Biology subjects

Sharma, R.

Publications and source records attributed to Sharma, R..

13 recordsLinked to original sources

bioRxiv2018

Biophysical basis of disruption in alpha rhythm in Alzheimer’s disease

Alpha is one of the most prominent rhythms (7.5-12.5 Hz) detected in electroencephalography (EEG) during wakeful relaxation with closed eyes. In response to elevated ambient acetylcholine levels, a subclass of thalamic pacemaker cells generate alpha. This rhythm is intrinsic to the cell and is robustly orchestrated by an interplay of hyperpolarization activated cyclic nucleotide gated channels(HCN) and calcium-ion channels. It has been shown that decreased expression of HCN channels is correlated to Alzheimer's Diseased (AD). In early stages of AD, alpha is known to be down-regulated and lowered in coherence. We use this well characterized and quantified rhythm to understand the changes in ion channel properties that lead to disruption of alpha as seen in AD in a biophysically detailed network model of the thalamo-cortical circuit that generates the alpha-rhythm. Our computational model allows us to explore the causal links between alpha rhythms, HCN channels and amyloid-beta aggregation. The most commonly used drugs(acetylcholinesterase inhibitors) in AD increase the duration and level of acetylcholine and provide temporary symptomatic relief in some cases. Our simulations show how increasing acetylcholine can provide rescue for a small range of aberrant HCN expression. We hypothesize that reduced alpha rhythm frequency and coherence is a result of down-regulated HCN expression, rather then compromised cholinergic modulation(as is currently thought). The model predicts that lowering of the alpha-rhythm can modify the network activity in the thalamo-cortical circuit and lead to an increase in the inhibitory drive to the thalamus.

neuroscience↗

bioRxiv2018

N-1-naphthylphthalamic acid stimulates tomato hypocotyl elongation, elevates ethylene levels and alters metabolic homeostasis

One sentence summaryN-1-naphthylphthalamic acid (NPA) treatment stimulates tomato hypocotyl elongation likely by elevating ethylene emission and lowering indole-3-butyric acid levels in the seedlings.\n\nAbstractIn higher plants, phytohormone indole-3-acetic acid is characteristically transported from the apex towards the base of the plant, termed as polar auxin transport (PAT). Among the inhibitors blocking PAT, N-1-naphthylphthalamic acid (NPA) that targets ABCB transporters is most commonly used. NPA-treated light-grown Arabidopsis seedlings show severe inhibition of hypocotyl and root elongation. In light-grown tomato seedlings, NPA inhibited root growth, but contrary to Arabidopsis stimulated hypocotyl elongation. The NPA-stimulation of hypocotyl elongation was milder in blue, red, and far-red light-grown seedlings. The NPA-treatment stimulated emission of ethylene from the seedlings. The scrubbing of ethylene by mercuric perchlorate reduced NPA-stimulated hypocotyl elongation. NPA action on hypocotyl elongation was antagonized by 1-methylcyclopropene, an inhibitor of ethylene action. NPA-treated seedlings had reduced levels of indole-3-butyric acid and higher levels of zeatin in the shoots. NPA did not alter indole-3-acetic levels in shoots. The analysis of metabolic networks indicated that NPA-treatment induced moderate shifts in the networks compared to exogenous ethylene that induced a drastic shift in metabolic networks. Our results indicate that in addition to ethylene, NPA-stimulated hypocotyl elongation in tomato may also involve zeatin and indole-3-butyric acid. Our results indicate that NPA-mediated physiological responses may vary in a species-specific fashion.

plant biology↗

bioRxiv2018

Structural, functional and molecular dynamics analysis of cathepsin B gene SNPs associated with tropical calcific pancreatitis, a rare disease of tropics.

Tropical Calcific Pancreatitis (TCP) is a neglected juvenile form of chronic non-alcoholic pancreatitis. Cathepsin B (CTSB), a lysososmal protease involved in cellular degradation process, is recently been studied as a potential candidate gene in the pathogenesis of TCP. According to cathepsin B hypothesis, mutated CTSB can lead to premature intracellular activation of trypsinogen, which is a key regulatory mechanism in pancreatitis. So far, CTSB mutations have been studied in pancreatitis and neurodegenerative disorders but little is known about the structural and functional effect of variants in CTSB. In this study, we investigated the effect of single nucleotide variants (SNVs) associated with TCP, using molecular dynamics and simulation algorithms. There were two non-synonymous variants in the coding region (L26V and S53G) of CTSB, located in the propeptide region. We tried to predict the effect of these variants on structure and function using multiple algorithms: SIFT, Polyphen2, Panther, SDM sever, i-Mutant2.0 suite, mCSM algorithm and Vadar. Further, using databases like miRdbSNP, PolymiRTS and miRNASNP, two SNPs in 3UTR region were predicted to affect the miRNA binding sites. Structural mutated models of nsSNP mutants (L26V and S53G) were prepared by MODELLER v9.15 and evaluated using TM-Align, Verify 3D, ProSA and Ramachandran plot. The results showed that the models (L26V and S53G) were of high accuracy. The 3D mutated structures were simulated using GROMACS 5.0 to predict the impact of these SNPs on protein stability. The results from in silico analysis and molecular dynamics simulations suggested that these variants in the propeptide region of cathepsin B could lead to structural and functional changes in the protein. Hence, the structural and functional analysis results have given interim conclusions that these variants can have deleterious effect in TCP and thus should be screen in samples from all TCP patients to decipher its distribution in patient population.

bioinformatics↗

bioRxiv2018

Intronic non-coding RNAs within ribosomal protein coding genes can regulate biogenesis of yeast ribosome

The genome of the budding yeast (Saccharomyces cerevisiae) has selectively retained introns in ribosomal protein coding genes. The function of these introns has remained elusive in spite of experimental evidence that they are required for the fitness of yeast. Here, we computationally predict novel small RNAs that arise from the intronic regions of ribosomal protein (RP) coding genes in Saccharomyces cerevisiae. Further, we experimentally validated the presence of seven intronic small RNAs (isRNAs). Computational predictions suggest that these isRNAs potentially bind to the ribosomal DNA (rDNA) locus or the corresponding rRNAs. Several isRNA candidates can also interact with transcripts of transcription factors and small nucleolar RNAs (snoRNAs) involved in the regulation of rRNA expression. We propose that the isRNAs derived from intronic regions of ribosomal protein coding genes may regulate the biogenesis of the ribosome through a feed-forward loop, ensuring the coordinated regulation of the RNA and protein components of the ribosomal machinery. Ribosome biogenesis and activity are fine-tuned to the conditions in the cell by integrating nutritional signals, stress response and growth to ensure optimal fitness. The enigmatic introns of ribosomal proteins may prove to be a novel and vital link in this regulatory balancing act.

bioinformatics↗

bioRxiv2018

Detection of GBA missense mutations and other variants using the Oxford Nanopore MinION

PurposeMutations in GBA cause Gaucher disease when biallelic, and are strong risk factors for Parkinsons disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA on the Oxford Nanopore MinION.\n\nMethodsWe sequenced an 8.9 kb amplicon from DNA samples of 17 individuals, including patients with Parkinsons and Gaucher disease, on older and current (R9.4) flow cells. These included samples with known mutations, assessed in a blinded fashion on the R9.4 data. We used NanoOK for quality metrics, two different aligners (Graphmap and NGMLR), Nanopolish and Sniffles to call variants, and Whatshap for phasing.\n\nResultsWe detected all known mutations, including the common p.N409S (N370S) and p.L483P (L444P), and three rarer ones, at the correct zygosity, as well as intronic SNPs. In a sample with the complex RecNciI allele, we detected an additional coding mutation, and a 55-base pair deletion. We confirmed compound heterozygosity where relevant. False positives were easily identified.\n\nConclusionThe Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantage of phasing and intronic analysis. It can be used as an efficient diagnostic tool.

genetics↗

bioRxiv2018

Olfactory and Vomeronasal Receptor Feedback Employ Divergent Mechanisms of PERK Activation

Mutually-exclusive chemoreceptor expression in olfactory and vomeronasal sensory neurons (OSNs and VSNs) enables odorant discrimination. This configuration involves chemoreceptor mediated activation of the endoplasmic reticulum (ER)-resident kinase PERK. PERK drives translation of the transcription factor ATF5 to preclude additional chemoreceptor expression. ATF5 translation is transient in OSNs but persistent in VSNs, suggesting chemoreceptor-specific modes of PERK activation. Herein, we showed that the ER-lumenal domain (LD) of PERK recognized vomeronasal receptor (VR)-derived peptides, suggesting direct PERK activation drives persistent ATF5 translation in VSNs. In contrast, PERK LD did not recognize olfactory receptor (OR)-derived peptides in vitro, and facilitating OR maturation in vivo prevented PERK activation, suggesting that ORs activate PERK indirectly through a failure to exit the ER. Importantly, impairing or prolonging ATF5 expression drove specific chemoreceptor repertoire biases. Together, these results demonstrate mechanistic divergence in chemoreceptor feedback and establish that differences in PERK activation promote qualitatively different gene regulatory results.

cell biology↗

bioRxiv2017

NAC-NOR mutations in tomato Penjar accessions attenuate multiple metabolic processes and prolong the fruit shelf life

Several Penjar accessions of tomato (Solanum lycopersicum), widely grown in the Mediterranean region, exhibit prolonged shelf life, and harbor alcobaca mutation with valine-106-aspartic acid substitution in the NAC-NOR protein. To uncover the metabolic basis underlying the prolonged shelf life, we compared four Penjar accessions to Ailsa Craig (AC). Three accessions bore alcobaca mutation, whereas fourth was a novel NAC-NOR allele with only six amino acids in the encoded protein. The cuticle composition among Penjars varied widely during the progression of fruit ripening. All Penjars exhibited delayed ripening, prolonged on-vine and off-vine shelf life, low ethylene emission and carotenoid levels albeit with accession-specific differences. Metabolic profiling revealed shifts in Krebs cycle intermediates, amino acids, and {beta}-aminobutyric acid levels indicating the attenuation of respiration in Penjars during post-harvest storage. The prolonged shelf life of Penjar fruits was associated with a concerted downregulation of a number of cell-wall modifying genes and cell-wall-related metabolites. The accumulation of higher ABA and sucrose levels at the onset of senescence in Penjar fruits likely contribute to reduced water loss. Our analyses reveal that in addition to specialized cuticle composition, the attenuation of various metabolic processes by NAC-NOR mutation likely prolongs the shelf life of Penjar fruits.\n\nHighlightThe prolonged shelf life of tomato Penjar accessions bearing mutations in NAC-NOR transcription factor appears to be regulated by a combined effect of attenuation of respiration, altered cuticle composition, enhanced ABA and sucrose levels in fruits and downregulation of cell wall modification

plant biology↗

bioRxiv2017

Isolation of phosphate solubilizing Pseudomonas strains from apple rhizosphere in the Trans Himalayan region of Himachal Pradesh, India

Total fifteen phosphate solubilizing bacteria were isolated from rhizosphere soil of apple tree on Kings B medium belonging to genus Pseudomonas spp. They were characterized on the basis of morphological and biochemical characteristics. Preliminary selection as phosphate solubilizing bacteria were done on the basis of formation of transparent zone around the colony on Pikovskayas agar medium containing 0.5% triclcium phosphate (TCP). Maximum in vitro phosphate solubilization on Pikovskayas agar plates after 72h incubation at 28{degrees}C was shown by An-15-Mg (46 mm), whereas in case of broth, again this strain showed maximum tricalcium phosphate (TCP) solubilization (76 g/ml). The pH of each inoculated broth was recorded daily and dropped significantly (pH 7.0-3.99). All the Pseudomonas isolates were further evaluated for overall plant growth promoting traits. Greatest siderophore activity was exhibited by An-14-Mg (71.23 %SU) followed by An-15-Mg (70.12 %SU) which were statiscally at par with each other. Whereas, maximum IAA production was observed again in An-15-Mg (95 g/ml). This isolate also showed the maximum production of HCN and ammonia. 16S rDNA and phylogenetic analysis showed that strain An-15-Mg exhibits 99% level of similarity with Pseudomonas aeruginosa. Therefore, it was designated as Pseudomonas aeruginosa strain An-15-Mg. HPLC analysis showed that the Pseudomonas aeruginosa strain An-15-Mg produced maximum concentration of succinic acid, malonic, citric and malic acid with small amounts of schimic, quinic, tartaric, fumaric and lactic acids. The strain An-15-Mg possessed phosphate solubilization as major PGP trait along with different PGP traits. The potential phosphate solubilizing strain of Pseudomonas aeruginosa was reported first time as PGPR which lives in close association with apple tree without harming the plant, therefore could be used as a promising phosphate solubilizer and biofertilizer in apple crop grown in high hills of Himachal Pradesh.\n\nAuthors contributionAll authors made significant efforts towards the completion of this research work and preparation of manuscript timely. Author Ranjna Sharma has done collection of soil samples of apple tree from two districts, isolation and characterization of phosphate solubilizing Pseudomonas isolates and their screening for different PGPTs, phenotypic characteriazation, 16S rRNA analysis and statistical analysis. Author S P Singh has done HPLC analysis at their institute. Author Ankita has done sequence analysis and phylogenetic tree preparation. Author Mohinder Kaur is my mentor and generated idea regarding this work. She guided me in completing this work successfully as well as manuscript writing.

microbiology↗

bioRxiv2017

Crystal structure of BrlR reveals a potential pyocyanin binding site

The transcriptional regulator BrlR from Pseudomonas aeruginosa is a member of the MerR family of multidrug transport activators. Studies have shown BrlR plays an important role in high level drug tolerance of P. aeruginosa in biofilm. Its drug tolerance ability can be enhanced by 3',5'-cyclic diguanylic acid (c-di-GMP). Here, we show the apo structure of BrlR and the direct binding between GyrI-like domain of BrlR and P. aeruginosa toxin pyocyanin. Furthermore, pyocyanin can enhance the binding between BrlR and DNA in vitro. These findings suggest BrlR can serve as the binding partner for both c-di-GMP and pyocyanin.

biochemistry↗

bioRxiv2017

Ponatinib shows potent antitumor activity in small cell carcinoma of the ovary hypercalcemic type (SCCOHT) through multi-kinase inhibition

Structured AbstractPurpose: Subunits of the SWI/SNF chromatin-remodeling complex are tumor suppressors inactivated in [~]20% of all cancers. Yet, few targeted treatments for SWI/SNF-mutant cancers exist. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits, SMARCA4 and SMARCA2. Given poor two-year survival rates for these women, a great need exists for effective targeted therapies.\n\nExperimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches comprehensively profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two PDX models and one cell line xenograft model of SCCOHT.\n\nResults: FGFRs and PDGFRs were overlapping hits between screens and the receptor tyrosine kinase (RTK) family was enriched in the siRNA screen hits. Evaluation of eleven RTK inhibitors in three SCCOHT cell lines identified ponatinib, an inhibitor of multiple RTKs, as the most effective clinically approved agent. Proteomics approaches confirmed inhibition of known targets of ponatinib and more than 20 non-canonical ponatinib targets. Ponatinib also delayed tumor doubling time 4-fold in SCCOHT-1 xenografts and reducing final tumor volumes in two SCCOHT patient-derived xenograft (PDX) models by 58.6% and 42.5%.\n\nConclusion: Ponatinib is an effective agent for SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted.\n\nAdditional InformationThis work was supported by research funds from the Canadian Cancer Society Research Institute 34 (#703458, D.G.H.), the National Institutes of Health (R01 CA195670-01, B.E.W., D.G.H., and 35 J.M.T., and T32 HL007106-39 to E.M.C), the Terry Fox Research Institute Initiative New Frontiers Program in Cancer (#1021, D.G.H.), the British Columbia Cancer Foundation (D.G.H.), the VGH & UBC Foundation (D.G.H.), the Anne Rita Monahan Foundation (P.R.), the Marsha Rivkin Center for Ovarian Cancer Research (J.M.T.), the Ovarian Cancer Alliance of Arizona (J.M.T.), the Small Cell Ovarian Cancer Foundation (P.R., J.D.L., B.V., and J.M.T.), and philanthropic support to the TGen Foundation (J.M.T.).\n\nCOI disclosure statement: The authors declare no potential conflicts of interest.

cancer biology↗

bioRxiv2017

Learning Edge Rewiring in EMT from Single Cell Data

Cellular regulatory networks are not static, but continuously reconfigure in response to stimuli via alterations in gene expression and protein confirmations. However, typical computational approaches treat them as static interaction networks derived from a single experimental time point. Here, we provide a method for learning the dynamic modulation, or rewiring of pairwise relationships (edges) from a static single-cell data. We use the epithelial-to-mesenchymal transition (EMT) in murine breast cancer cells as a model system, and measure mass cytometry data three days after induction of the transition by TGF{beta}. We take advantage of transitional rate variability between cells in the data by deriving a pseudo-time EMT trajectory. Then we propose methods for visualizing and quantifying time-varying edge behavior over the trajectory and use these methods: TIDES (Trajectory Imputed DREMI scores), and measure of edge dynamism (3DDREMI) to predict and validate the effect of drug perturbations on EMT.

systems biology↗

bioRxiv2017

MutS-Homolog2 Silencing Generates Tetraploid Meiocytes In Tomato (Solanum lycopersicum)

MSH2 is the core protein of MutS-homolog family involved in recognition and repair of the errors in the DNA. While other members of MutS-homolog family reportedly regulate mitochondrial stability, meiosis, and fertility, MSH2 is believed to participate mainly in mismatch repair. The search for polymorphism in MSH2 sequence in tomato accessions revealed both synonymous and nonsynonymous SNPs; however, SIFT algorithm predicted that none of the SNPs influenced MSH2 protein function. The silencing of MSH2 gene expression by RNAi led to phenotypic abnormalities in highly-silenced lines, particularly in the stamens with highly reduced pollen formation. MSH2 silencing exacerbated formation of UV-B induced thymine dimers and blocked light-induced repair of the dimers. The MSH2 silencing also affected the progression of male meiosis to a varying degree with either halt of meiosis at zygotene stage or formation of diploid tetrads. The immunostaining of male meiocytes with centromere localized CENPC (Centromere protein C) protein antibody showed the presence of 48 univalent along with 24 bivalent chromosomes suggesting abnormal tetraploid meiosis. The mitotic cells of root tips of silenced lines showed diploid nuclei but lacked intervening cell plates leading to cells with syncytial nuclei. Thus we speculate that tetraploid pollen mother cells may have arisen due to the fusion of syncytial nuclei before the onset of meiosis. It is likely that in addition to Mismatch repair (MMR), MSH2 may have an additional role in regulating ploidy stability.

plant biology↗

bioRxiv2017

MAGIC: A diffusion-based imputation method reveals gene-gene interactions in single-cell RNA-sequencing data

Single-cell RNA-sequencing is fast becoming a major technology that is revolutionizing biological discovery in fields such as development, immunology and cancer. The ability to simultaneously measure thousands of genes at single cell resolution allows, among other prospects, for the possibility of learning gene regulatory networks at large scales. However, scRNA-seq technologies suffer from many sources of significant technical noise, the most prominent of which is dropout due to inefficient mRNA capture. This results in data that has a high degree of sparsity, with typically only ~10% non-zero values. To address this, we developed MAGIC (Markov Affinity-based Graph Imputation of Cells), a method for imputing missing values, and restoring the structure of the data. After MAGIC, we find that two- and three-dimensional gene interactions are restored and that MAGIC is able to impute complex and non-linear shapes of interactions. MAGIC also retains cluster structure, enhances cluster-specific gene interactions and restores trajectories, as demonstrated in mouse retinal bipolar cells, hematopoiesis, and our newly generated epithelial-to-mesenchymal transition dataset.

bioinformatics↗