Biology subjects

Sharma, P.

Publications and source records attributed to Sharma, P..

At least 199 records · Page 11Linked to original sources

bioRxiv2020

Single-cell Characterization of Acute Myeloid Leukemia and its Microenvironment Following PD-1 Blockade Based Therapy

Acute myeloid leukemia (AML) and effector cells of immune checkpoint blockade (ICB) therapy co-reside in a complex bone marrow (BM) milieu. The interplay of tumor intrinsic and microenvironment (TME) mechanisms that influences the response to ICB-based therapies in AML have not been elucidated. Here we report our analyses of single cell RNA profiling of more than 127,000 BM cells from healthy donors and relapsed/refractory (R/R) AML patients at pre/post treatment with azacitidine/nivolumab, paired with single cell T cell receptor (TCR) repertoire profiles, to uncover factors impacting response and resistance. Loss of chromosome 7/7q conferred an immunosuppressive TME and was associated with resistance to ICB-based therapy in R/R AML. Our trajectory analysis revealed a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B (GZMB) and GZMK. GZMK expression defined a BM residing memory CD8+ T cell subset with stem-like properties likely an intermediary between naive and cytotoxic lymphocytes. Responses to ICB-based therapy were primarily driven by novel and expanded T cell clonotypes. Our findings support an adaptable T cell plasticity in response to PD-1 blockade in AML. Disentangling AML cells from their complex, immune-rich microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities. SignificanceDetermining the cellular and molecular underpinnings of response and resistance to PD-1 blockade based therapy in AML can guide immune-based therapeutic strategies. Our results reveal AML intrinsic characteristics (chromosome 7/7q status and oxidative stressors) and tumor microenvironment to modulate responses to checkpoint blockers. CD8 cells exist in the bone marrow in a continuum with GZMK expression defining a memory, stem-like T cell population that could play a role in response to therapy.

cancer biology↗

bioRxiv2020

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 {micro}g/L, 1.08 mg/L, 1.77 {micro}g/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

microbiology↗

bioRxiv2020

Integrated genomic view of SARS-CoV-2 in India

India first detected SARS-CoV-2, causal agent of COVID-19 in late January-2020, imported from Wuhan, China. March-2020 onwards; importation of cases from rest of the countries followed by seeding of local transmission triggered further outbreaks in India. We used ARTIC protocol based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT from Oxford Nanopore Technology to understand introduction and local transmission. The analyses revealed multiple introductions of SARS-CoV-2 from Europe and Asia following local transmission. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, here, proposed as A4-clade based on its divergence within A-cluster. The viral haplotypes may link their persistence to geo-climatic conditions and host response. Despite the effectiveness of non-therapeutic interventions in India, multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.

microbiology↗

bioRxiv2020

Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

A cytoskeletal protein keratin 19 (K19) is highly expressed in breast cancer but its effects on breast cancer cell mechanics are unclear. Using KRT19 knockout (KO) cells and cells where K19 expression was rescued, we found that K19 is required to maintain rounded epithelial-like shape and tight cell-cell adhesion of MCF7 cells. A loss of K19 resulted in a lower level of plakoglobin and internalization of E-cadherin in early and recycling endosomes. Inhibiting internalization restored cell-cell adhesion of KRT19 KO cells, suggesting E-cadherin internalization contributes to defective adhesion. Ultimately, while K19 inhibited cell migration, it was required for cells to form colonies in suspension. Our results suggest that K19 stabilizes E-cadherin complexes at the cell membrane to maintain cell-cell adhesion which inhibits cell migration but provides growth and survival advantages for circulating tumor cells. These findings provide context-dependent roles of K19 during metastasis.

cell biology↗

bioRxiv2020

Chromosome scale reference genome of Cluster bean (Cyamopsis tetragonoloba (L.) Taub.)

Clusterbean (Cyamopsis tetragonoloba (L.) Taub.), also known as Guar is a widely cultivated dryland legume of Western India and parts of Africa. Apart from being a vegetable crop, it is also an abundant source of a natural hetero-polysaccharide called guar gum or galactomannan which is widely used in cosmetics, pharmaceuticals, food processing, shale gas drilling etc. Here, for the first time we are reporting a chromosome-scale reference genome assembly of clusterbean, from a high galactomannan containing popular guar cultivar, RGC-936, by combining sequenced reads from Illumina, 10x Chromium and Oxford Nanopore technologies. The initial assembly of 1580 scaffolds with an N50 value of 7.12 Mbp was generated. Then, the final genome assembly was obtained by anchoring these scaffolds to a high density SNP map. Finally, a genome assembly of 550.31 Mbp was obtained in 7 pseudomolecules corresponding to 7 chromosomes with a very high N50 of 78.27 Mbp. We finally predicted 34,680 protein-coding genes in the guar genome. The high-quality chromosome-scale cluster bean genome assembly will facilitate understanding of the molecular basis of galactomannan biosynthesis and aid in genomics-assisted breeding of superior cultivars.

genomics↗

bioRxiv2020

Genetic differences between extreme and composite constitution types from whole exome sequences reveal actionable variations

Personalized medicine relies on successful identification of genome-wide variations that governs inter-individual differences in phenotypes and system level outcomes. In Ayurveda, assessment of composite constitution types "Prakriti" forms the basis for risk stratification, predicting health and disease trajectories and personalized recommendations. Here, we report a novel method for identifying pleiotropic genes and variants that associate with healthy individuals of three extreme and contrasting "Prakriti" constitutions through exome sequencing and state-of-the-art computational methods. Exome Seq of three extreme Prakriti types from 108 healthy individuals 54 each from genetically homogeneous populations of North India (NI, Discovery cohort) and Western India (VADU, Replication cohort) were evaluated. Fishers Exact Test was applied between Prakriti types in both cohorts and further permutation based p-value was used for selection of exonic variants. To investigate the effect of sample size per genetic association test, we performed power analysis. Functional impact of differentiating genes and variations were inferred using diverse resources -Toppfun, GTEx, GWAS, PheWAS, UK Biobank and mouse knockdown/knockout phenotype (MGI). We also applied supervised machine learning approach to evaluate the association of exonic variants with multisystem phenotypes of Prakriti. Our targeted investigation into exome sequencing from NI (discovery) and VADU (validation) cohorts datasets provide ~7,000 differentiating SNPs. Closer inspection further identified a subset of SNPs (2407 (NI) and 2393 (VADU)), that mapped to an overlapping set of 1181 genes. This set can robustly stratify the Prakriti groups into three distinct clusters with distinct gene ontological (GO) enrichments. Functional analysis further strengthens the potential pleiotropic effects of these differentiating genes/variants and multisystem phenotypic consequences. Replicated SNPs map to some very prominent genes like FIG4, EDNRA, ANKLE1, BCKDHA, ATP5SL, EXOCS5, IFIT5, ZNF502, PNPLA3 and IL6R. Lastly, multivariate analysis using random forest uncovered rs7244213 within urea transporter SLC14A2, that associate with an ensemble of features linked to distinct constitutions. Our results reinforce the concept of integration of Prakriti based deep phenotypes for risk stratification of healthy individuals and provides markers for early actionable interventions.

genomics↗

bioRxiv2020

Developing a Coarse-Grained Model for Bacterial Cell Walls and Evaluating Mechanical Properties and Free Energy Barriers

The bacterial cell envelope of Gram-negative bacteria is a complex biological barrier with multiple layers consisting of the inner membrane, periplasm of peptidoglycan and the outer membrane with lipopolysaccharides (LPS). With rising antimicrobial resistance there is increasing interest in understanding interactions of small molecules with the cell membrane to aid in the development of novel drug molecules. Hence suitable representations of the bacterial membrane are required to carry out meaningful molecular dynamics simulations. Given the complexity of the cell envelope, fully atomistic descriptions of the cell membrane with explicit solvent are computationally prohibitive, allowing limited sampling with small system sizes. However coarse-grained (CG) models such as MARTINI allow one to study phenomena at physiologically relevant length and time scales. Although MARTINI models for lipids and the LPS are available in literature, a suitable CG model of peptidoglycan is lacking. In this manuscript we develop a CG model of the peptidoglycan network within the MARTINI framework using an all-atom model developed by Gumbart et al. 1. The model is parametrized to reproduce the structural properties of the glycan strands, such as the end-to-end distance, equilibrium angle between adjacent peptides along the strands and area per disaccharide. Mechanical properties such as the area compressibility and the bending modulus are accurately reproduced. While developing novel antibiotics it is important to assess barrier properties of the peptidogylcan network. We evaluate and compare the free energy of insertion for a thymol molecule using umbrella sampling on both the MARTINI and all-atom peptidoglycan models. The insertion free energy was found to be less than kBT for both the MARTINI and all-atom models. Additional restraint free simulations reveal rapid translocation of thymol across peptidogylcan. We expect that the proposed MARTINI model for peptidoglycan will be useful in understanding phenomena associated with bacterial cell walls at larger length and time scales, thereby overcoming the current limitations of all-atom models.

biophysics↗

bioRxiv2020

A Comprehensive Survey on the Nature of Ring:Ring Nucleobase Stacking Interactions in RNA: Occurrence, Structural Variability and Classification of the Associated Contacts

The astonishing diversity in folding patterns of RNA 3D structures is crafted by myriads of noncovalent contacts, of which base pairing and stacking are the most prominent. Although the classification scheme proposed by Leontis and Westhof (RNA (2001), 7, 499) has been widely accepted for the annotation of RNA base pairs, the absence of an unambiguous classification system for base stacks appears to be a roadblock for exploring the stacking diversity in RNA. Here we provide an unambiguous and structurally-intuitive scheme for a geometry cum topology based classification of base stacking, where a stack is essentially classified in terms of the topology of the interacting nucleobase faces and the geometry described by the relative orientation of the glycosidic bonds. For heterodimeric stacks, this generates eight basic stacking geometric families, whereas for homodimeric stacks, this generates six of those. Further annotation in terms of the identity of the bases and the region of involvement of purines (5-membered, 6-membered or both rings), leads to the enumeration of 384 distinct RNA base stacks. Based on our classification scheme, we also present an algorithm for automated identification of stacks in RNA crystal structures. Overall, the work described here is expected to greatly facilitate structure-based RNA research.

bioinformatics↗

bioRxiv2020

The WD and linker domains of ATG16L1 required for non canonical autophagy limit lethal influenza A virus infection at epithelial surfaces.

Respiratory viruses such as influenza A virus (IAV) and SARS-CoV-2 (Covid-19) cause pandemic infections where cytokine storm syndrome, lung inflammation and pneumonia lead to high mortality. Given the high social and economic cost of these viruses, there is an urgent need for a comprehensive understanding of how the airways defend against virus infection. Viruses entering cells by endocytosis are killed when delivered to lysosomes for degradation. Lysosome delivery is facilitated by non-canonical autophagy pathways that conjugate LC3 to endo-lysosome compartments to enhance lysosome fusion. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that non-canonical autophagy protects mice from lethal IAV infection of the airways. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity murine-adapted IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV infection was controlled within epithelial barriers where non-canonical autophagy slowed fusion of IAV with endosomes and reduced activation of interferon signalling. This was consistent with conditional mouse models and ex vivo analysis showing that protection against IAV infection of lung was independent of phagocytes and other leukocytes. This establishes non-canonical autophagy pathways in airway epithelial cells as a novel innate defence mechanism that can restrict IAV infection and lethal inflammation at respiratory surfaces.

microbiology↗

bioRxiv2019

Human follicular helper T cell promoter connectomes reveal novel genes and regulatory elements at SLE GWAS loci

Systemic lupus erythematosus (SLE) is a complex inflammatory disease mediated by autoreactive antibodies that damages multiple tissues in children and adults. Genome-wide association studies (GWAS) have statistically implicated hundreds of loci in the susceptibility to human disease, including SLE, but the majority have failed to identify the causal variants or the effector genes. As a physicochemical approach to detecting functional variants and connecting them to target genes, we generated comprehensive, high-resolution maps of SLE variant accessibility and gene connectivity in the context of the three-dimensional chromosomal architecture of human tonsillar follicular helper T cells (TFH), a cell type required for the production of anti-nuclear antibodies characteristic of SLE. These spatial epigenomic maps identified a shortlist of over 400 potentially functional variants across 48 GWAS-implicated SLE loci. Twenty percent of these variants were located in open promoters of highly-expressed TFH genes, while 80% reside in non-promoter genomic regions that are connected in 3D to genes that likewise tend to be highly expressed in TFH. Importantly, we find that 90% of SLE-associated variants exhibit spatial proximity to genes that are not nearby in the 1D sequence of the genome, and over 60% of variants skip the nearest gene to physically interact only with the promoters of distant genes. Gene ontology confirmed that genes in spatial proximity to SLE variants reside in highly SLE-relevant networks, including accessible variants that loop 200-1000 kb to interact with the promoters of the canonical TFH genes BCL6 and CXCR5. CRISPR-Cas9 genome editing confirmed that these variants reside in novel, distal regulatory elements required for normal BCL6 and CXCR5 expression by T cells. Furthermore, SLE-associated SNP-promoter interactomes implicated a set of novel genes with no known role in TFH or SLE disease biology, including the homeobox-interacting protein kinase HIPK1 and the Ste kinase homolog MINK1. Targeting these kinases in primary human TFH cells inhibited production of IL-21, a requisite cytokine for production of class-switched antibodies by B cells. This 3D-variant-to-gene mapping approach gives mechanistic insight into the SLE-associated regulatory architecture of the human genome.

genomics↗

bioRxiv2019

Strain-level identification of bacterial tomato pathogens directly from metagenomic sequences

Routine strain-level identification of plant pathogens directly from symptomatic tissue could significantly improve plant disease control and prevention. Here we tested the Oxford Nanopore Technologies (ONT) MinION sequencer for metagenomic sequencing of tomato plants either artificially inoculated with a known strain of the bacterial speck pathogen Pseudomonas syringae pv. tomato (Pto), or collected in the field and showing bacterial spot symptoms caused by either one of four Xanthomonas species. After species-level identification using ONTs WIMP software and the third party tools Sourmash and MetaMaps, we used Sourmash and MetaMaps with a custom database of representative genomes of bacterial tomato pathogens to attempt strain-level identification. In parallel, each metagenome was assembled and the longest contigs were used as query with the genome-based microbial identification Web service LINbase. Both the read-based and assembly-based approaches correctly identified Pto strain T1 in the artificially inoculated samples. The pathogen strain in most field samples was identified as a member of Xanthomonas perforans group 2. This result was confirmed by whole genome sequencing of colonies isolated from one of the samples. Although in our case, metagenome-based pathogen identification at the strain-level was achieved, caution still needs to be exerted when interpreting strain-level results because of the challenges inherent to assigning reads to specific strains and the error rate of nanopore sequencing.

microbiology↗

bioRxiv2019

Genetic changes of P. vivax tempers host tissue-specific responses in Anopheles stephensi

In our preceding study (Sharma et al., 2019; BioRxiv) we showed that in the gut lumen Plasmodium vivax follows a unique strategy of immuno-suppression by disabling gut flora proliferation. Here, we further demonstrate that post gut invasion, a shrewd molecular relationship with individual tissues such as midgut, hemocyte, salivary glands, and strategic changes in the genetic makeup of P. vivax favors its survival in the mosquito host. A transient suppression of metabolic machinery by early oocysts, and increased immunity against late oocysts suggested a unique mechanism of gut homeostasis restoration and Plasmodium population regulation. Though a hyper immune response of hemocyte was a key to remove free circulating sporozoites, but a strong suppression of salivary metabolic activities, may favor successful survival of invaded sporozoites. Finally, genetic alteration of P. vivax ensures evasion of mosquito responses. Conclusively, our system-wide RNAseq analysis provides first genetic evidences of direct mosquito-Plasmodium interaction and establishes a functional correlation.\n\nAuthor SummaryMalaria transmission dynamics is heavily influenced by mosquito -parasite interaction. When passing through tissue specific barriers, Plasmodium have to compromise by losing its own population, but genetic relation is unknown. To win the developmental race Plasmodium need to overcome two important immuno-physiological barriers. First one accounts an indirect 24-30hr long pre-invasive gut-microbe-parasite interaction in the gut lumen. And second one follows a direct post gut invasive 14-18 days interaction with midgut, hemocyte and salivary glands. During pre-invasive phase of interaction, we showed Plasmodium vivax follows immuno-suppression strategy by restricting microbial growth in the gut lumen. Here, we demonstrate that switch of parasite from one stage to another stage within mosquito vector is accompanied by genetic changes of parasite. Our data suggests genetic makeup change enables the parasite to manipulate the metabolism of mosquito tissues. This strategy not only clear off multifaceted mosquitos tissue specific immune responses, but also favors Plasmodium own survival and transmission. Comprehending this tissue specific interaction between host and parasite at molecular level could provide new tool to intervene the plasmodium life cycle within vector.

genomics↗

bioRxiv2019

Microbiome-Gut-Brain-Axis communication regulates metabolic switch in the mosquito Anopheles culicifacies

Periodic ingestion of a protein-rich blood meal by adult female mosquitoes causes a drastic metabolic change in their innate physiological status, which is referred to as metabolic switch. Although the down-regulation of olfactory factors is key to restrain host-attraction, how the gut metabolic switch modulates brain functions, and resilience physiological homeostasis remains unexplored. Here, we demonstrate that the protein-rich diet induces mitochondrial function and energy metabolism, possibly shifting the brains engagement to manage organismal homeostasis. A dynamic expression pattern of neuro-signaling and neuro-modulatory genes in both the brain and gut indicates an optimal brain-distant organ communication. Even after decapitation, significant modulation of the neuro-modulator receptor genes as well as quantitative estimation of neurotransmitters (NTs), together confer the guts ability to serve as a second brain. Finally, data on comparative metagenomic analysis and altered NTs dynamics of naive and aseptic mosquitoes provide the initial evidence that gut-endosymbionts are key modulators for the synthesis of major neuroactive molecules. Conclusively, our data establish a new conceptual understanding of microbiome-gut-brain-axis communication in mosquitoes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/774430v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@4fed8corg.highwire.dtl.DTLVardef@17494f3org.highwire.dtl.DTLVardef@df1cd7org.highwire.dtl.DTLVardef@1de7923_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG HighlightsO_LIHighly proteinaceous blood meal uptake causes gut metabolic switch activity in mosquitoes. C_LIO_LIGuts calling shifts the brains administrative function from external communication to inter-organ management. C_LIO_LI Gut, as a Second brain plays a crucial role in the maintenance of physiological homeostasis. C_LIO_LIMetabolic switch and proliferation of symbiotic bacteria establish microbiome-gut-brain axis communication in mosquitoes. C_LI

animal behavior and cognition↗

bioRxiv2019

Altered gut microbiota and immunity defines Plasmodium vivax survival in Anopheles stephensi

Blood feeding-enriched gut-microbiota boosts mosquitoes anti-Plasmodium immunity. Here, we ask how Plasmodium vivax alters microbiota, anti-Plasmodial immunity and impact tripartite Plasmodium-mosquito-microbiota interactions in the gut lumen. Using a metagenomics analysis, we predominantly detect Elizabethkingia meningitis and Pseudomonas sps. in naive mosquitoes. Naive blood fed gut shows a heightened presence of Elizabethkingia, Pseudomonas and Serratia. A parallel RNAseq analysis of blood-fed midguts identify Elizabethkingia-transcripts, which may have role in iron metabolism. Post, a Plasmodium vivax infected blood-meal, however, we do not detect bacterial until circa 36 hours. Intriguingly, transcriptional expression of a selected array of antimicrobial arsenal cecropins 1-2, defensin-1 and gambicin remains low during the first 36 hours-a time frame when ookinietes/early oocysts invade gut. We conclude during the preinvasive phase, Plasmodium vivax outcompetes midgut-microbiota. Suppression of important immune factors, likely due to altered microbiota, may enhance Plasmodium vivax survival. Additional finding of a novel Wolbachia association warrants further research to design paratransgenesis tools for malaria control.\n\nAuthor SummarySuccessful malaria transmission relies on the competitive interactions of Plasmodium and mosquitos tissue specific immune potential. Within 24hrs of blood meal gut-microbiota grows exponentially and lead to robust enhancement of mosquito immune response, which is detrimental to parasite survival and development. But the mechanism how Plasmodium manages to evade this pre-invasive immune barrier is not well known. We investigated the influence of tripartite gut-microbiome-parasite interaction on human malaria parasite Plasmodium vivax in its natural/native vector Anopheles stephensi. Surprisingly we found that infectious blood meal lead to dramatic suppression in gut-bacteria population, a plausible strategy of P. vivax ookinetes to avoid immune responses. Our study suggests that for its own survival Plasmodium vivax causes early suppression of bacterial population, possibly by scavenging Fe from the blood meal which is indispensable for bacterial growth. Disruption and manipulation of this gut-microbe-interaction may help to design new paratransgenesis molecular tool for malaria control.

genomics↗

bioRxiv2019

The translation inhibitor cycloheximide affects ribosome profiling data in a species-specific manner

Ribosome profiling provides genome-wide snapshots of translation dynamics by determining ribosomal positions at sub-codon resolution. To maintain this positional information, the translation inhibitor cycloheximide (CHX) has been widely used to arrest translating ribosomes prior to library preparation. Several studies have reported CHX-induced biases in yeast data casting uncertainty about its continued use and questioning the accuracy of many ribosome profiling studies. However, the presence of these biases has not been investigated comprehensively in organisms other than Saccharomyces cerevisiae. Here, we use a highly standardized and optimized protocol to compare different CHX-treatment conditions in yeast and human cells. Our data suggest that unlike in S. cerevisiae, translating ribosomes in human cells are not susceptible to conformational restrictions by CHX. Furthermore, CHX-induced codon-specific effects on ribosome occupancy are not detectable in human cells nor in other model organisms including Schizosaccharomyces pombe and Candida albicans. In fact, we find that even in S. cerevisiae most biases can be avoided by omitting CHX pre-treatment, indicating that other parameters of library generation contribute to differences between ribosome profiling experiments. Together our findings provide a framework for researchers who plan their own ribosome profiling experiments or who analyze published datasets to draw judicious conclusions.

molecular biology↗

bioRxiv2019

Pairing interactions between nucleobases and ligands in aptamer:ligand complexes of riboswitches: Crystal structure analysis, classification, optimal structures and accurate interaction energies

In the present work, sixty-seven crystal structures of the aptamer domains of RNA riboswitches, are chosen for analysis of the structure and strength of hydrogen bonding (pairing) interactions between nucleobases constituting the aptamer binding pockets and the bound ligands. A total of eighty unique base:ligand hydrogen-bonded pairs containing at least two hydrogen bonds were identified through visual inspection. Classification of these contacts in terms of the interacting edge of the aptamer nucleobase revealed that interactions involving the Watson-Crick edge are the most common, followed by the sugar edge of purines and the Hoogsteen edge of uracil. Alternatively, classification in terms of the chemical constitution of the ligand yields five unique classes of base:ligand pairs: base:base, base:amino acid, base:sugar, base:phosphate and base:other. Further, quantum mechanical (QM) geometry optimizations revealed that sixty seven out of eighty pairs exhibit stable geometries and optimal deviations from their macromolecular crystal occurrences. This indicates that these contacts are well-defined RNA aptamer:ligand interaction motifs. QM calculated interaction energies of base:ligand pairs reveal rich hydrogen bonding landscape, ranging from weak interactions (base:other, -3 kcal/mol) to strong (base:phosphate, -48 kcal/mol) contacts. The analysis was further extended to study the biological importance of base:ligand interactions in the binding pocket of the tetrahydrofolate riboswitch and thiamine pyrophosphate riboswitch. Overall, our study helps in understanding the structural and energetic features of base:ligand pairs in riboswitches, which could aid in developing meaningful hypotheses in context of RNA:ligand recognition. This can, in turn contribute towards current efforts to develop antimicrobials that target RNAs.

biophysics↗

bioRxiv2019

The TLR5 agonist flagellin modifies phenotypical and enhances functional activation of lung mucosal antigen presenting cells in neonatal mice

Intranasal mucosal vaccines are of interest in that they may induce protective mucosal immune responses. Activation of lung antigen presenting cells (APCs), a phenotyoically and functionally heterogeneous cell population located at distinct mucosal sites, may be key to the immunogenicity of such vaccines. Characterizing responsiveness of newborn lung APCs to adjuvants may inform design of efficacious intranasal vaccines for early life, when most infections occur. We characterized APCs from neonatal (<7 days of life) and adult (6-8 weeks of age) mice. Neonatal mice displayed a relatively high abundance of alveolar macrophages (AMs), with lower percentages of plasmacytoid dendritic cells (pDCs), CD103+ (cDC1) and CD11b+ (cDC2) DCs. Furthermore, neonatal CD103+ and CD11b+ DC subsets demonstrated an inverse expression of maturation markers as compared to adult mice. Upon stimulation of lung APC subsets with a panel of pattern recognition receptor (PRR), including TLR and STING, agonists, CD11c+ enriched cells from neonatal and adult mice lungs demonstrated distinct maturation profiles. The TLR5 ligand, flagellin, was most effective at activating neonatal lung APCs, inducing significantly higher expression of maturation markers on CD103+ (cDC1) and CD11b+ (cDC2) subsets. Intranasal administration of flagellin induced a distinct migration of CD103+ and CD11b+ DC subsets to the mediastinal lymph nodes (mLNs) of neonatal mice. Overall, these findings highlight age specific differences in the maturation and responsiveness of lung APC subsets to different PRR agonists. The unique efficacy of flagellin in enhancing lung APC activity suggests that it may serve as an effective adjuvant for early life mucosal vaccines.

immunology↗

bioRxiv2019

White Matter Hyperintensity Quantification in Large-Scale Clinical Acute Ischemic Stroke Cohorts - The MRI-GENIE Study

White matter hyperintensity (WMH) burden is a critically important cerebrovascular phenotype linked to prediction of diagnosis and prognosis of diseases, such as acute ischemic stroke (AIS). However, current approaches to its quantification on clinical MRI often rely on time intensive manual delineation of the disease on T2 fluid attenuated inverse recovery (FLAIR), which hinders high-throughput analyses such as genetic discovery. In this work, we present a fully automated pipeline for quantification of WMH in clinical large-scale studies of AIS. The pipeline incorporates automated brain extraction, intensity normalization and WMH segmentation using spatial priors. We first propose a brain extraction algorithm based on a fully convolutional deep learning architecture, specifically designed for clinical FLAIR images. We demonstrate that our method for brain extraction outperforms two commonly used and publicly available methods on clinical quality images in a set of 144 subject scans across 12 acquisition centers, based on dice coefficient (median 0.95; inter-quartile range 0.94-0.95) and Pearson correlation of total brain volume (r=0.90). Subsequently, we apply it to the large-scale clinical multi-site MRI-GENIE study (N=2783) and identify a decrease in total brain volume of -2.4cc/year. Additionally, we show that the resulting total brain volumes can successfully be used for quality control of image preprocessing. Finally, we obtain WMH volumes by building on an existing automatic WMH segmentation algorithm that delineates and distinguishes between different cerebrovascular pathologies. The learning method mimics expert knowledge of the spatial distribution of the WMH burden using a convolutional auto-encoder. This enables successful computation of WMH volumes of 2,533 clinical AIS patients. We utilize these results to demonstrate the increase of WMH burden with age (0.950 cc/year) and show that single site estimates can be biased by the number of subjects recruited.

neuroscience↗