Viral RNA-dependent RNA polymerase mutants display an altered mutation spectrum resulting in attenuation in both mosquito and vertebrate hosts.

The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks.

Matrix metalloproteinases cleave membrane-bound PD-L1 on CD90+ (myo-)fibroblasts in Crohn's disease and regulate Th1/Th17 cell responses.

Increased T helper (Th)1/Th17 immune responses are a hallmark of Crohn's disease (CD) immunopathogenesis. CD90+ (myo-)fibroblasts (MFs) are abundant cells in the normal (N) intestinal mucosa contributing to mucosal tolerance via suppression of Th1 cell activity through cell surface membrane-bound PD-L1 (mPD-L1). CD-MFs have a decreased level of mPD-L1. Consequently, mPD-L1-mediated suppression of Th1 cells by CD-MFs is decreased, yet the mechanism responsible for the reduction in mPDL-1 is unknown. Increased expression of matrix metalloproteinases (MMPs) has been reported in CD. Herein we observed that when compared to N- and ulcerative colitis (UC)-MFs, CD-MFs increase in LPS-inducible levels of MMP-7 and -9 with a significant increase in both basal and inducible MMP-10. A similar pattern of MMP expression was observed in the CD-inflamed mucosa. Treatment of N-MFs with a combination of recombinant human MMP-7, -9 and -10 significantly decreased mPD-L1. In contrast, inhibition of MMP activity with MMP inhibitors or anti-MMP-10 neutralizing antibodies restores mPD-L1 on CD-MFs. CD-MFs demonstrated reduced capacity to suppress Th1 and Th17 responses from activated CD4+ T cells. By contrast, supplementation of the CD-MF:T-cell co-cultures with MMP inhibitors or anti-MMP neutralizing antibodies restored the CD-MF-mediated suppression. Our data suggest that (i) increased MMP-10 expression by CD-MFs and concomitant cleavage of PD-L1 from the surface of CD-MFs are likely to be one of the factors contributing to the decrease of mPD-L1-mediated suppression of Th1/Th17 cells in CD; and (ii) MMPs are likely to have a significant role in the intestinal mucosal immune responses.

Comparative transcriptomic analysis of Rickettsia conorii during in vitro infection of human and tick host cells.

BACKGROUND: Pathogenic Rickettsia species belonging to the spotted fever group are arthropod-borne, obligate intracellular bacteria which exhibit preferential tropism for host microvascular endothelium in the mammalian hosts, resulting in disease manifestations attributed primarily to endothelial damage or dysfunction. Although rickettsiae are known to undergo evolution through genomic reduction, the mechanisms by which these pathogens regulate their transcriptome to ensure survival in tick vectors and maintenance by transovarial/transstadial transmission, in contrast to their ability to cause debilitating infections in human hosts remain unknown. In this study, we compare the expression profiles of rickettsial sRNAome/transcriptome and determine the transcriptional start sites (TSSs) of R. conorii transcripts during in vitro infection of human and tick host cells. RESULTS: We performed deep sequencing on total RNA from Amblyomma americanum AAE2 cells and human microvascular endothelial cells (HMECs) infected with R. conorii. Strand-specific RNA sequencing of R. conorii transcripts revealed the expression 32 small RNAs (Rc_sR's), which were preferentially expressed above the limit of detection during tick cell infection, and confirmed the expression of Rc_sR61, sR71, and sR74 by quantitative RT-PCR. Intriguingly, a total of 305 and 132 R. conorii coding genes were differentially upregulated (> 2-fold) in AAE2 cells and HMECs, respectively. Further, enrichment for primary transcripts by treatment with Terminator 5'-Phosphate-dependent Exonuclease resulted in the identification of 3903 and 2555 transcription start sites (TSSs), including 214 and 181 primary TSSs in R. conorii during the infection to tick and human host cells, respectively. Seventy-five coding genes exhibited different TSSs depending on the host environment. Finally, we also observed differential expression of 6S RNA during host-pathogen and vector-pathogen interactions in vitro, implicating an important role for this noncoding RNA in the regulation of rickettsial transcriptome depending on the supportive host niche. CONCLUSIONS: In sum, the findings of this study authenticate the presence of novel Rc_sR's in R. conorii, reveal the first evidence for differential expression of coding transcripts and utilization of alternate transcriptional start sites depending on the host niche, and implicate a role for 6S RNA in the regulation of coding transcriptome during tripartite host-pathogen-vector interactions.

Broom: application for non-redundant storage of high throughput sequencing data.

Motivation: The data generation capabilities of high throughput sequencing (HTS) instruments have exponentially increased over the last few years, while the cost of sequencing has dramatically decreased allowing this technology to become widely used in biomedical studies. For small labs and individual researchers, however, storage and transfer of large amounts of HTS data present a significant challenge. The recent trends in increased sequencing quality and genome coverage can be used to reconsider HTS data storage strategies. Results: We present Broom, a stand-alone application designed to select and store only high-quality sequencing reads at extremely high compression rates. Written in C++, the application accepts single and paired-end reads in FASTQ and FASTA formats and decompresses data in FASTA format. Availability and implementation: C++ code available at https://scsb.utmb.edu/labgroups/fofanov/broom.asp. Supplementary information: Supplementary data are available at Bioinformatics online.

Detection of multi-dimensional co-exclusion patterns in microbial communities.

Motivation: Identification of complex relationships among members of microbial communities is key to understand and control the microbiota. Co-exclusion is arguably one of the most important patterns reflecting micro-organisms' intolerance to each other's presence. Knowing these relations opens an opportunity to manipulate microbiotas, personalize anti-microbial and probiotic treatments as well as guide microbiota transplantation. The co-exclusion pattern however, cannot be appropriately described by a linear function nor its strength be estimated using covariance or (negative) Pearson and Spearman correlation coefficients. This manuscript proposes a way to quantify the strength and evaluate the statistical significance of co-exclusion patterns between two, three or more variables describing a microbiota and allows one to extend analysis beyond micro-organism abundance by including other microbiome associated measurements such as, pH, temperature etc., as well as estimate the expected numbers of false positive co-exclusion patterns in a co-exclusion network. Results: The implemented computational pipeline (CoEx) tested against 2380 microbial profiles (samples) from The Human Microbiome Project resulted in body-site specific pairwise co-exclusion patterns. Availability and implementation: C++ source code for calculation of the score and P-value for two, three and four dimensional co-exclusion patterns as well as source code and executable files for the CoEx pipeline are available at https://scsb.utmb.edu/labgroups/fofanov/co-exclusion_in_microbial_communities.asp. Supplementary information: Supplementary data are available at Bioinformatics online.

Expression Profiling of Long Noncoding RNA Splice Variants in Human Microvascular Endothelial Cells: Lipopolysaccharide Effects In Vitro.

Endothelial cell interactions with lipopolysaccharide (LPS) involve both activating and repressing signals resulting in pronounced alterations in their transcriptome and proteome. Noncoding RNAs are now appreciated as posttranscriptional and translational regulators of cellular signaling and responses, but their expression status and roles during endothelial interactions with LPS are not well understood. We report on the expression profile of long noncoding (lnc) RNAs of human microvascular endothelial cells in response to LPS. We have identified a total of 10,781 and 8310 lncRNA transcripts displaying either positive or negative regulation of expression, respectively, at 3 and 24 h posttreatment. A majority of LPS-induced lncRNAs are multiexonic and distributed across the genome as evidenced by their presence on all chromosomes. Present among these are a total of 44 lncRNAs with known regulatory functions, of which 41 multiexonic lncRNAs have multiple splice variants. We have further validated splice variant-specific expression of EGO (NONHSAT087634) and HOTAIRM1 (NONHSAT119666) at 3 h and significant upregulation of lnc-IL7R at 24 h. This study illustrates the genome-wide regulation of endothelial lncRNA splice variants in response to LPS and provides a foundation for further investigations of differentially expressed lncRNA transcripts in endothelial responses to LPS and pathophysiology of sepsis/septic shock.

The ability of human nuclear DNA to cause false positive low-abundance heteroplasmy calls varies across the mitochondrial genome.

BACKGROUND: Low-abundance mutations in mitochondrial populations (mutations with minor allele frequency ≤ 1%), are associated with cancer, aging, and neurodegenerative disorders. While recent progress in high-throughput sequencing technology has significantly improved the heteroplasmy identification process, the ability of this technology to detect low-abundance mutations can be affected by the presence of similar sequences originating from nuclear DNA (nDNA). To determine to what extent nDNA can cause false positive low-abundance heteroplasmy calls, we have identified mitochondrial locations of all subsequences that are common or similar (one mismatch allowed) between nDNA and mitochondrial DNA (mtDNA). RESULTS: Performed analysis revealed up to a 25-fold variation in the lengths of longest common and longest similar (one mismatch allowed) subsequences across the mitochondrial genome. The size of the longest subsequences shared between nDNA and mtDNA in several regions of the mitochondrial genome were found to be as low as 11 bases, which not only allows using these regions to design new, very specific PCR primers, but also supports the hypothesis of the non-random introduction of mtDNA into the human nuclear DNA. CONCLUSION: Analysis of the mitochondrial locations of the subsequences shared between nDNA and mtDNA suggested that even very short (36 bases) single-end sequencing reads can be used to identify low-abundance variation in 20.4% of the mitochondrial genome. For longer (76 and 150 bases) reads, the proportion of the mitochondrial genome where nDNA presence will not interfere found to be 44.5 and 67.9%, when low-abundance mutations at 100% of locations can be identified using 417 bases long single reads. This observation suggests that the analysis of low-abundance variations in mitochondria population can be extended to a variety of large data collections such as NCBI Sequence Read Archive, European Nucleotide Archive, The Cancer Genome Atlas, and International Cancer Genome Consortium.

Bacterial small RNAs in the Genus Rickettsia.

BACKGROUND: Rickettsia species are obligate intracellular Gram-negative pathogenic bacteria and the etiologic agents of diseases such as Rocky Mountain spotted fever (RMSF), Mediterranean spotted fever, epidemic typhus, and murine typhus. Genome sequencing revealed that R. prowazekii has ~25 % non-coding DNA, the majority of which is thought to be either "junk DNA" or pseudogenes resulting from genomic reduction. These characteristics also define other Rickettsia genomes. Bacterial small RNAs, whose biogenesis is predominantly attributed to either the intergenic regions (trans-acting) or to the antisense strand of an open reading frame (cis-acting), are now appreciated to be among the most important post-transcriptional regulators of bacterial virulence and growth. We hypothesize that intergenic regions in rickettsial species encode for small, non-coding RNAs (sRNAs) involved in the regulation of its transcriptome, leading to altered virulence and adaptation depending on the host niche. RESULTS: We employed a combination of bioinformatics and in vitro approaches to explore the presence of sRNAs in a number of species within Genus Rickettsia. Using the sRNA Identification Protocol using High-throughput Technology (SIPHT) web interface, we predicted over 1,700 small RNAs present in the intergenic regions of 16 different strains representing 13 rickettsial species. We further characterized novel sRNAs from typhus (R. prowazekii and R. typhi) and spotted fever (R. rickettsii and R. conorii) groups for their promoters and Rho-independent terminators using Bacterial Promoter Prediction Program (BPROM) and TransTermHP prediction algorithms, respectively. Strong σ70 promoters were predicted upstream of all novel small RNAs, indicating the potential for transcriptional activity. Next, we infected human microvascular endothelial cells (HMECs) with R. prowazekii for 3 h and 24 h and performed Next Generation Sequencing to experimentally validate the expression of 26 sRNA candidates predicted in R. prowazekii. Reverse transcriptase PCR was also used to further verify the expression of six putative novel sRNA candidates in R. prowazekii. CONCLUSIONS: Our results yield clear evidence for the expression of novel R. prowazekii sRNA candidates during infection of HMECs. This is the first description of novel small RNAs for a highly pathogenic species of Rickettsia, which should lead to new insights into rickettsial virulence and adaptation mechanisms.

Metagenomic analysis of the airborne environment in urban spaces.

The organisms in aerosol microenvironments, especially densely populated urban areas, are relevant to maintenance of public health and detection of potential epidemic or biothreat agents. To examine aerosolized microorganisms in this environment, we performed sequencing on the material from an urban aerosol surveillance program. Whole metagenome sequencing was applied to DNA extracted from air filters obtained during periods from each of the four seasons. The composition of bacteria, plants, fungi, invertebrates, and viruses demonstrated distinct temporal shifts. Bacillus thuringiensis serovar kurstaki was detected in samples known to be exposed to aerosolized spores, illustrating the potential utility of this approach for identification of intentionally introduced microbial agents. Together, these data demonstrate the temporally dependent metagenomic complexity of urban aerosols and the potential of genomic analytical techniques for biosurveillance and monitoring of threats to public health.

The presence of nitrate dramatically changed the predominant microbial community in perchlorate degrading cultures under saline conditions.

BACKGROUND: Perchlorate contamination has been detected in both ground water and drinking water. An attractive treatment option is the use of ion-exchange to remove and concentrate perchlorate in brine. Biological treatment can subsequently remove the perchlorate from the brine. When nitrate is present, it will also be concentrated in the brine and must also be removed by biological treatment. The primary objective was to obtain an in-depth characterization of the microbial populations of two salt-tolerant cultures each of which is capable of metabolizing perchlorate. The cultures were derived from a single ancestral culture and have been maintained in the laboratory for more than 10 years. One culture was fed perchlorate only, while the other was fed both perchlorate and nitrate. RESULTS: A metagenomic characterization was performed using Illumina DNA sequencing technology, and the 16S rDNA of several pure strains isolated from the mixed cultures were sequenced. In the absence of nitrate, members of the Rhodobacteraceae constituted the prevailing taxonomic group. Second in abundance were the Rhodocyclaceae. In the nitrate fed culture, the Rhodobacteraceae are essentially absent. They are replaced by a major expansion of the Rhodocyclaceae and the emergence of the Alteromonadaceae as a significant community member. Gene sequences exhibiting significant homology to known perchlorate and nitrate reduction enzymes were found in both cultures. CONCLUSIONS: The structure of the two microbial ecosystems of interest has been established and some representative strains obtained in pure culture. The results illustrate that under favorable conditions a group of organisms can readily dominate an ecosystem and yet be effectively eliminated when their advantage is lost. Almost all known perchlorate-reducing organisms can also effectively reduce nitrate. This is certainly not the case for the Rhodobacteraceae that were found to dominate in the absence of nitrate, but effectively disappeared in its presence. This study is significant in that it reveals the existence of a novel group of organisms that play a role in the reduction of perchlorate under saline conditions. These Rhodobacteraceae especially, as well as other organisms present in these communities may be a promising source of unique salt-tolerant enzymes for perchlorate reduction.

Epigenetic alteration by DNA-demethylating treatment restores apoptotic response to glucocorticoids in dexamethasone-resistant human malignant lymphoid cells.

BACKGROUND: Glucocorticoids (GCs) are often included in the therapy of lymphoid malignancies because they kill several types of malignant lymphoid cells. GCs activate the glucocorticoid receptor (GR), to regulate a complex genetic network, culminating in apoptosis. Normal lymphoblasts and many lymphoid malignancies are sensitive to GC-driven apoptosis. Resistance to GCs can be a significant clinical problem, however, and correlates with resistance to several other major chemotherapeutic agents. METHODS: We analyzed the effect of treatment with the cytosine analogue 5 aza-2' deoxycytidine (AZA) on GC resistance in two acute lymphoblastic leukemia (T or pre-T ALL) cell lines- CEM and Molt-4- and a (B-cell) myeloma cell line, RPMI 8226. Methods employed included tissue culture, flow cytometry, and assays for clonogenicity, cytosine extension, immunochemical identification of proteins, and gene transactivation. High throughput DNA sequencing was used to confirm DNA methylation status. CONCLUSIONS: Treatment of these cells with AZA resulted in altered DNA methylation and restored GC-evoked apoptosis in all 3 cell lines. In CEM cells the altered epigenetic state resulted in site-specific phosphorylation of the GR, increased GR potency, and GC-driven induction of the GR from promoters that lie in CpG islands. In RPMI 8226 cells, expression of relevant coregulators of GR function was altered. Activation of p38 mitogen-activated protein kinase (MAPK), which is central to a feed-forward mechanism of site-specific GR phosphorylation and ultimately, apoptosis, occurred in all 3 cell lines. These data show that in certain malignant hematologic B- and T-cell types, epigenetically controlled GC resistance can be reversed by cell exposure to a compound that causes DNA demethylation. The results encourage studies of application to in vivo systems, looking towards eventual clinical applications.

Where does Neisseria acquire foreign DNA from: an examination of the source of genomic and pathogenic islands and the evolution of the Neisseria genus.

BACKGROUND: Pathogenicity islands (PAIs) or genomic islands (GEIs) are considered to be the result of a recent horizontal transfer. Detecting PAIs/GEIs as well as their putative source can provide insight into the organism's pathogenicity within its host. Previously we introduced a tool called S-plot which provides a visual representation of the variation in compositional properties across and between genomic sequences. Utilizing S-plot and new functionality developed here, we examined 18 publicly available Neisseria genomes, including strains of both pathogenic and non-pathogenic species, in order to identify regions of unusual compositional properties (RUCPs) using both a sliding window as well as a gene-by-gene approach. RESULTS: Numerous GEIs and PAIs were identified including virulence genes previously found within the pathogenic Neisseria species. While some genes were conserved amongst all species, only pathogenic species, or an individual species, a number of genes were detected that are unique to an individual strain. While the majority of such genes have an origin unknown, a number of putative sources including pathogenic and capsule-containing bacteria were determined, indicative of gene exchange between Neisseria spp. and other bacteria within their microhabitat. Furthermore, we uncovered evidence that both N. meningitidis and N. gonorrhoeae have separately acquired DNA from their human host. Data suggests that all three Neisseria species have received horizontally transferred elements post-speciation. CONCLUSIONS: Using this approach, we were able to not only find previously identified regions of virulence but also new regions which may be contributing to the virulence of the species. This comparative analysis provides a means for tracing the evolutionary history of the acquisition of foreign DNA within this genus. Looking specifically at the RUCPs present within the 18 genomes considered, a stronger similarity between N. meningitidis and N. lactamica is observed, suggesting that N. meningitidis arose before N. gonorrhoeae.

Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics.

Understanding the fine-structure molecular architecture of bacterial epidemics has been a long-sought goal of infectious disease research. We used short-read-length DNA sequencing coupled with mass spectroscopy analysis of SNPs to study the molecular pathogenomics of three successive epidemics of invasive infections involving 344 serotype M3 group A Streptococcus in Ontario, Canada. Sequencing the genome of 95 strains from the three epidemics, coupled with analysis of 280 biallelic SNPs in all 344 strains, revealed an unexpectedly complex population structure composed of a dynamic mixture of distinct clonally related complexes. We discovered that each epidemic is dominated by micro- and macrobursts of multiple emergent clones, some with distinct strain genotype-patient phenotype relationships. On average, strains were differentiated from one another by only 49 SNPs and 11 insertion-deletion events (indels) in the core genome. Ten percent of SNPs are strain specific; that is, each strain has a unique genome sequence. We identified nonrandom temporal-spatial patterns of strain distribution within and between the epidemic peaks. The extensive full-genome data permitted us to identify genes with significantly increased rates of nonsynonymous (amino acid-altering) nucleotide polymorphisms, thereby providing clues about selective forces operative in the host. Comparative expression microarray analysis revealed that closely related strains differentiated by seemingly modest genetic changes can have significantly divergent transcriptomes. We conclude that enhanced understanding of bacterial epidemics requires a deep-sequencing, geographically centric, comparative pathogenomics strategy.

Identification of multivariable Boolean patterns in microbiome and microbial gene composition data.

Virtually every biological system is governed by complex relations among its components. Identifying such relations requires a rigorous or heuristics-based search for patterns among variables/features of a system. Various algorithms have been developed to identify two-dimensional (involving two variables) patterns employing correlation, covariation, mutual information, etc. It seems obvious, however, that comprehensive descriptions of complex biological systems need also to include more complicated multivariable relations, which can only be described using patterns that simultaneously embrace 3, 4, and more variables. The goal of this manuscript is to (a) introduce a novel type of associations (multivariable Boolean patterns) that can be manifested between features of complex systems but cannot be identified (described) by traditional pair-vise metrics; (b) propose patterns classification method, and (c) provide a novel definition of the pattern's strength (pattern's score) able to accommodate heterogeneous multi-omics data. To demonstrate the presence of such patterns, we performed a search for all possible 2-, 3-, and 4-dimensional patterns in historical data from the Human Microbiome Project (15 body sites) and collection of H. pylori genomes associated with gastric ulcers, gastritis, and duodenal ulcers. In all datasets under consideration, we were able to identify hundreds of statistically significant multivariable patterns. These results suggest that such patterns can be common in microbial genomics/microbiomics systems.

Slim-filter: an interactive Windows-based application for illumina genome analyzer data assessment and manipulation.

BACKGROUND: The emergence of Next Generation Sequencing technologies has made it possible for individual investigators to generate gigabases of sequencing data per week. Effective analysis and manipulation of these data is limited due to large file sizes, so even simple tasks such as data filtration and quality assessment have to be performed in several steps. This requires (potentially problematic) interaction between the investigator and a bioinformatics/computational service provider. Furthermore, such services are often performed using specialized computational facilities. RESULTS: We present a Windows-based application, Slim-Filter designed to interactively examine the statistical properties of sequencing reads produced by Illumina Genome Analyzer and to perform a broad spectrum of data manipulation tasks including: filtration of low quality and low complexity reads; filtration of reads containing undesired subsequences (such as parts of adapters and PCR primers used during the sample and sequencing libraries preparation steps); excluding duplicated reads (while keeping each read's copy number information in a specialized data format); and sorting reads by copy numbers allowing for easy access and manual editing of the resulting files. Slim-Filter is organized as a sequence of windows summarizing the statistical properties of the reads. Each data manipulation step has roll-back abilities, allowing for return to previous steps of the data analysis process. Slim-Filter is written in C++ and is compatible with fasta, fastq, and specialized AS file formats presented in this manuscript. Setup files and a user's manual are available for download at the supplementary web site ( https://www.bioinfo.uh.edu/Slim_Filter/). CONCLUSION: The presented Windows-based application has been developed with the goal of providing individual investigators with integrated sequencing reads analysis, curation, and manipulation capabilities.

Rare target enrichment for ultrasensitive PCR detection using cot-rehybridization and duplex-specific nuclease.

Nucleic acid detection by polymerase chain reaction (PCR) is invaluable for the detection of dilute and rare sequences, including pathogens and infrequent species in complex clinical and environmental backgrounds. The presence of excess complex background nucleic acid can reduce sensitivity and specificity. This is because mispriming can cause failure of the amplification reaction. Here we describe a new approach to ultrasensitive PCR detection, using enrichment of rare target nucleic acid from abundant background by combining the classic technique of cot-rehybridization to convert the abundant background to double-stranded form, with the use of a newly described, highly processive duplex-specific crab nuclease. We show that trace sequences in a vast excess of background DNA can be undetectable by PCR, independent of the amount of the mixture added to the PCR, and that these sequences can be made detectable by background suppression using this method.

Ehrlichia chaffeensis TRP120 binds a G+C-rich motif in host cell DNA and exhibits eukaryotic transcriptional activator function.

Ehrlichia chaffeensis is an obligately intracellular bacterium that modulates host cell gene transcription in the mononuclear phagocyte, but the host gene targets and mechanisms involved in transcriptional modulation are not well-defined. In this study, we identified a novel tandem repeat DNA-binding domain in the E. chaffeensis 120-kDa tandem repeat protein (TRP120) that directly binds host cell DNA. TRP120 was observed by immunofluorescent microscopy in the nucleus of E. chaffeensis-infected host cells and was detected in nuclear extracts by Western immunoblotting with TRP120-specific antibody. The TRP120 binding sites and associated host cell target genes were identified using high-throughput deep sequencing (Illumina) of immunoprecipitated DNA (chromatin immunoprecipitation and high-throughput DNA sequencing). Multiple em motif elicitation (MEME) analysis of the most highly enriched TRP120-bound sequences revealed a G+C-rich DNA motif, and recombinant TRP120 specifically bound synthetic oligonucleotides containing the motif. TRP120 target gene binding sites were mapped most frequently to intersecting regions (intron/exon; 49%) but were also identified in upstream regulatory regions (25%) and downstream locations (26%). Genes targeted by TRP120 were most frequently associated with transcriptional regulation, signal transduction, and apoptosis. TRP120 targeted inflammatory chemokine genes, CCL2, CCL20, and CXCL11, which were strongly upregulated during E. chaffeensis infection and were also upregulated by direct transfection with recombinant TRP120. This study reveals that TRP120 is a novel DNA-binding protein that is involved in a host gene transcriptional regulation strategy.

Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.

The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.

Impairment of Tissue-Resident Mesenchymal Stem Cells in Chronic Ulcerative Colitis and Crohn's Disease.

BACKGROUND AND AIMS: Little is known about the presence and function of tissue-resident mesenchymal stem cells [MtSCs] within the gastrointestinal mucosa in health and inflammatory bowel disease [IBD]. The contribution of MtSCs to the generation of inflammatory fibroblasts during IBD is also poorly understood. We hypothesized that IBD-MtSCs are impaired and contribute to the generation of the pathological myofibroblasts in IBD. METHODS: In a cohort of clinically and endoscopically active IBD patients and normal controls, we used quantitative RT-PCR and stem cell differentiation assays, as well as confocal microscopy, to characterize MtSCs. RESULTS: Expression of two stem cell markers, Oct4 and ALDH1A, was increased in the inflamed IBD colonic mucosa and correlated with an increase of the mesenchymal lineage marker Grem1 in ulcerative colitis [UC], but not Crohn's disease [CD]. Increased proliferation and aberrant differentiation of Oct4+Grem1+ MtSC-like cells was observed in UC, but not in CD colonic mucosa. In contrast to normal and UC-derived MtSCs, CD-MtSCs lose their clonogenic and most of their differentiation capacities. Our data also suggest that severe damage to these cells in CD may account for the pathological PD-L1low phenotype of CD myofibroblasts. In contrast, aberrant differentiation of MtSCs appears to be involved in the appearance of pathological partially differentiated PD-L1high myofibroblasts within the inflammed colonic mucosa in UC. CONCLUSION: Our data show, for the first time, that the progenitor functions of MtSCs are differentially impaired in CD vs UC, providing a scientific rationale for the use of allogeneic MSC therapy in IBD, and particularly in CD.

Identification of multidimensional Boolean patterns in microbial communities.

BACKGROUND: Identification of complex multidimensional interaction patterns within microbial communities is the key to understand, modulate, and design beneficial microbiomes. Every community has members that fulfill an essential function affecting multiple other community members through secondary metabolism. Since microbial community members are often simultaneously involved in multiple relations, not all interaction patterns for such microorganisms are expected to exhibit a visually uninterrupted pattern. As a result, such relations cannot be detected using traditional correlation, mutual information, principal coordinate analysis, or covariation-based network inference approaches. RESULTS: We present a novel pattern-specific method to quantify the strength and estimate the statistical significance of two-dimensional co-presence, co-exclusion, and one-way relation patterns between abundance profiles of two organisms as well as extend this approach to allow search and visualize three-, four-, and higher dimensional patterns. The proposed approach has been tested using 2380 microbiome samples from the Human Microbiome Project resulting in body site-specific networks of statistically significant 2D patterns as well as revealed the presence of 3D patterns in the Human Microbiome Project data. CONCLUSIONS: The presented study suggested that search for Boolean patterns in the microbial abundance data needs to be pattern specific. The reported presence of multidimensional patterns (which cannot be reduced to a combination of two-dimensional patterns) suggests that multidimensional (multi-organism) relations may play important roles in the organization of microbial communities, and their detection (and appropriate visualization) may lead to a deeper understanding of the organization and dynamics of microbial communities. Video Abstract.