similarity). The MD simulation result revealed good binding stability of ligand ZINC05528590 with both KPC-3 and KPC-15, whereas ligand ZINC76060350 showed good binding stability to KPC-3. ConclusionThe ligand ZINC05528590 could be taken forward to develop a new drug against a multi-resistant- Klebsiella pneumoniae infection. At the same time, ZINC76060350 can be considered to develop a new drug against KPC-15 resistant Klebsiella pneumoniae.

bioinformatics↗

bioRxiv2019

Differential rapid plasticity in auditory and visual responses in the primarily multisensory orbitofrontal cortex

In a dynamic environment with rapidly changing contingencies, the orbitofrontal cortex (OFC) guides flexible behavior through coding of stimulus value. Although stimulus-evoked responses in the OFC are known to convey outcome, baseline sensory response properties in the mouse OFC are poorly understood. To understand mechanisms involved in stimulus value/outcome encoding it is important to know the response properties of single neurons in the mouse OFC, purely from a sensory perspective. Ruling out effects of behavioral state, memory and others, we studied the anesthetized mouse OFC responses to auditory, visual and audiovisual/multisensory stimuli, multisensory associations and sensory-driven input organization to the OFC. Almost all, OFC single neurons were found to be multisensory in nature, with sublinear to supralinear integration of the component unisensory stimuli. With a novel multisensory oddball stimulus set, we show that the OFC receives both unisensory as well as multisensory inputs, further corroborated by retrograde tracers showing labeling in secondary auditory and visual cortices, which we find to also have similar multisensory integration and responses. With long audiovisual pairing/association, we show rapid plasticity in OFC single neurons, with a strong visual bias, leading to a strong depression of auditory responses and effective enhancement of visual responses. Such rapid multisensory association driven plasticity is absent in the auditory and visual cortices, suggesting its emergence in the OFC. Based on the above results we propose a hypothetical local circuit model in the OFC that integrates auditory and visual information which participates in computing stimulus value in dynamic multisensory environments. Significance StatementProperties and modification of sensory responses of neurons in the orbitofrontal cortex (OFC) involved in flexible behavior through stimulus value/outcome encoding are poorly understood. Such responses are critical in providing the framework for the encoding of stimulus value based on behavioral context while also directing plastic changes in sensory regions. The mouse OFC is found to be primarily multisensory with varied nonlinear interactions, explained by unisensory and multisensory inputs. Audio-visual associations cause rapid plastic changes in the OFC, which bias visual responses while suppressing auditory responses. Similar plasticity was absent in the sensory cortex. Thus the observed intrinsic visual bias in the OFC weighs visual stimuli more than associated auditory stimuli in value encoding in a dynamic multisensory environment.

neuroscience↗

bioRxiv2019

Integrative analysis and machine learning based characterization of single circulating tumor cells

We collated publicly available single-cell expression profiles of circulating tumor cells (CTCs) and showed that CTCs across cancers lie on a near-perfect continuum of epithelial to mesenchymal (EMT) transition. Integrative analysis of CTC transcriptomes also highlighted the inverse gene expression pattern between PD-L1 and MHC, which is implicated in cancer immunotherapy. We used the CTCs expression profiles in tandem with publicly available peripheral blood mononuclear cell (PBMC) transcriptomes to train a classifier that accurately recognizes CTCs of diverse phenotype. Further, we used this classifier to validate circulating breast tumor cells captured using a newly developed microfluidic systems for label-free enrichment of CTCs.

genomics↗