Evidence of an Increased Pathogenic Footprint in the Lingual Microbiome of Untreated HIV Infected Patients
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ABSTRACT: Opportunistic oral infections are ultimately presented in a vast majority of HIV-infected patients, often causing debilitating lesions that also contribute to deterioration in nutritional health. Although appreciation for the role that the microbiota is likely to play in the initiation and/or enhancement of oral infections has grown considerably in recent years, little is known about the impact of HIV infection on host-microbe interactions within the oral cavity. In the current study, we characterize modulations in the bacterial composition of the lingual microbiome in patients with treated and untreated HIV infection. Bacterial species profiles were elucidated by microarray assay and compared between untreated HIV infected patients, HIV infected patients receiving antiretroviral therapy, and healthy HIV negative controls. The relationship between clinical parameters (viral burden and CD4+ T cell depletion) and the loss or gain of bacterial species was evaluated in each HIV patient group. Characterization of modulations in the dorsal tongue (lingual) microbiota that are associated with chronic HIV infection.
Project description:Analysis of gingival crevicular fluid (GCF) samples may give information of the identity of unattached (planktonic) subgingival bacteria, the 35 forefront candidates for systemic dispersal via ulcerated periodontal pocket epithelium. Our study represents the first one targeting the identity of bacteria in gingival crevicular fluid. Methodology/Principal findings: We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla 45 were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, 46 per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. Conclusions/Significance: Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms. The microbial profiles of GCF and subgingival plaque were analyzed from 17 subjects with periodontal disease.
Project description:The purpose of this study is to describe the effects of allogeneic stem cell transplant on oral microbiota and to examine differences in those patients who acquired respiratory complications. Forty-five patients were consented for the study and followed for 100 days post-transplant. Eleven patients represented by 115 speciment had specimens collected before and after transplant were subjected to further analysis. The Human Oral Microbe Identification Microarray was used for this analysis. In these 11 patients, five developed respiraotry complications after transplant and six did not develop this complication. Cluster analysis was used to identify patterns in the data. 115 specimens are included from 11 patients. These specimens were collected before and after transplant. There are no duplicate samples.
Project description:RNA transcripts are distributed non-uniformly in the oocytes of many animals, such that newly-divided embryo cells (blastomeres) inherit distinct transcriptomes following fertilization. In animals such as the frog, Xenopus laevis, programmed transcript regionalization directs early embryonic axis formation and is essential for normal development. However, it is unknown whether such transcriptome asymmetry directs embryogenesis in mammals, or indeed, whether it occurs at all. We here address this by transcript profiling of matching sub-cellular structures and single cells in mouse oocytes and early embryos and analytical strategies exploiting the paired data structure. Spindle samples contained a set of transcripts that was distinguishable from that of the unfertilized metaphase II (mII) oocytes from which each spindle was microsurgically dissected. Immediately following fertilization, cytokinesis produces a 1-cell embryo (zygote) and associated spindle-enriched second polar body (Pb2) whose transcript profiles also differed one from the other, partially reflecting the enrichment of spindle-associated transcripts. Non-uniform transcript distribution within zygotes did not lead to programmed transcriptome asymmetry between the blastomeres of nascent two-cell embryos, or between the second mitotic products of 3-cell embryos. These findings suggest that mammalian oocytes and zygotes exhibit transcript regionalization without subsequent transcriptome asymmetries between respective early mitotic products. This contrasts the situation in Xenopus and places constraints on the ability of maternal transcriptomic prepatterning to prescribe early mammalian development. 16 Zygote vs. Polar body pairs, 10 spindle vs. oocyte pairs, 22 2-cell embryos and 8 3-cell embryos were analysed. Although the raw data are two channel, only the Cy5 signal of each file was analyzed. The Cy5 channel for each gene is normalized to the average Cy5 intensity of the gene across all samples.
Project description:To understand the relationship between gene expression and capsule formation, H99 cells were cultured overnight at 37ºC in the following eight conditions: low iron medium with or without both 500 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM bathophenanthroline disulfonate (BPDS); phosphate-buffered saline (PBS) with or without 10% v/v fetal bovine serum; Dulbecco's Modified Eagle's Medium (from Sigma) in ambient air or 5% CO2; and Littman's medium with either 0.01 µg/ml or 1 µg/ml thiamine. All samples were hybridized against a common reference pool of total RNA. Three biological replicates were performed for each growth condition, with the exception of growth in DMEM.
Project description:We have used mouse embryonic stem cells (ESCs) as a model to study the signaling mechanisms that regulate self-renewal and commitment to differentiation. We hypothesized that genes critical to stem cell fate would be dynamically regulated at the initiation of commitment. Time course microarray analysis following initiation of commitment led us to propose a model of ESC maintenance in which highly regulated transcription factors and chromatin remodeling genes (down-regulated in our time course) maintain repression of genes responsible for cell differentiation, morphogenesis and development (up-regulated in our time course). Microarrays of Oct4, Nanog and Sox2 shRNA knockdown cell lines confirmed predicted regulation of target genes. shRNA knockdowns of candidate genes were tested in a novel high throughput screen of self-renewal, confirming their role in ESC pluripotency. We have identified genes that are critical for self-renewal and those that initiate commitment and developed draft transcriptional networks that control self-renewal and early development. Keywords: genetic modification Gene expression in Oct4 knockdown, Sox2 knockdown and their empty vector contol ES cells was analyzed.
Project description:We report dynamics of X-chromosome upregulation (XCU) along X-chromosome inactivation (XCI) in mESCs as they differentiate into EpiSCs. F1 hybrid C57BL6/J × CAST/EiJ male and female mESCs were adapted to 2i/LIF and female cells grown in serum/LIF conditions were differentiated using Fgf2 and Activin A for 1, 2, 4 and 7 days to induce random XCI. scRNA-seq was performed using the Smart-seq3 protocol, providing full-length coverage together with molecular counting using UMIs. Allelic resolution is achieved using strain-specific SNPs in the data. We reveal dynamic balancing of X alleles as cells undergo XCI to compensate dosage imbalances between sexes as well as between X and autosomes. Furthermore, we reveal that female naïve mESCs with two active X chromosomes lack XCU on both alleles which has major implications for reprogramming studies. Finally, we estimate allelic transcriptional burst kinetics from the data and find that progressively increased burst frequencies underlies the XCU process.
Project description:Technical control for allelic detection using Smart-seq3. Liver RNA from pure C57BL6/J and CAST/EiJ strains was combined at varying ratios (0:1, 1:7, 1:3, 3:5, 1:1, 5:3, 3:1, 7:1, 1:0) for a total of 200 pg RNA per sample.
Project description:The aim of the experiment is to identify candidate genes to phenotype epitope-specific autoreactive B cells. These genes could serve as markers to delineate further populations of autoreactive B cells that were shown to be key players in regulating autoimmunity and in explaining B cell selection.
Project description:We report dynamics of X-chromosome upregulation (XCU) along X-chromosome inactivation (XCI) in mESCs as they differentiate into EpiSCs. F1 hybrid C57BL6/J × CAST/EiJ male and female mESCs were grown in serum/LIF conditions were differentiated using Fgf2 and Activin A for 1, 2, 4 and 7 days to induce random XCI in female cells. Multi-modal single-cell sequencing was performed using scATAC on nuclei and Smart-seq3 to assay chromatin accessibility and poly-A+ RNA expression, respectively. Allelic resolution is achieved using strain-specific SNPs in the data. We reveal dynamic balancing of X alleles as cells undergo XCI to compensate dosage imbalances between sexes as well as between X and autosomes. Furthermore, we reveal that female naïve mESCs with two active X chromosomes lack XCU on both alleles which has major implications for reprogramming studies. Finally, we estimate allelic transcriptional burst kinetics from the data and find that progressively increased burst frequencies underlies the XCU process.
Project description:Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs), enhancers and silencers. While enhancers have been thoroughly characterized, the properties and mechansisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs, as they mostly lack detectable DNA accessibility. The silencer-binding TF CG11247, which we term Saft, safeguards cell fate decisions in vivo and functions via a highly-conserved domain we term ZAC and the corepressor G9a, independently of G9a’s H3K9-methyltransferase activity. Overall, our identification of silencers with unexpected properties and mechanisms has important implications for the understanding and future study of repressive CREs, as well as the functional annotation of animal genomes.