Biology subjects

Ganguly, S.

Publications and source records attributed to Ganguly, S..

32 records · Page 2Linked to original sources

bioRxiv2022

Gene model correction for PVRIG in single cell and bulk sequencing data enables accurate detection and study of its functional relevance

Single cell RNA sequencing (scRNA-seq) has gained increased popularity in recent years and has revolutionized the study of cell populations; however, this technology presents several caveats regarding specific gene expression measurement. Here we examine the expression levels of several immune checkpoint genes, which are currently assessed in clinical studies. We find that unlike in most bulk sequencing studies, PVRIG, a novel immune-modulatory receptor in the DNAM-1 axis, suffers from poor detection in 10x Chromium scRNA-seq and other types of assays that utilize the GENCODE transcriptomic reference (gene model). We show that the default GENCODE gene model, typically used in the analysis of such data, is incorrect in the PVRIG genomic region and demonstrate that fixing the gene model recovers genuine PVRIG expression levels. We explore computational strategies for resolving multi-gene mapped reads, such as those implemented in RSEM and STARsolo and find that they provide a partial solution to the problem. Our study provides means to better interrogate the expression of PVRIG in scRNA-seq and emphasizes the importance of optimizing gene models and alignment algorithms to enable accurate gene expression measurement in scRNA-seq and bulk sequencing. The methodology applied here for PVRIG can be applied to other genes with similar issues.

bioinformatics↗

bioRxiv2022

Rosetta 's Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery

Fragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da that weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments in in silico screening. Here we test RosettaLigand by docking a series of fragments to a cysteine-depleted variant of the TIM-barrel protein, HisF. We compare the computational results with experimental NMR spectroscopy screens. NMR spectroscopy gives details on binding affinities of individual ligands, which allows assessment of the ligand-ranking ability by RosettaLigand, and also provides feedback on the location of the binding pocket, which serves as a reliable test of RosettaLigands ability to identify plausible binding poses. From a library screen of 3456 fragments, we identified a set of 31 ligands with intrinsic affinities to HisF with dissociation constants as low as 400 {micro}M. The same library of fragments was blindly screened in silico. RosettaLigand was able to rank binders before non-binders with an area under the curve (AUC) of the receiver operating characteristics (ROC) of 0.74. The docking poses observed for binders agreed with the binding pocket identified by NMR chemical shift perturbations for all fragments. Taken together, these results provide a baseline performance of RosettaLigand in a fragment-based drug discovery setting.

biochemistry↗

bioRxiv2022

Pseudocontact shift NMR data obtained from a non-canonical amino acid-linked lanthanide tag improves integral membrane protein structure prediction

A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction. We demonstrate this approach using the disulfide bond formation protein B (DsbB), an -helical IMP. We attached a cyclen-based paramagnetic lanthanide tag to an engineered noncanonical amino acid (ncAA) using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction. Using this tagging strategy, we collected 203 backbone HN pseudocontact shifts (PCSs) for three different labeling sites and used these as input to guide de novo membrane protein structure prediction protocols in Rosetta. We find that this sparse PCS dataset combined with 44 long-range NOEs as restraints in our calculations improves structure prediction of DsbB by enhancements in model accuracy, sampling, and scoring. The most accurate DsbB models generated in this case gave C-RMSD values over the transmembrane region of 2.11 [A] (best-RMSD) and 3.23 [A] (best-scoring).

biophysics↗

bioRxiv2022

Helminth egg derivatives as pro-regenerative immunotherapies

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4 expressing T helper cells, eosinophils, and regulatory T cells, and decreased expression of IL-17A in gamma delta ({gamma}{delta}) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a new regenerative immunotherapy approach using natural helminth-derivatives. One-Sentence SummaryHelminth-derived soluble egg antigen regenerative immunotherapies promote tissue repair in multiple injury models.

bioengineering↗

bioRxiv2022

First crystal structure of a non-canonical amino acid linked to a paramagnetic lanthanide tag facilitates protein structure determination using NMR-derived restraints

Site-directed spin labeling of proteins via non-canonical amino acids (ncAAs) is a non-traditional method for the measurement of pseudocontact shifts (PCSs) by nuclear magnetic resonance (NMR) spectroscopy. PCSs provide long-range distance and orientational information between a paramagnetic center and protein nuclei that can be used as restraints for computational structural modeling techniques. Here, we present the first experimental structure of an ncAA chemically linked to a lanthanide tag conjugated to the protein, T4-Lysozyme (T4L). T4L was crystallized with a cyclen-based C3 tag coordinated to the paramagnetic ion terbium (Tb3+). The paramagnetic C3-lanthanide tag generated PCSs measured at four different ncAA sites. We show that the addition of these restraints improves structure prediction protocols for T4L using the RosettaNMR framework. Generated models provide insight into T4L conformational flexibility sampled in solution. This integrative modeling protocol is readily transferable to larger proteins. Methods to predict protein structures are advancing into an exciting arena such that reliable experimental data will play important roles for evaluating the biophysical relevance of predicted structural models. Our contribution here caters to the growing interest in using ncAAs for a range of biophysical studies, and these methods can be readily transferred to larger protein systems of interest.

biophysics↗

bioRxiv2021

Leishmania Hijacks microRNA Import-Export Machinery of Infected Macrophage and Survives by Cross-Communicating Host miR-146a to Subjugate HuR and miR-122 in Neighbouring cells

Leishmania donovani, the causative agent of visceral leishmaniasis, infects and resides within tissue macrophage cells of the mammalian host. It is not clear how the parasite infected cells cross-talk with the non-infected cells in the infection niche to regulate the infection process. Interestingly, miRNAs, the regulatory small RNAs of the host, could get trafficked into and out of infected cells as part of extracellular vesicles to ensure exchange of the epigenetic signals and can regulate the expression of their target genes in both donor and recipient cells. Leishmania, for its survival in host macrophage, adopts a dual strategy to regulate the intercellular transport of host miRNAs. The parasite, by preventing mitochondrial function of the host cells, restricts the entry of liver cell derived miR-122 containing extracellular vesicles in infected macrophage to curtail the inflammatory response by miR-122. The parasite reciprocally upregulates the extracellular export of anti-inflammatory miR-146a from the infected cells. The exported miR-146a restricts miR-122 production in liver cells and polarizes neighbouring naive macrophages to the M2 state. miR-146a upregulates IL-10 in neighbouring macrophages where miR-146a dominates the RNA binding and miRNA suppressor protein HuR to inhibit the expression of proinflammatory cytokine mRNAs having HuR-interacting AU-rich elements and polarized the recipient cells to M2 stage. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/459146v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@32e32dorg.highwire.dtl.DTLVardef@e0d3c6org.highwire.dtl.DTLVardef@6f77e6org.highwire.dtl.DTLVardef@289e49_HPS_FORMAT_FIGEXP M_FIG C_FIG Key FindingsO_LILeishmania stops vesicular entry of inflammatory miR-122 in infected cells by causing mitochondrial depolarization C_LIO_LILeishmania secrets miR-146a from infected cells to stop miR-122 production in neighbouring hepatocytes C_LIO_LImiR-146a containing vesicles secreted by infected cells stops inflammatory response in recipient naive macrophage C_LIO_LImiR-146 targets RNA binding protein HuR to stop inflammatory cytokine production C_LI

immunology↗

bioRxiv2021

Age-related Immune-Stromal Networks Inhibit Response to Regenerative Immunotherapies

Aging is associated with immunological changes that compromise response to infections and vaccines, exacerbate inflammatory diseases and could potentially mitigate tissue repair. Even so, age-related changes to the immune response to tissue damage and regenerative medicine therapies remain unknown. Here, we characterized how aging induces senescence and immunological changes that inhibit tissue repair and therapeutic response to a clinical regenerative biological scaffold derived from extracellular matrix. Tissue signatures of inflammation and interleukin (IL)-17 signaling increased with injury and treatment in aged animals, and computational analysis uncovered age-associated senescent-T cell communication that promotes type 3 immunity in T cells. Local inhibition of type 3 immune activation using IL17-neutralizing antibodies improved healing and restored therapeutic response to the regenerative biomaterial, promoting muscle repair in older animals. These results provide insights into tissue immune dysregulation that occurs with aging that can be targeted to rejuvenate repair.

bioengineering↗

bioRxiv2021

Ergonomic risk factors and Work-related Musculoskeletal Disorders among Fireworks workers in West Bengal, India: A cross-sectional study

ObjectiveFireworks industries are very old, unorganized cottage industries in West Bengal mainly confined in South 24 Parganas. The present investigation was intended to investigate the prevalence of work-related musculoskeletal disorders among the workers and to identify the causative factors behind it. MethodIn this present study, 152 male fireworks workers from different age groups and 100 control subjects were investigated. Modified Nordic questionnaire were used to identify the region wise disorders. Hand Grip strength of both groups were also estimated. ResultAmong the fireworks workers posture related musculoskeletal disorders were severely observed in the lower back which was aggravated with the advancement of age and working experience. Pain and stiffness were also reported in neck, upper back, wrist, elbow, knee and ankle. Lower back rest with support at lumber region was strongly recommended. ConclusionAfter analysis of ergonomic factors and results, it can be concluded that the fire workers who are working with awkward postures have high risk of developing WMSDs specially affecting the upper limbs and both upper and lower back.

physiology↗

bioRxiv2021

Morph-specific patterns of sex organ positions in species with style length polymorphism

In style length polymorphism, morph-specific sequence of encountering male and female sex organs within a flower by pollinators can cause differences in the need to avoid self-pollination and encourage inter-morph pollination. We asked if this difference can lead to disparity in stigma-anther separation (herkogamy) between morphs and spatial match between sex organs of complementary morphs (reciprocity). Further, we tested if herkogamy, and hence the level of selfing, is fairly constant among individuals of a population. Additionally, we examined the relationship between herkogamy and reciprocity among individuals of a population to understand functional interactions between these two morphological traits. Using data on sex organ heights for >200 heterostylous species from the literature, we observed that the short-styled morph had higher herkogamy as compared to the long-styled morph indicating a higher need to avoid selfing. Reciprocity did not show a consistent difference between the upper and lower sex organs implying a strong influence of local ecological factors. In most populations, allometric relationships suggested that herkogamy and hence the level of selfing remains constant. Finally, we observed that herkogamy and reciprocity can be related among individuals of a population, sometimes indicating a potential trade-off between avoidance of self-pollination and facilitation of inter-morph pollination.

ecology↗

bioRxiv2021

Quantitative estimates of deciduousness in woody species from a seasonally dry tropical forest are related to leaf functional traits and the timing of leaf flush

Leaf phenology based classification of woody species into discrete evergreen and deciduous categories is widely used in ecology, but these categories hide important variation in leaf phenological behaviour. Few studies have examined the continuous nature of deciduousness and our understanding of variation in quantitative estimates of leaf shedding behaviour and the causes and consequences of this is limited. In this study we monitored leaf phenology in 75 woody species from a seasonally dry tropical forest to quantify three quantitative measures of deciduousness, namely: maximum canopy loss, duration of deciduousness, and average canopy loss. Based on proposed drought tolerance and drought avoidance strategies of evergreen and deciduous species, respectively, we tested whether the quantitative measures of deciduousness were related to leaf functional traits. Additionally, to understand the functional consequences of variation in deciduousness we examined relationships with the timing of leaf flushing and senescing. We found wide and continuous variation in quantitative measures of deciduousness in these coexisting species. Variation in deciduousness was related to leaf function traits, and the timing of leaf flushing. Along a continuous axis ranging from evergreen to deciduous species, increasing deciduousness was associated with more acquisitive leaf functional traits, with lower leaf mass per area and leaf dry matter content, and greater leaf nitrogen content. These results indicate that the continuous nature of deciduousness is an important component of resource acquisition strategies in woody species from seasonally dry forests.

ecology↗

bioRxiv2021

Mapping the Promiscuous Binding Interface of HOX-A11 with KIX by Experimentally Guided in-silico docking

Transcription factors (TFs) regulate levels of transcription through a complex array of protein-protein interactions, thereby controlling key physiological processes such as development, stress response and cell growth. The transcription factor HOXA11 contains an intrinsically disordered regions (IDR) through which it interacts with CREB binding protein (CBP) and regulates endometrial development and function in eutherian mammals. The interaction between the IDR of HOXA11 and CBP was analyzed using computational docking guided by experimental constraints. HOXA11 IDR interacts with the KIX domain of CBP at two discrete sites - MLL and cMyb, mediated by sticky hydrophobic grooves on the surface of KIX. A five residue motif FDQFF on HOXA11 can interact both at cMyb and MLL site of KIX resulting in a promiscuous binding. Author SummaryWe demonstrate how the intrinsically disordered region (IDR) of transcription factor HOXA11 interacts at two distinct sites of the transcription coactivator CREB binding protein (CBP). By combining computational docking with limited experimental data we construct models of the complex of the KIX domain within CBP and a short helical segment within the IDR of HOXA11. The interaction between HOXA11 and CBP is believed to trigger the downstream expression of genes important in embryonic development.

biophysics↗

bioRxiv2020

Herkogamy but not reciprocity is a better predictor of legitimate pollen transfer and fruit set in Jasminum malabaricum, a self-compatible species with stigma height dimorphism.

Premise Reciprocity and herkogamy, morphological traits that define style length polymorphisms, are thought to be critical in determining legitimate inter-morph pollen transfer in plants with style length polymorphism. However, the consequences of individual-level variation in these traits for pollen transfer and reproductive success have rarely been examined, and the relationship between these two fundamental traits remains unexplored.Methods We quantified individual-level estimates of herkogamy and reciprocity and tested the assumption that higher herkogamy and reciprocity result in higher legitimate pollen transfer and reproductive success in natural populations of Jasminum malabaricum, a species that exhibits stigma-height dimorphism. Additionally, we examined the relationship between herkogamy and reciprocity to understand potential consequences for avoiding self-pollination and encouraging legitimate pollen deposition.Results Surprisingly, reciprocity was not related to pollen load, legitimate pollen fraction or reproductive success. In contrast, herkogamy was positively associated with legitimate pollen fraction and fruit set in the long-styled morph. Interestingly, we observed a negative relationship between herkogamy and reciprocity in the long-styled morph.Conclusions Herkogamy was more important than reciprocity in increasing legitimate pollen transfer and reproductive success in J. malabaricum. Herkogamy might be particularly important in stabilising species with stigma-height dimorphism and other such polymorphic intermediates with low reciprocity, and this may allow the evolution of reciprocal arrangement of sex organs at a later stage in the pathway towards distyly. The negative relationship between herkogamy and reciprocity suggests a trade-off between avoidance of self-pollen deposition and promotion of legitimate pollen deposition.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗

bioRxiv2020

Molecular modelling predicts SARS-CoV-2 ORF8 protein and human complement Factor 1 catalytic domain sharing common binding site on complement C3b

Pathogens are often known to use host factor mimicry to take evolutionary advantage. As the function of the non-structural ORF8 protein of SARS-CoV-2 in the context of host-pathogen relationship is still obscure, we investigated its role in host factor mimicry using computational protein modelling techniques. Modest sequence similarity of ORF8 of SARS-CoV-2 with the substrate binding site within the C-terminus serine-protease catalytic domain of human complement factor 1 (F1; PDB ID: 2XRC), prompted us to verify their resemblance at the structural level. The modelled ORF8 protein was found to superimpose on the F1 fragment. Further, protein-protein interaction simulation confirmed ORF8 binding to C3b, an endogenous substrate of F1, via F1-interacting region on C3b. Docking results suggest ORF8 to occupy the binding groove adjacent to the conserved "arginine-serine" (RS) F1-mediated cleavage sites on C3b. Comparative H-bond interaction dynamics indicated ORF8/C3b binding to be of higher affinity than the F1/C3b interaction. Hence, ORF8 is predicted to inhibit C3b proteolysis by competing with F1 for C3b binding using molecular mimicry with a possibility of triggering unregulated complement activation. This could offer a mechanistic premise for the unrestrained complement activation observed in large number of SARS-CoV-2 infected patients.

bioinformatics↗

bioRxiv2020

Increased variation in sex organ positions can increase reciprocity and pollination success in heterostylous plant populations

AimsThe reciprocal position of sexual organs in complementary floral morphs is central to our understanding of heterostyly. Reciprocity indices are used to quantify the spatial match between complementary sex organs, but previous indices fail to appropriately account for variation in sex organ positions among individuals in a population. The objective of this study was to examine how reciprocity and consequently reproductive success change with an increase in intra-population variation in sex organ heights. To this end, we formulated a reciprocity index that incorporates variation in sex organ positions among individuals in a population and asked if estimates of reciprocity can predict reproductive success in naturally occurring heterostylous populations. MethodsWe developed a reciprocity index that assumed pollen transfer success equalled one for a perfectly matched stigma-anther pair, and decreased to zero with increasing mismatch. Reciprocity was quantified as the average pollen transfer success for all pair-wise combinations of complementary sex organs in the population. We examined the relationship between intra-population variation and reciprocity using simulated populations that varied in the distribution of sex organ positions, and with empirical data from natural populations. We compared previously proposed indices using the simulated and natural populations, and for a subset of natural populations we tested the ability of the indices to predict reproductive success. Important FindingsIn both simulated and natural populations we observed that when differences between mean anther and stigma heights of complementary morphs are small, increasing intra-population variation in heights resulted in a monotonous decrease in reciprocity. However, when differences between mean complementary anther and stigma heights are larger, reciprocity increased, reached a peak, and then decreased with increasing variation. Previous indices failed to capture this behaviour and were largely insensitive to variation or differences in mean complementary sex organ heights. Seed set was consistently positively related to reciprocity for our index, and for two of the four previous indices. These results highlight the importance of incorporating intra-population variation in sex organ dimensions in quantifying reciprocity, and challenge the current understanding that increasing variation will always decrease reciprocity in heterostylous populations. These results may help explain why heterostylous systems exhibit, and tolerate high amounts of intra-population variation in sex organ heights.

ecology↗