Biology subjects

Khan, R.

Publications and source records attributed to Khan, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

scLongTree: an accurate computational tool to infer the longitudinal tree for scDNAseq data

Longitudinal single-cell DNA sequencing (scDNA-seq) refers to single-cell data sequenced at different time points providing more knowledge of the order of mutations than scDNA-seq taken at only one time point. The technique can facilitate the inference of subclonal trees that depict the evolution of cancer cells and facilitate understanding of how cancer grows, with implications for prognosis and treatment. There is currently a scarcity of tools that can infer subclonal trees based on longitudinal scDNA-seq, and existing tools are limited in accuracy and scale. We therefore introduce scLongTree, a computational tool that can accurately infer a subclonal tree based on longitudinal scDNA-seq. ScLongTree is scalable to hundreds of mutations, and outperforms state-of-the-art tools such as LACE, SCITE, and SiCloneFit on a comprehensive simulated dataset. Tests on a real dataset, SA501, showed that scLongTree can more accurately interpret the progressive growth of the tumor than LACE, and is more robust to different numbers of mutations being used. Tests on a large AML dataset AML107, which has 4,617 cells, show that scLongTree is scalable to thousands of cells. ScLongTree is freely available on https://github.com/compbio-mallory/sc_longitudinal_infer. Key pointsO_LIWe propose scLongTree that can infer the subclonal longitudinal tree for cancer given single-cell DNA sequencing data, and thus can facilitate the study of cancer evolution given the dataset from multiple time points. C_LIO_LIMultiple simulated data show that scLongTree is more accurate than existing state-of-the-art methods such as LACE, SCITE and SiCloneFit. C_LIO_LIScLongTree has been shown to have a higher scalability than LACE and thus can be applicable to the datasets that have hundreds of mutations. C_LIO_LIThe experiment on a real SA501 shows that scLongTree is more robust to the number of mutations than LACE. It consistently generates the same longitudinal tree even under different sets of mutations. C_LIO_LIThe experiment on AML107 shows that scLongTree is scalable to thousands of cells. C_LI

bioinformatics↗

bioRxiv2023

Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample

Ancient DNA (aDNA) sequencing analysis typically involves alignment to a modern reference genome assembly from a related species. Since aDNA molecules are fragmentary, these alignments yield information about small-scale differences, but provide no information about larger features such as the chromosome structure of ancient species. We report the genome assembly of a female Late Pleistocene woolly mammoth (Mammuthus primigenius) with twenty-eight chromosome-length scaffolds, generated using mammoth skin preserved in permafrost for roughly 52,000 years. We began by creating a modified Hi-C protocol, dubbed PaleoHi-C, optimized for ancient samples, and using it to map chromatin contacts in a woolly mammoth. Next, we developed "reference-assisted 3D genome assembly," which begins with a reference genome assembly from a related species, and uses Hi-C and DNA-Seq data from a target species to split, order, orient, and correct sequences on the basis of their 3D proximity, yielding accurate chromosome-length scaffolds for the target species. By means of this reference-assisted 3D genome assembly, PaleoHi-C data reveals the 3D architecture of a woolly mammoth genome, including chromosome territories, compartments, domains, and loops. The active (A) and inactive (B) genome compartments in mammoth skin more closely resemble those observed in Asian elephant skin than the compartmentalization patterns seen in other Asian elephant tissues. Differences in compartmentalization between these skin samples reveal sequences whose transcription was potentially altered in mammoth. We observe a tetradic structure for the inactive X chromosome in mammoth, distinct from the bipartite architecture seen in human and mouse. Generating chromosome-length genome assemblies for two other elephantids (Asian and African elephant), we find that the overall karyotype, and this tetradic Xi structure, are conserved throughout the clade. These results illustrate that cell-type specific epigenetic information can be preserved in ancient samples, in the form of DNA geometry, and that it may be feasible to perform de novo genome assembly of some extinct species.

genomics↗

bioRxiv2023

Polymer biodegradation by Halanaerobium promotes reservoir souring during hydraulic fracturing.

Hydraulically fractured shale reservoirs have facilitated studies of unexplored niches in the continental deep biosphere. Members of the genus Halanaerobium are ubiquitous in these environments. Polymers like guar gum used as gelling agents in hydraulic fracturing fluids are known to be fermentable substrates, but metabolic pathways encoding these processes have not been characterized. To explore this, produced water samples from the Permian Basin were incubated at 30{degrees}C to simulate above-ground storage conditions, and at 60{degrees}C to simulate subsurface reservoir conditions. Guar metabolism coincided with Halanaerobium enrichment only at 30{degrees}C, revealing genes for polymer biodegradation through the mixed-acid fermentation pathway in different metagenome-assembled genomes (MAGs). Whereas thiosulfate reduction to sulfide is often invoked to explain the dominance of Halanaerobium in these settings, Halanaerobium genomes did not uncover genes for this metabolism. Sulfide production was observed in 60{degrees}C incubations, with corresponding enrichment of Desulfohalobium and Desulfovibrionaceae that possess complete pathways for coupling mannose and acetate oxidation to sulfate reduction. These findings outline how production of fermentation intermediates (mannose, acetate) by Halanaerobium in topside settings can result in reservoir souring when these metabolites are introduced into the subsurface through produced water re-use. ImportanceHydraulically fractured shale oil reservoirs are ideal for studying extremophiles such as thermohalophiles. During hydraulic fracturing, reservoir production water is stored in surface ponds prior to re-use. Microorganisms in these systems therefore need to withstand various environmental changes such as the swing between warm downhole oil reservoir temperatures and cooler surface conditions. This study follows this water cycle during fracking and the associated microbial metabolic potential. Of particular interest are members of the genus Halanaerobium, that have been reported to reduce thiosulfate contributing to souring of oil reservoirs. Here, we show that some Halanaerobium strains were unable to grow under oil reservoir temperatures and do not possess genes for thiosulfate reduction. Rather, it is likely that these organisms metabolize complex organics in fracking fluids at lower temperatures, thereby generating substrates that support reservoir souring by thermophilic sulfate-reducing bacteria at higher temperatures.

microbiology↗

bioRxiv2023

Heterogeneity of foam cell biogenesis across diseases

Foam cells are dysfunctional, lipid-laden macrophages associated with chronic inflammation of diverse origin. The long-standing paradigm that foam cells are cholesterol-laden derives from atherosclerosis research. We previously showed that, in tuberculosis, foam cells surprisingly accumulate triglycerides. Here, we utilized bacterial (Mycobacterium tuberculosis), fungal (Cryptococcus neoformans), and human papillary renal cell carcinoma (pRCC) models to address the need for a new explanation of foam cell biogenesis. We applied mass spectrometry-based imaging to assess the spatial distribution of storage lipids relative to foam-cell-rich areas in lesional tissues, and we characterized lipid-laden macrophages generated under corresponding in vitro conditions. The in vivo data and the in vitro findings showed that cryptococcus-infected macrophages accumulate triglycerides, while macrophages exposed to pRCC- conditioned-medium accumulated both triglycerides and cholesterol. Moreover, cryptococcus- and mycobacterium-infected macrophages accumulated triglycerides in different ways. Collectively, the data show that the molecular events underlying foam cell formation are specific to disease and microenvironment. Since foam cells are potential therapeutic targets, recognizing that their formation is disease-specific opens new biomedical research directions.

immunology↗

bioRxiv2023

DBDA matrix increases ion abundance of fatty acids and sulfatides in MALDI-TOF and mass spectrometry imaging studies

MALDI-TOF MS is a powerful tool to analyze biomolecules owing to its soft ionization nature and generally results in simple spectra of singly charged ions. Moreover, implementation of the technology in imaging mode provides a means to spatially map analytes in situ. Recently, a new matrix, DBDA (N1,N4-dibenzylidenebenzene-1,4-diamine) was reported to facilitate the ionization of free fatty acids in the negative ion mode. Building on this finding, we sought to implement DBDA for MALDI mass spectrometry imaging studies in brain tissue and successfully map oleic acid, palmitic acid, stearic acid, docosahexaenoic acid and arachidonic acid using mouse brain sections. Moreover, we hypothesized that DBDA would provide superior ionization for sulfatides, a class of sulfolipids, with multiple biological functions. Herein we also demonstrate that DBDA is ideal for MALDI mass spectrometry imaging of fatty acids and sulfatides in brain tissue sections. Additionally, we show enhanced ionization of sulfatides using DBDA compared to three different traditionally used MALDI matrices. Together these results provide new opportunities for studies to measure sulfatides by MALDI-TOF MS including in imaging modes.

biochemistry↗

bioRxiv2022

A Chromosome-length Assembly of the Black Petaltail (Tanypteryx hageni) Dragonfly

We present a chromosome-length genome assembly and annotation of the Black Petaltail dragonfly (Tanypteryx hageni). This habitat specialist diverged from its sister species over 70 million years ago, and separated from the most closely related Odonata with a reference genome 150 million years ago. Using PacBio HiFi reads and Hi-C data for scaffolding we produce one of the most high quality Odonata genomes to date. A scaffold N50 of 206.6 Mb and a BUSCO score of 96.8% indicate high contiguity and completeness. SignificanceWe provide a chromosome-length assembly of the Black Petaltail dragonfly (Tanypteryx hageni), the first genome assembly for any non-libelluloid dragonfly. The Black Petaltail diverged from its sister species over 70 million years ago. T. hageni, like its confamilials, occupies fen habitats in its nymphal stage, a life history uncommon in the vast majority of dragonflies. We hope that the availability of this assembly will facilitate research on T. hageni and other petaltail species, to better understand their ecology and support conservation efforts.

genomics↗

bioRxiv2022

Assessing the Performance of Methods for Cell Clustering from Single-cell DNA Sequencing Data

BackgroundMany cancer genomes have been known to contain more than one subclone inside one tumor, the phenomenon of which is called intra-tumor heterogeneity (ITH). Characterizing ITH is essential in designing treatment plans, prognosis as well as the study of cancer progression. Single-cell DNA sequencing (scDNAseq) has been proven effective in deciphering ITH. Cells corresponding to each subclone are supposed to carry a unique set of mutations such as single nucleotide variations (SNV). While there have been many studies on the cancer evolutionary tree reconstruction, not many have been proposed that simply characterize the subclonality without tree reconstruction. While tree reconstruction is important in the study of cancer evolutionary history, typically they are computationally expensive in terms of running time and memory consumption due to the huge search space of the tree structure. On the other hand, subclonality characterization of single cells can be converted into a cell clustering problem, the dimension of which is much smaller, and the turnaround time is much shorter. Despite the existence of a few state-of-the-art cell clustering computational tools for scDNAseq, there lacks a comprehensive and objective comparison under different settings. ResultsIn this paper, we evaluated six state-of-the-art cell clustering tools-SCG, BnpC, SCClone, RobustClone, SCITE and SBMClone-on simulated data sets given a variety of parameter settings and a real data set. We designed a simulator specifically for cell clustering, and compared these methods performances in terms of their clustering accuracy, specificity and sensitivity and running time. For SBMClone, we specifically designed an ultra-low coverage large data set to evaluate its performance in the face of an extremely high missing rate. ConclusionFrom the benchmark study, we conclude that BnpC and SCGs clustering accuracy are the highest and comparable to each other. However, BnpC is more advantageous in terms of running time when cell number is high (> 1500). It also has a higher clustering accuracy than SCG when cluster number is high (> 16). SCClones accuracy in estimating the number of clusters is the highest. RobustClone and SCITEs clustering accuracy are the lowest for all experiments. SCITE tends to over-estimate the cluster number and has a low specificity, whereas RobustClone tends to under-estimate the cluster number and has a much lower sensitivity than other methods. SBMClone produced reasonably good clustering (V-measure > 0.9) when coverage is >= 0.03 and thus is highly recommended for ultra-low coverage large scDNAseq data sets. Author summaryCancer cells evolve by gaining new mutations. Different cancer cells may gain different mutations. Thus even inside the tumor of one cancer patient, there could be multiple clones of cancer cells, each having its unique set of mutations. Characterization of the clonality of a tumor can improve cancer treatment and prognosis. Single-cell DNA sequencing, or scDNAseq, refers to the DNA sequencing technology that can sequence each cell separately. ScDNAseq has been widely used to characterize the clonality of a tumor. In this study, we benchmarked six existing computational tools that characterize the clonality of the cancer cells, which are SCG, SCClone, BnpC, RobustClone, SCITE and SBMClone. To accomplish this study, we modified our simulator and simulated eight sets of simulation data for testing the first five methods, and one set of simulation data for testing SBMClone considering that SBMClone was specifically designed for highly sparse data set. We also tested SCG, SCClone, BnpC and RobubstClone on a real data set CRC2. We recommend SCG and BnpC due to their high accuracy and low running time. In the face of highly sparse data, we highly recommend SBMClone, the only method that can deal with such type of data.

bioinformatics↗

bioRxiv2021

Colon Innervating TRPA1 Positive Nociceptors Influence Mucosal Health In Mice

IntroductionTransient receptor potential ankyrin-1 positive (TRPA1+ve) nociceptors, primarily present as peptidergic neuronal afferents in the colon are sensors of disturbance in lower gastrointestinal tract including pain induced by different pathologies. Their therapeutic role in the alleviation of chronic pain (receptor antagonism and receptor desensitization) associated with inflammatory bowel diseases (IBD) is reported. However, there is limited literature available about their role in formation and sustenance of the mucosal layer, and its interaction with host physiology as well as luminal microbial community. The aim of this study focuses on the effects of nociceptive TRPA1 channel desensitization on colonic mucus production and gut health. MethodsTRPA1+ve nociceptors were desensitized by rectal administration of capsazepine. Ileum, colon was harvested and cecum content was collected. We performed morphological/histological analysis, gut permeability alteration, gene expression changes, colon metabolite profiling, and gut microbial abundance in these animals. ResultsWe found that presence of TRPA1-positive nociceptors is required for mucus layer integrity, using an intra-rectal capsazepine-induced TRPA1 desensitization model. Desensitization of TRPA1 positive nociceptors resulted in damaged mucosal lining, resultant increase in gut permeability and altered transcriptional profile of genes for goblet cell markers, mucus regulation, immune response and tight junction proteins. The damage to mucosal lining prevented its role in enterosyne (short chain fatty acids) actions. ConclusionThese results suggest that caution may need to be exercised before employing TRPA1 desensitization as a therapeutic option to alleviate pain caused due to IBD.

pharmacology and toxicology↗

bioRxiv2021

De novo chromosome level assembly of the mule deer (Odocoileus hemionus) genome

The mule deer (Odocoileus hemionus) is an ungulate species that ranges from western Canada to central Mexico. Mule deer are an essential source of food for many predators, are relatively abundant, and commonly make broad migration movements. A clearer understanding of the mule deer genome can help facilitate knowledge of its population genetics, movements, and demographic history, aiding in conservation efforts. While mule deer are excellent candidates for population genomic studies because of their large population size, continuous distribution, and diversity of habitat, few genomic resources are currently available for this species. Here, we sequence and assemble the mule deer genome into a highly contiguous chromosome-length assembly for use in future research using long-read sequencing and Hi-C. We also provide a genome annotation and compare demographic histories of the mule deer and white-tail deer using PSMC. We expect this assembly to be a valuable resource in the continued study and conservation of mule deer.

genomics↗

bioRxiv2021

Chromosome size affects sequence divergence between species through the interplay of recombination and selection

The structure of the genome shapes the distribution of genetic diversity and sequence divergence. To investigate how the relationship between chromosome size and recombination rate affects sequence divergence between species, we combined empirical analyses and evolutionary simulations. We estimated pairwise sequence divergence among 15 species from three different Mammalian clades - Peromyscus rodents, Mus mice, and great apes - from chromosome-level genome assemblies. We found a strong significant negative correlation between chromosome size and sequence divergence in all species comparisons within the Peromyscus and great apes clades, but not the Mus clade, suggesting that the dramatic chromosomal rearrangements among Mus species may have masked the ancestral genomic landscape of divergence in many comparisons. Our evolutionary simulations showed that the main factor determining differences in divergence among chromosomes of different size is the interplay of recombination rate and selection, with greater variation in larger populations than in smaller ones. In ancestral populations, shorter chromosomes harbor greater nucleotide diversity. As ancestral populations diverge, diversity present at the onset of the split contributes to greater sequence divergence in shorter chromosomes among daughter species. The combination of empirical data and evolutionary simulations revealed that chromosomal rearrangements, demography, and divergence times may also affect the relationship between chromosome size and divergence, and deepen our understanding of the role of genome structure on the evolution of species divergence.

evolutionary biology↗

bioRxiv2020

Agricultural production and CO2 emissions causes in the developing and developed countries: New insights from quantile regression and decomposition analysis

Agriculture is the dominant economic activity of the economies. The developing and developed countries are responsible for the most greenhouse gasses emitted in the developing areas. Are there heterogeneous determinants of environmental degradation and CO2 emitters in developing and developed countries? and estimating the significance of agricultural production, renewable energy consumption, the industrial revolution, and economic growth. In this study, 22 countries environmental degradation analyze by two (per-capita and liquid) sources of CO2 emissions and using panel data from 1991 through 2016. This study adopts a panel regression (non-additive effects) and quantile regression techniques to explore the connection between agriculture and economic factors. And the extent of the CO2 emitter gap between developing and developed countries. The outcome of agriculture has a positive and significant influence on CO2 emission from liquid with a 36.75% increase in environmental degradation and a negative impact on CO2 emission in the total emissions by 19.12%. The agriculture-related activities negatively influence the environment, such as deforestation for feed cropping, burning of biomass, and deep soil cropping in the developing countries. Furthermore, the quantiles decomposition procedure in agriculture production is signifying heterogeneity of the determinants of environmental degradation, low and high CO2 emitters.

ecology↗

bioRxiv2020

New mechanisms of radioiodide uptake revealed via a novel high throughput drug screening approach in thyroid cancer

New combinatorial drug strategies are urgently needed to improve radioiodide (RAI) uptake and efficiently ablate thyroid cancer cells, thereby addressing recurrent and metastatic disease. Cellular iodide uptake is accomplished solely by the sodium iodide symporter (NIS), but the complexity of NIS functional regulation and a lack of amenable high-throughput screening assays has impeded progress. We utilised mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide for [~]1200 FDA-approved drugs, allowing us to appraise the impact of 73 leading compounds at 10 doses on 125I uptake in thyroid cancer cell lines. Subsequent mechanistic analysis suggests three predominant modes of drug action: Firstly, a number of drugs inhibited specific regulation of NIS function by the protein VCP. Secondly, some drugs enhanced transcriptional or post-transcriptional regulation of NIS expression. Thirdly, several drugs strongly implicated proteasomal degradation and the unfolded protein response in the cellular processing of NIS. Exploiting these mechanistic insights, multiple compounds gave striking increases in radioiodide uptake when combined with the drug SAHA. Importantly, our new drug combination strategies were also effective in human primary thyrocytes, suggesting they target endogenous NIS physiology. In patients with papillary thyroid cancer, genes involved in proteostasis were remarkably altered and predicted significantly worse outcome, but only in those patients who received RAI therapy. Collectively, we therefore propose a new model of intracellular NIS processing, and identify key nodes which may now be druggable in patients with aggressive thyroid cancer. SUMMARYOur data identify FDA-approved drugs that enhance radioiodide uptake outside of the canonical pathways of NIS processing, leading to a new mechanistic understanding of endogenous NIS function which is subverted in cancer.

cancer biology↗

bioRxiv2020

Cigarette Smoke and Nicotine-Containing E-cigarette Vapor Downregulate Lung WWOX Expression Which is Associated with Increased Severity of Murine ARDS

RATIONALEA history of chronic cigarette smoking is known to increase risk for ARDS, but the corresponding risks associated with chronic e-cigarette use are largely unknown. The chromosomal fragile site gene, WWOX, is highly susceptible to genotoxic stress from environmental exposures, and thus an interesting candidate gene for the study of exposure-related lung disease. METHODS AND RESULTSLungs harvested from current versus former/never smokers exhibited a 47% decrease in WWOX mRNA levels. Exposure to nicotine-containing e-cigarette vapor resulted in an average 57% decrease in WWOX mRNA levels relative to vehicle treated controls. In separate studies, endothelial (EC)-specific WWOX KO versus wild type mice were examined under ARDS-producing conditions. EC WWOX KO mice exhibited significantly greater levels of vascular leak and histologic lung injury. ECs were isolated from digested lungs of untreated EC WWOX KO mice using sorting by flow cytometry for CD31+CD45- cells. These were grown in culture, confirmed to be WWOX-deficient by RT-PCR and Western blotting, and analyzed by electric cell impedance sensing (ECIS) as well as a FITC dextran transwell assay for their barrier properties during MRSA or LPS exposure. WWOX KO ECs demonstrated significantly greater declines in barrier function relative to wild type cells during either MRSA or LPS treatment as measured by both ECIS and the transwell assay. CONCLUSIONThe increased risk for ARDS observed in chronic smokers may be mechanistically linked, at least in part, to lung WWOX downregulation, and this phenomenon may also manifest in the near future in chronic users of e-cigarettes.

molecular biology↗

bioRxiv2020

Multiple evolutionary pathways to achieve thermal adaptation in small mammals

Phenotypic plasticity enables an immediate response to changing conditions, but for most species, evolutionary change through adaptation will be more important for long-term survival. Warming climate and increasing desertification urges the identification of genes involved in heat-and dehydration-tolerance to better inform and target biodiversity conservation efforts. Comparisons among extant desert adapted species can highlight parallel or convergent patterns of genome evolution through the identification of shared signatures of selection. We generate chromosome-level genome assembly for the canyon mouse (Peromyscus crinitus) and test for signature of parallel evolution by comparing signatures of selective sweeps across population-level genomic resequencing data from another desert specialist deer mouse (P. eremicus) and a widely-distributed habitat generalist (P. maniculatus), that may locally adapted to arid conditions. We identify few shared candidate loci involved in desert adaptation and do not find support for a shared pattern of parallel evolution. Instead, we hypothesize divergent molecular mechanisms of desert adaptation among deer mice, potentially tied to species-specific historical demography, which may limit or enhance adaptation. We identify a number of candidate loci experiencing selective sweeps in the P. crinitus genome that are implicated in osmoregulation (Trypsin, Prostasin) and metabolic regulation (Kallikrein, eIF2-alpha kinase GCN2, APPL1/2), which may be important to accommodating hot and dry environmental conditions.

evolutionary biology↗

bioRxiv2020

Prognostic significance of Substance P/ Neurokinin 1 receptor and its association with hormonal receptors in breast carcinoma

To evaluate the expression and Immunolocalization of Substance P (SP)/ Neurokinin-1 Receptor (NK-1R) in Breast Carcinoma (BC) patients and its association with routine proliferative markers (ER, PR, HER2/ neu and Ki-67). MethodsA cross-sectional study was performed on 34 cases of BC. There were 23 cases of group A (Grade III), 8 of group B (Grade II) and only 3 cases of group C (Grade I). Age range comprised of patients from 20-80 years and the mean age of patients was 45.74 years. HE, ER, PR, HER2 and Ki-67 staining was performed as routine biomarkers. Samples were then processed for immunomarkers study of Substance P and NK-1R immunohistochemistry was performed for few cases. Results14/23 cases (61%) of group A, 7/8 cases (88%) of group B and 2/3 (67%) cases of group C were SP positive. Overall, strong staining ([≥] 10% tumors cells), labeled as "3+", was observed in 9/14 (64.2%) cases of group A and 1/8 (12.5%) case of group B. Moderate staining labelled as "2+" (in [≥] 10% tumor cells) was observed in 3/14 (21.4%) cases of group A, 4/8 (50%) cases of group B. weak positive staining "1+" was observed in only 2/14 (14.28%) cases of group A, 2/8 (25%) cases of group B and all 2/2 (100%) cases of group C. ConclusionsSP and NK-1R is overexpressed in breast carcinomas and there is significant association between grade of tumor and their overexpression. It may serve as a novel biomarker for grading of BC. We also suggest that NK-1R antagonists as a potential therapeutic strategy to inhibit and manage BC. Key PointsO_LIImmunohistochemical expression of Substance P and Neurokinin 1 Receptor in breast carcinoma tissue was evaluated C_LIO_LIIt was strongly expressed in grade III, with maximum intensity C_LIO_LIIt may be investigated further for its role as prognostic and diagnostic marker C_LIO_LITherapeutic potential of Neurokinin-1 Receptor antagonists must be explored C_LI

pathology↗

bioRxiv2020

Genes and pathways implicated in tetralogy of Fallot revealed by ultra-rare variant burden analysis in 231 genome sequences

Recent genome-wide studies of rare genetic variants have begun to implicate novel mechanisms for tetralogy of Fallot (TOF), a severe congenital heart defect (CHD). To provide statistical support for case-only data without parental genomes, we re-analyzed genome sequences of 231 individuals with TOF or related CHD. We adapted a burden test originally developed for de novo variants to assess singleton variant burden in individual genes, and in gene-sets corresponding to functional pathways and mouse phenotypes, accounting for highly correlated gene-sets, and for multiple testing. The gene burden test identified a significant burden of deleterious missense variants in NOTCH1 (Bonferroni-corrected p-value <0.01). These NOTCH1 variants showed significant enrichment for those affecting the extracellular domain, and especially for disruption of cysteine residues forming disulfide bonds (OR 39.8 vs gnomAD). Individuals with NOTCH1 variants, all with TOF, were enriched for positive family history of CHD. Other genes not previously implicated in TOF had more modest statistical support and singleton missense variant results were non-significant for gene-set burden. For singleton truncating variants, the gene burden test confirmed significant burden in FLT4. Gene-set burden tests identified a cluster of pathways corresponding to VEGF signaling (FDR=0%), and of mouse phenotypes corresponding to abnormal vasculature (FDR=0.8%), that suggested additional candidate genes not previously identified (e.g., WNT5A and ZFAND5). Analyses using unrelated sequencing datasets supported specificity of the findings for CHD. The findings support the importance of ultra-rare variants disrupting genes involved in VEGF and NOTCH signaling in the genetic architecture of TOF. These proof-of-principle data indicate that this statistical methodology could assist in analyzing case-only sequencing data in which ultra-rare variants, whether de novo or inherited, contribute to the genetic etiopathogenesis of a complex disorder. Author summaryWe analyzed the ultra-rare nonsynonymous variant burden for genome sequencing data from 231 individuals with congenital heart defects, most with tetralogy of Fallot. We adapted a burden test originally developed for de novo variants. In line with other studies, we identified a significant truncating variant burden for FLT4 and deleterious missense burden for NOTCH1, both passing a stringent Bonferroni multiple-test correction. For NOTCH1, we observed frequent disruption of cysteine residues establishing disulfide bonds in the extracellular domain. We also identified genes with BH-FDR <10% that were not previously implicated. To overcome limited power for individual genes, we tested gene-sets corresponding to functional pathways and mouse phenotypes. Gene-set burden of truncating variants was significant for vascular endothelial growth factor signaling and abnormal vasculature phenotypes. These results confirmed previous findings and suggested additional candidate genes for experimental validation in future studies. This methodology can be extended to other case-only sequencing data in which ultra-rare variants make a substantial contribution to genetic etiology.

genomics↗

bioRxiv2019

Conserved pheromone production, response and degradation by Streptococcus mutans

Streptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses, but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM, but can also stimulate bacteriocin genes as a late response. Inter-connections between the pathways activated by the two pheromones have been characterized in certain detail in S. mutans UA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin gene cipB and the alternative sigma factor sigX to investigate various S. mutans isolates for production and response to CSP and XIP pheromones in CDM. Similar to S. mutans UA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using the cipB reporter, we discovered that the acivity of exogenous CSP used as a standard was reduced over time during S. mutans growth. Using a FRET-CSP reporter peptide, we found that S. mutans UA159 was indeed able to degrade CSP, and that such proteolytic activity was not significantly different in isogenic mutants with deletion of the protease gene htrA, or the competence genes sigX, oppD, and comR. CSP proteolysis was also detected in all the wild type strains, indicating that such activity is conserved in S. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 nM and 26000 nM. Transformation in the presence of exogenous XIP was detected in all, but one, of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as proteolytic activity leading to the inactivation of CSP are conserved functions among different S. mutans isolates. Understanding the functionality and conservation of pheromone systems in S. mutans may lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favour diseases.

microbiology↗

bioRxiv2019

Mouse IgG2a antibodies specific for the commensal Streptococcus mitis show stronger cross-reactivity with Streptococcus pneumoniae than IgG1 antibodies

Here we show that mouse IgG2a and IgG1 antibodies specific for the commensal Streptococcus mitis cross-react with the pathogen Streptococcus pneumoniae, although the cross-reactivity conferred by IgG2a is stronger than IgG1 antibodies. These findings may have implications for designing S. mitis-based vaccines against pneumococcal infections.

immunology↗