Biology subjects

Ramakrishnan, S.

Publications and source records attributed to Ramakrishnan, S..

44 records · Page 3Linked to original sources

bioRxiv2020

Lung Bronchial Epithelial Cells are HIV Targets for Proviral Genomic Integration

In the era of highly active anti-retroviral therapy (HAART), obstructive lung diseases (OLDs) are common among the people living with HIV (PLWH); however, the mechanism by which HIV induces OLDs is unclear. Although human bronchial epithelial cells (HBECs) express HIV coreceptors and are critical in regulating lung immune responses, their role in transmitting HIV remains unclear. Herein, we present evidence that HIV-1 infects normal HBECs and the viral DNA is integrated in the genome to establish the viral latency. To prove that HIV productively infects HBECs, we demonstrate: (a) along with CXCR4, HBECs express the HIV-receptor CD4, and are induced to express CCR5 by IL-13 treatment; (b) following infection with HIV, HBECs produce HIV-p24 and contain the latent HIV provirus, which is activated by endotoxin and/or vorinostat; (c) DNA from HIV-1 infected HBECs contains the HIV-specific gag and nef genes, along with Alu sequences, confirming the integration of HIV in the host DNA; (d) the lung epithelial cells of HIV-infected subjects and SHIV-infected cynomolgus macaques are positive for HIV-specific transcripts. Thus, these studies suggest that HIV establishes latency in lung epithelial cells, making them potential HIV reservoirs. The long-living lung epithelial cells, activated by commonly encountered lung infections, might represent an ideal HIV target/reservoir, contributing to OLDs and other HIV-associated lung comorbidities.

physiology↗

bioRxiv2020

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor

Introductory paragraphGut colonization by the pathogen Klebsiella pneumoniae (Kp) is consistently associated with subsequent Kp disease1-5, and patients are predominantly infected with their colonizing strain1,2. However, colonizing strains likely vary in their potential to cause infection. We previously identified the plasmid-encoded tellurium resistance (ter) operon as highly associated with infection when compared to asymptomatic colonization in hospitalized patients1. The ter operon bestows resistance to the toxic compound tellurite oxide (TeO3-2), but this is unlikely to be its physiological function, as tellurium and TeO3-2 are exceedingly rare. Here we show that terC is necessary and terZABCDEF is sufficient for phenotypic TeO3-2 resistance. Next, we demonstrate that ter is encoded on a diverse group of plasmids without known plasmid-encoded virulence genes, suggesting an independent role in infection. Finally, our studies indicate that ter is a gut fitness factor, and its fitness advantage is conferred only when specific gut microbiota constituents are present. Collectively, these data reveal the Kp ter operon that is highly associated with human infection likely acts early in pathogenesis as a horizontally-transferrable fitness factor promoting robust gut colonization in the presence of the indigenous microbiota.

microbiology↗

bioRxiv2019

Independent Yet Synergistic Roles of Synaptotagmin-1 and Complexin in Calcium Regulated Neuronal Exocytosis

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved, namely the vesicular fusion machinery (SNARE proteins) and the regulatory proteins (Synaptotagmin-1 and Complexin) are well-known. Here, we use a reconstituted single-vesicle fusion assay to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act independently but synergistically to establish Ca2+-regulated fusion. Under physiologically-relevant conditions, we find that Synaptotagmin-1 oligomers bind and clamp a limited number of central SNARE complexes via the primary binding interface, to introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to independently arrest the remaining free peripheral SNAREpins to produce stably clamped vesicles. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.

neuroscience↗

bioRxiv2019

Fast and accurate reference-guided scaffolding of draft genomes

BackgroundAs the number of new genome assemblies continues to grow, there is increasing demand for methods to coalesce contigs from draft assemblies into pseudomolecules. Most current methods use genetic maps, optical maps, chromatin conformation (Hi-C), or other long-range linking data, however these data are expensive and analysis methods often fail to accurately order and orient a high percentage of assembly contigs. Other approaches utilize alignments to a reference genome for ordering and orienting, however these tools rely on slow aligners and are not robust to repetitive contigs.\n\nResultsWe present RaGOO, an open-source reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in just minutes. With the pseudomolecules constructed, RaGOO identifies structural variants, including those spanning sequencing gaps that are not reported by alternative methods. We show that RaGOO accurately orders and orients contigs into nearly complete chromosomes based on de novo assemblies of Oxford Nanopore long-read sequencing from three wild and domesticated tomato genotypes, including the widely used M82 reference cultivar. We then demonstrate the scalability and utility of RaGOO with a pan-genome analysis of 103 Arabidopsis thaliana accessions by examining the structural variants detected in the newly assembled pseudomolecules. RaGOO is available open-source with an MIT license at https://github.com/malonge/RaGOO.\n\nConclusionsWe demonstrate that with a highly contiguous assembly and a structurally accurate reference genome, reference-guided scaffolding with RaGOO outperforms error-prone reference-free methods and enable rapid pan-genome analysis.

bioinformatics↗

bioRxiv2018

Natural Secretory Immunoglobulins Enhance Norovirus Infection

Secretory immunoglobulins (SIg) are a first line of mucosal defense by the host. They are secreted into the gut lumen via the polymeric immunoglobulin receptor (pIgR) where they bind to antigen and are transported back across the FAE via M cells. Noroviruses are highly prevalent, enteric pathogens that cause significant morbidity, mortality and economic losses worldwide. Murine norovirus (MNV) exploits microfold (M) cells to cross the lymphoid follicle-associated epithelium (FAE) and infect the underlying population of immune cells. However, whether natural, innate SIg can protect against norovirus infection remains unknown. To investigate the role of natural SIg during murine norovirus pathogenesis, we used pIgR-deficient animals, which lack SIg in the intestinal lumen. Contrary to other enteric pathogens, acute MNV replication was significantly reduced in the gastrointestinal tract of pIgR-deficient animals compared to controls, despite increased numbers of dendritic cells, macrophages, and B cells in the Peyers patch, established MNV target cell types. Also, natural SIg did not alter MNV FAE binding or FAE crossing into the lymphoid follicle. Instead, further analysis revealed enhanced baseline levels of the antiviral molecules interferon gamma (IFN{gamma}) and inducible nitric oxide synthase (iNOS) in the small intestine of naive pIgR-deficient animals compared to controls. Removing the microbiota equalized IFN{gamma} and iNOS transcript levels as well as MNV viral loads in germ-free pIgR KO mice compared to germ-free controls. These data are consistent with a model whereby SIg sensing reduces pro-inflammatory, antiviral molecules, which facilitates intestinal homeostasis but thereby promotes MNV infection. In conclusion, these findings demonstrate that natural SIg are not protective during norovirus infection in mice and represent another example of indirect modulation of enteric virus pathogenesis by the microbiota.

microbiology↗

bioRxiv2017

No major flaws in "Identification of individuals by trait prediction using whole-genome sequencing data"

In a recently published PNAS article, we studied the identifiability of genomic samples using machine learning methods [Lippert et al., 2017]. In a response, Erlich [2017] argued that our work contained major flaws. The main technical critique of Erlich [2017] builds on a simulation experiment that shows that our proposed algorithm, which uses only a genomic sample for identification, performed no better than a strategy that uses demographic variables. Below, we show why this comparison is misleading and provide a detailed discussion of the key critical points in our analyses that have been brought up in Erlich [2017] and in the media. Further, not only faces may be derived from DNA, but a wide range of phenotypes and demographic variables. In this light, the main contribution of Lippert et al. [2017] is an algorithm that identifies genomes of individuals by combining multiple DNA-based predictive models for a myriad of traits.

genomics↗

bioRxiv2017

Reference Quality Assembly of the 3.5 Gb genome of Capsicum annuum from a Single Linked-Read Library

BackgroundLinked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Capsicum annuum) genome with a single Linked-Read library. Plant genomes, including pepper, are characterized by long, highly similar repetitive sequences. Accordingly, significant effort is used to ensure the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus. With a phased assembly approach, we targeted a heterozygous F1 derived from a wide cross to assess the ability to derive both haplotypes for a pungency gene characterized by a large insertion/deletion.\n\nResultsThe Supernova software generated a highly ordered, more contiguous sequence assembly than all currently available C. annuum reference genomes. Eighty-four percent of the final assembly was anchored and oriented using four de novo linkage maps. A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly. The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5 kb insertion/deletion haplotypes of the PUN1 locus in the F1 sample that represents pungent and non-pungent peppers.\n\nConclusionsThe most contiguous pepper genome assembly to date has been generated through this work which demonstrates that Linked-Read library technology provides a rapid tool to assemble de novo complex highly repetitive heterozygous plant genomes. This technology can provide an opportunity to cost-effectively develop high-quality reference genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction.

genomics↗

bioRxiv2017

Precision Medicine Screening Using Whole Genome Sequencing And Advanced Imaging To Identify Disease Risk In Adults

BACKGROUNDProgress in science and technology have created the capabilities and alternatives to symptom-driven medical care. Reducing premature mortality associated with age-related chronic diseases, such as cancer and cardiovascular disease, is an urgent priority we address using advanced screening detection.\n\nMETHODSWe enrolled active adults for early detection of risk for age-related chronic disease associated with premature mortality. Whole genome sequencing together with: global metabolomics, 3D/4D imaging using non-contrast whole body magnetic resonance imaging and echocardiography, and 2-week cardiac monitoring were employed to detect age-related chronic diseases and risk for diseases.\n\nRESULTSWe detected previously unrecognized age-related chronic diseases requiring prompt (<30 days) medical attention in 17 (8%, 1:12) of 209 study participants, including 4 participants with early stage neoplasms (2%, 1:50). Likely mechanistic genomic findings correlating with clinical data were identified in 52 participants (25%. 1:4). More than three-quarters of participants (n=164, 78%, 3:4) had evidence of age-related chronic diseases or associated risk factors.\n\nCONCLUSIONSPrecision medicine screening using genomics with other advanced clinical data among active adults identified unsuspected disease risks for age-related chronic diseases associated with premature mortality. This technology-driven phenotype screening approach has the potential to extend healthy life among active adults through improved early detection and prevention of age-related chronic diseases. Our success provides a scalable strategy to move medical practice and discovery toward risk detection and disease modification thus achieving healthier extension of life.\n\nSIGNIFICANCE STATEMENTAdvances in science and technology have enabled scientists to analyze the human genome cost-effectively and to combine genome sequencing with noninvasive imaging technologies for alternatives to symptom-driven medical care. Using whole genome sequencing and noninvasive 3D/4D imaging technologies we screened 209 adults to detect age-related chronic diseases, such as cancer and cardiovascular disease. We found unrecognized age-related chronic diseases requiring prompt (<30 days) medical attention in 1:12 study participants, likely genomic findings correlating with clinical data in 1:4 participants, and evidence of age-related chronic diseases or associated risk factors in more than 3 of 4 participants. These results demonstrate that genome sequencing with clinical imaging data can be used for screening and early detection of diseases associated with premature mortality.

genomics↗