Biology subjects

Roy, S.

Publications and source records attributed to Roy, S..

At least 307 records · Page 17Linked to original sources

bioRxiv2020

Protein induced membrane phase transition facilitates leishmania infection

Although host membrane is known to play critical roles in the internalization of leishmania parasites inside macrophages (M{phi}), any detailed mechanistic understanding is missing. We show here that KMP-11, a small immunogenic protein of Leishmania Donovani (LD) facilitates the infection process by binding to M{phi} membrane through its N-terminal domain (1-19AA). This binding results in a membrane phase transition that occurs at a threshold protein/lipid ratio, which is linked to the change in membrane tension. KMP-11 induced phase transition is also associated with lipid raft disruption and T-cell deactivation. Finally, using a combination of tryptophan-scanning mutagenesis and synthesized peptides, we develop a mathematical exposition, which demonstrates that hydrophobic moment (H) and the number of residues involved in a mirror sequence (N) at the interacting N-terminal are governing factors for the membrane phase transition, which facilitates infection process.

biophysics↗

bioRxiv2020

A Numerical Representation and Classification of Codons to Investigate Codon Alternation Patterns during Genetic Mutations on Disease Pathogenesis

Alteration of amino acids is possible due to mutation in codons that could have potential reasons to occur disease. Single nucleotide substitutions (SNS) in genetic codon thus have prime importance for their ability to occur mutations that may be deleterious indeed. Effective mutation analysis can help to predict the fate of the diseased individual which can be validated later by in-vitro experiments. Hence in this present study, we try to investigate the codon alteration patterns and their impact during mutation for the genes known to be responsible for a particular disease. We use a numerical representation of four nucleotides based on the number of hydrogen bonds in their chemical structures and make a classification of 64 codons as well as corresponding 20 amino acids into three different classes (Strong, Weak and Transitional). The entire analysis has been carried out based on these classifications. For our current study, we consider two neurodegenerative diseases, Parkinsons disease, and Glaucoma. Several evidences claim similarities between both the diseases but proper pathogenetic factors are still unknown. The analysis reveals that the strong class of codons is highly mutated followed by the weak and transitional class. We observe that most of the mutations occur in the first or second positions in the codon rather than the third and mutations that occurred at the second place of codons are majorly deleterious. In most cases, the change in the determinative degree of codon due to mutation is directly proportional to the physical density property. Furthermore, we derive a determinative degree of five wild-type amino acid sequences, which can help biologists to understand the evolutionary relationship among them based on amino acid occurrence frequencies in proteins. In this regard we proposed an alignment-free method SSADDA (Sequence Similarity Analysis using Determinative Degree of Amino acid). Thus, our scheme gives a more microscopic and alternative representation of the existing codon table that helps in deciphering interesting codon alteration patterns during mutations in disease pathogenesis.

bioinformatics↗

bioRxiv2020

Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets

Clathrin has established roles in endocytosis, with clathrin-cages enclosing membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin synthesized in cell-bodies is conveyed into axons and synapses via slow axonal transport; as shown by classic pulse-chase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining live-imaging, mass-spectrometry (MS), Apex-labeled EM-tomography and super-resolution, we found that unlike dendrites where clathrin transiently assembles/disassembles as expected, axons contain stable transport-packets that move intermittently with an anterograde bias; with actin/myosin-VI as putative tethers. Transport-packets are unrelated to endocytosis, and the overall kinetics generate a slow biased flow of axonal clathrin. Synapses have integer-numbers of clathrin-packets circumferentially abutting the synaptic-vesicle cluster, advocating a model where delivery of clathrin-packets by slow axonal transport generates a radial organization of clathrin at synapses. Our experiments reveal novel trafficking mechanisms, and an unexpected nanoscale organization of synaptic clathrin.

neuroscience↗

bioRxiv2020

Emergence of ribozyme and tRNA-like structures from mineral-rich muddy pools on prebiotic earth

AO_SCPLOWBSTRACTC_SCPLOWThe RNA world hypothesis, although a viable one regarding the origin of life on earth, has so far failed to provide a compelling explanation for the synthesis of RNA molecules with catalytic functions, from free nucleotides via abiotic processes. To tackle this long-standing problem, we develop a realistic model for the onset of the RNA world, using experimentally determined rates for polymerization reactions. We start with minimal assumptions about the initial state that only requires the presence of short oligomers or just free nucleotides and consider the effects of environmental cycling by dividing a day into a dry, semi-wet and wet phases that are distinguished by the nature of reactions they support. Long polymers, with maximum lengths sometimes exceeding 100 nucleotides, spontaneously emerge due to a combination of non-enzymatic, non-templated polymer extension and template-directed primer extension processes. The former helps in increasing the lengths of RNA strands, whereas the later helps in producing complementary copies of the strands. Strands also undergo hydrolysis in a structure-dependent manner that favour breaking of bonds connecting unpaired nucleotides. We identify the most favourable conditions needed for the emergence of ribozyme and tRNA-like structures and double stranded RNA molecules, classify all RNA strands on the basis of their secondary structures and determine their abundance in the population. Our results indicate that under suitable environmental conditions, non-enzymatic processes would have been sufficient to lead to the emergence of a variety of ribozyme-like molecules with complex secondary structures and potential catalytic functions.

biophysics↗

bioRxiv2020

A Grain of Salt

We quantified grain sodium (Na+) content across a barley GWAS panel grown under optimal conditions. We identified a strong association with a region containing two low and one high Na+ accumulating haplotypes of a Class 1 HIGH-AFFINITY POTASSIUM TRANSPORTER (HKT1;5) known to be involved in regulating plant Na+ homeostasis. The haplotypes exhibited an average 1.8-fold difference in grain Na+ content. We show that an L189P substitution disrupts Na+ transport in the high Na+ lines, disturbs the plasma membrane localisation typical of HKT1;5 and induces a conformational change in the protein predicted to compromise function. Under NaCl stress, lines containing P189 accumulate high levels of Na+, but show no significant difference in biomass. P189 increases in frequency from wild-species to elite cultivars leading us to speculate that the compromised haplotype is undergoing directional selection possibly due to the value of Na+ as a functional nutrient in non-saline environments.

genetics↗

bioRxiv2020

Identification of QTLs for high grain yield and component traits in New Plant Types of rice

A panel of 60 genotypes consisting of New Plant Types (NPTs) along with indica, tropical and temperate japonica genotypes were phenotypically evaluated for four seasons in irrigated situation for grain yield per se and component traits. Twenty NPT genotypes were found to be promising with an average grain yield of 5.45 to 8.8 t/ha. A total of 85 SSR markers were used in the study to identify QTLs associated with grain yield per se and related traits. Sixty-six (77.65%) markers were found to be polymorphic. The PIC values varied from 0.516 to 0.92 with an average of 0.704. A moderate level of genetic diversity (0.39) was detected among genotypes. Variation to the tune of 8% within genotypes, 68% among the genotypes within the population and 24% among the populations were observed (AMOVA). The association analysis using GLM and MLM models led to the identification of 30 and 10 SSR markers were associated with 70 and 16 QTLs, respectively. Thirty novel QTLs linked with 16 SSRs were identified to be associated with eleven traits, namely, tiller number (qTL-6.1, qTL-11.1, qTL-4.1), panicle length (qPL-1.1, qPL-5.1, qPL-7.1, qPL-8.1), flag leaf length (qFLL-8.1, qFLL-9.1), flag leaf width (qFLW-6.2, qFLW-5.1, qFLW-8.1, qFLW-7.1), total no. of grains (qTG-2.2, qTG-a7.1), thousand-grain weight (qTGW-a1.1, qTGW-a9.2, qTGW-5.1, qTGW-8.1), fertile grains (qFG-7.1), seed length-breadth ratio (qSlb-3.1), plant height (qPHT-6.1, qPHT-9.1), days to 50% flowering (qFD-1.1) and grain yield per se (qYLD-5.1, qYLD-6.1a, qYLD-11.1). This information could be useful for identification of highly potential parents for development of transgressive segregants. Moreover, super rice genotypes could be developed through pyramiding of these QTLS for important yield traits for prospective increment in yield potentiality and breaking yield ceiling.

plant biology↗

bioRxiv2019

GIV-Kindlin interaction is required for Kindlin-Mediated Integrin Recognition and Activation

Cells perceive and respond to the extracellular matrix (ECM) via integrin receptors; their dysregulation has been implicated in inflammation and cancer metastasis. Here we show that a guanine nucleotide exchange modulator of trimeric-GTPase Gi, GIV (a.k.a Girdin), directly binds the integrin adaptor Kindlin-2. A non-canonical short linear motif within GIVs C-terminus binds Kindlin-2-FERM3 domain at a site that is distinct from the binding site for the canonical NPxY motif on the -integrin tail. Binding of GIV to Kindlin-2 allosterically enhances Kindlin-2s affinity for {beta}1-integrin. Consequently, integrin activation and clustering are maximized, which augments cell adhesion, spreading and invasion. Findings elucidate how the GIV*Kindlin-2 complex has a two-fold impact: it allosterically synergizes integrin activation and enables {beta}1-integrins to indirectly access and modulate trimeric GTPases via the complex. Furthermore, Cox proportional-hazard models on tumor transcriptomics provide trans-scale evidence of synergistic interactions between GIV*Kindlin-2*{beta}1-integrin on time to progression to metastasis. The eTOC blurbIntegrins mediate cell adhesion to the extracellular matrix; their dysregulation fuels inflammation, cancer cell invasion and metastasis. Authors show how two pro-metastatic scaffold proteins, Kindlin and GIV/Girdin bind and cooperatively enhance their allosteric coupling to integrins, and their subsequent activation. Findings reveal novel interfaces in integrin signaling for pharmacologic manipulation. HIGHLIGHTSO_LIGIV and Kindlin(K2), two integrin adaptors that promote metastasis, bind each other C_LIO_LIBinding of GIV or integrin to K2 allosterically enhances GIV*K2*integrin complexes C_LIO_LIBinding is required for the maximal recruitment of GIV and K2 to active integrins C_LIO_LIBinding facilitates integrin clustering, activation, tumor cell adhesion, invasion. C_LI

cell biology↗

bioRxiv2019

Thrombospondin-1 Promotes Circuit-Specific Synapse Formation via β1-Integrin

Glial cells regulate synaptic connectivity during development, but whether they selectively instruct the formation of specific synaptic circuits is not known. Here we show that the major perisynaptic glia of the retina, the Muller glia (MG), control the proper establishment of the direction-selective (DS) circuit by a synaptogenic protein, Thrombospondin 1 (TSP1). We found that TSP1 promotes excitatory synapse formation specifically in on-off Direction-Selective retinal Ganglion Cells (ooDSGCs). Lack of TSP1 leads to reduced synapse formation within the inner plexiform sublayers containing DS-circuit, resulting in deficits of ooDSGC function. Even though pan-TSP receptor, 2{delta}-1, interaction is required for TSP1-induced synapse formation, the ooDSGC-subtype specificity of TSP1 is conferred by a second neuronal TSP1 receptor, {beta}1-Integrin. Furthermore, conditional deletion of {beta}1-Integrin in ooDSGCs results in diminished excitatory synapse formation without disturbing laminar organization showing that MG-secreted TSP1 controls circuit-specific synapse formation via {beta}1-Integrin.

neuroscience↗

bioRxiv2019

An allosteric hot-spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling

T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 domain and the doubly-phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly-phosphorylated ITAM-{zeta}1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of non-covalent interactions that allosterically couple the two SH2 domains during binding to doubly-phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells. SignificanceT-cell and B-cell signaling is initiated by the same family of non-receptor tyrosine kinases, ZAP-70 and Syk, respectively. ZAP-70 and Syk share homologous sequence and similar structural architecture, yet the two kinases differ in their mode of ligand recognition. ZAP-70 binds cooperatively to its ligand, whereas Syk binds uncooperatively. Spontaneous mutation (W165C) in the regulatory module of ZAP-70 impairs T-cell signaling causes autoimmune arthritis in SKG mice, the mechanism of which is unknown. We showed that ZAP-70 regulatory module undergoes a biphasic structural transition while binding to its ligand, which is fundamentally different from Syk. We presented a molecular mechanism of cooperativity in ZAP-70 regulatory module that explains altered ligand binding by ZAP-70 mutant found in SKG mice.

biochemistry↗

bioRxiv2019

Transcriptome-wide transmission disequilibrium analysis identifies novel risk genes for autism spectrum disorder

Recent advances in consortium-scale genome-wide association studies (GWAS) have highlighted the involvement of common genetic variants in autism spectrum disorder (ASD), but our understanding of their etiologic roles, especially the interplay with rare variants, is incomplete. In this work, we introduce an analytical framework to quantify the transmission disequilibrium of genetically regulated gene expression from parents to offspring. We applied this framework to conduct a transcriptome-wide association study (TWAS) on 7,805 ASD proband-parent trios, and replicated our findings using 35,740 independent samples. We identified 31 associations at the transcriptome-wide significance level. In particular, we identified POU3F2 (p=2.1e-7), a transcription factor (TF) mainly expressed in developmental brain. TF targets regulated by POU3F2 showed a 2.1-fold enrichment for known ASD genes (p=4.6e-5) and a 2.7-fold enrichment for loss-of-function de novo mutations in ASD probands (p=7.1e-5). These results provide a clear example of the connection between ASD genes affected by very rare mutations and an unlinked key regulator affected by common genetic variations.

genetics↗

bioRxiv2019

The γH2AX Peak in the S Phase After UV Irradiation Corresponds to the sites of DNA Replication and not DNA Damage

Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of γH2AX – the phosphorylated form of a histone variant H2AX – in the S phase within an asynchronous population of cells. γH2AX is often considered as a definitive marker of DNA damage inside a cell. In this report we show that γH2AX in the S phase cells after UV irradiation does not report on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers or on the extent of its secondary manifestations as DNA double strand breaks or in the inhibition of global transcription. Instead γH2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation – despite which, the cells complete the replication of their genomes and arrest within the G2 phase. Moreover, cells in all the phases of the cell cycle develop similar levels of DNA damage. Our study suggests that it is not DNA damage but the response elicited, which peaks in the S phase upon UV damage.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

CLICK-enabled analogues reveal pregnenolone interactomes in cancer and immune cells

Pregnenolone (P5) promotes prostate cancer cell growth, and de novo synthesis of intratumoural P5 is a potential cause of development of castration-resistance. Immune cells can also synthesize P5 de novo. Despite its biological importance, little is known about P5s mode of actions, which appears to be context-dependent and pleiotropic. A comprehensive proteome-wide spectrum of P5-binding proteins that are involved in its trafficking and functionality remains unknown. Here, we describe an approach that integrates chemical biology for probe synthesis with chemoproteomics to map P5-protein interactions in live prostate cancer cells and murine CD8+ T cells. We subsequently identified P5-binding proteins potentially involved in P5-trafficking, and in P5s non-genomic action that may drive the promotion of castrate-resistance prostate cancer and regulate CD8+ T cell function. We envisage that this methodology could be employed for other steroids to map their interactomes directly in a broad range of living cells, tissues and organisms.

biochemistry↗

bioRxiv2019

Human iPSC modeling elucidates mutation-specific responses to gene therapy in a genotypically diverse dominant maculopathy

Dominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients. SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.

genetics↗

bioRxiv2019

Influence of body weight at young adulthood on the epigenetic clock and lifespan in the BXD murine family

DNA methylation (DNAm) is shaped by genetic and environmental factors and modulated by aging. Here, we examine interrelations between epigenetic aging, body weight (BW), and lifespan in 12 inbred mouse strains from the BXD panel that exhibit over two-fold variation in longevity. Genome-wide DNAm was assayed in 70 liver specimens from mice ranging in age from 6 to 25 months that were maintained on normal chow or high fat diet (HFD). We defined subsets of CpG regions associated with age, BW at young adulthood, and strain-by-diet dependent life expectancy. The age associated differentially methylated CpG regions (age-DMRs) featured distinct genomic characteristics, with DNAm gains over time occurring in sites with high CpG density and low average methylation. CpG regions associated with BW were enriched in introns and generally showed lower methylation in mice with higher BW, and inversely correlated with gene expression such that mRNA was higher in mice with higher BW. Lifespan-associated regions featured no distinct genomic characteristics but were linked to genes involved in lifespan regulation, including the telomerase reverse transcriptase gene, Tert, which showed lower methylation and higher gene expression in long-lived strains. An epigenetic clock defined from the age-DMRs conveyed accelerated aging in mice belonging to strains with shorter lifespans. Both higher BW at young adulthood and HFD were associated with accelerated epigenetic aging. Our results highlight the age-accelerating effect of heavier body weight. Furthermore, the study demonstrates that the measure of epigenetic aging derived from age-DMRs can predict strain and diet-induced differences in lifespan.

genomics↗

bioRxiv2019

A Lamin Associated Chromatin Model for Chromosome Organization

We propose a simple model for chromatin organization based on the interaction of the chromatin fibres with Lamin proteins along the nuclear membrane. Lamin proteins are known to be a major factor that influences chromatin organization, and hence gene expression in the cells. Our polymer model explains the formation of lamin associated domains, and for heteropolymers with sequence control, can reproduce observed length distributions of LADs. In particular, lamin mediated interaction can enhance the formation of chromosome territories as well as the organization of chromatin into tightly packed heterochromatin and the loosely-packed gene-rich euchromatin regions.

biophysics↗

bioRxiv2019

Modulation of longevity by diet, and youthful body weight, but not by weight gain after maturity

Diet and environment profoundly modulate lifespan. We measured longevity as a function of diet and weight gain across a genetically diverse family of mice. We followed 1348 females from two parental strains--C57BL/6J and DBA/2J--and 146 cohorts of BXD isogenic progeny strains (n = 73) across their lifespan on a low fat chow diet (CD, 18% calories from fat) and on a high fat diet (HFD, 60% calories from fat). On average, HFD shortens lifespan by 85 days or 12%, roughly equivalent to an 8-10 year decrease in humans. However, strain variation in the response of diet on longevity is remarkably high, ranging from a longevity loss of 54% in BXD65 to a gain of 37% in BXD8. Baseline weights and early weight gain are both associated with a mean decrease in longevity of [~]4 days/g. By 500 days-of-age, cases fed HFD gained four times as much weight as control on average. However, strain-specific variation was substantial, thus weight gain did not correlate well with lifespan. In summary, high fat had a strong negative effect on longevity, but genetic interactions effects were even stronger. This highlights the unequivocal importance of genetic differences in making dietary recommendations.

genetics↗

bioRxiv2019

A low-cost and easy-to-use cell preservation reagent for 4{ring}C or room temperature sample storage

Current biospecimen storage and preservation methods no longer meet the demands of basic research and clinical diagnostics. Biospecimen preservation methodology has not advanced to accommodate cutting edge molecular analysis technologies that target single cells and full-length transcripts. Traditional methods, such as flash freezing and formalin-fixed paraffin-embedding (FFPE), were designed to provide information on cell structure and spatial relationships in whole tissues. These methods, however, do not maintain the integrity of proteins and nucleic acids. In this proof-of-concept study we examine preservation mechanisms utilized in nature for survival during cold seasons or periods of drought. Plants, brine shrimp, and tardigrades rely on components such as disaccharides or intrinsically disordered proteins to maintain cellular structure and biological activity in the settings of these environmental stresses. Our study demonstrates that these reagents aid in mammalian cell preservation for at least one week when a sample is stored in solution at 4{degrees}C, or as a dried sample in a low humidity desiccator.

pathology↗

bioRxiv2019

CpG-creating Mutations are Costly in Many Human Viruses

Mutations can occur throughout the virus genome and may be beneficial or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly that similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses.

evolutionary biology↗