Comparative Genomics of Cryptosporidium

Until recently, the apicomplexan parasites, Cryptosporidium hominis andC. parvum, were considered the same species. However, the two parasites, now considered distinct species, exhibit significant differences in host range, infectivity, and pathogenicity, and their sequenced genomes exhibit only 95–97% identity. The availability of the complete genome sequences of these organisms provides the potential to identify the genetic variations that are responsible for the phenotypic differences between the two parasites. We compared the genome organization and structure, gene composition, the metabolic and other pathways, and the local sequence identity between the genes of these two Cryptosporidium species. Our observations show that the phenotypic differences between C. hominisand C. parvum are not due to gross genome rearrangements, structural alterations, gene deletions or insertions, metabolic capabilities, or other obvious genomic alterations. Rather, the results indicate that these genomes exhibit a remarkable structural and compositional conservation and suggest that the phenotypic differences observed are due to subtle variations in the sequences of proteins that act at the interface between the parasite and its host

Comparative Genomics of Cryptosporidium

Until recently, the apicomplexan parasites, Cryptosporidium hominis and C. parvum, were considered the same species. However, the two parasites, now considered distinct species, exhibit significant differences in host range, infectivity, and pathogenicity, and their sequenced genomes exhibit only 95-97% identity. The availability of the complete genome sequences of these organisms provides the potential to identify the genetic variations that are responsible for the phenotypic differences between the two parasites. We compared the genome organization and structure, gene composition, the metabolic and other pathways, and the local sequence identity between the genes of these two Cryptosporidium species. Our observations show that the phenotypic differences between C. hominis and C. parvum are not due to gross genome rearrangements, structural alterations, gene deletions or insertions, metabolic capabilities, or other obvious genomic alterations. Rather, the results indicate that these genomes exhibit a remarkable structural and compositional conservation and suggest that the phenotypic differences observed are due to subtle variations in the sequences of proteins that act at the interface between the parasite and its host

Comprehensive comparison of monotherapies for psychiatric hospitalization risk in bipolar disorders.

OBJECTIVES: This study compared 29 drugs for risk of psychiatric hospitalization in bipolar disorders, addressing the evidence gap on the \u3e50 drugs used by US patients for treatment. METHODS: The Truven Health Analytics MarketScan RESULTS: Three drugs were associated with significantly lower risk of psychiatric hospitalization than lithium: valproate (relative risk [RR] = 0.80, P = 3.20 × 10 CONCLUSIONS: This largest reported retrospective observational study on bipolar disorders pharmacotherapy to date demonstrates that the majority of patients end monotherapy within 2 months after treatment start. The risk of psychiatric hospitalization varied almost two-fold across individual medications. The data add to the evidence favoring lithium and mood stabilizer use in short-term bipolar disorder management. The findings that the dopaminergic drugs aripiprazole and bupropion had better outcomes than other members of their respective classes and that antidepressant outcomes may vary by baseline mood polarity merit further investigation

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence

SrbB is a transcriptional regulator of genes involved in carbon metabolism, lipid metabolism, and heme biosynthesis.

<p>The FungiFun2 web server was utilized to assign FunCat and gene ontology enrichment in genes with transcript levels increased or decreased 4 fold in Δ<i>srbB</i> compared to the wild type strain. Statistically significant (P≤0.05) FunCat categories are presented at the respective time points in hypoxia.</p

Selected SrbA ChIP Peaks with flanking gene information.

<p>Selected SrbA ChIP Peaks with flanking gene information.</p

Binding of SrbB to the promoter of specific SrbA target genes and binding of SrbA in Δ<i>srbB</i>.

<p>(A) SrbB tagged with GFP was expressed in <i>A. fumigatus</i> wild type. The resulting strain was cultured in normoxia at 37°C, 250 rpm for 18 hours and shifted to hypoxia for additional incubation for 4 hours. ChIP was conducted using GFP antibody followed by ChIP-qPCR to study SrbB enrichment on the promoters of SrbA target genes. Compared to wild type control, SrbB enrichment was significant in SrbB:GFP for <i>srbA</i>, <i>srbB</i>, <i>erg25A</i>, <i>hem13</i>, and <i>alcC</i>, which suggest SrbB directly binds on the promoter of these genes for transcriptional regulation. In contrast, SrbB enrichment on the promoters of <i>erg11A</i> and <i>actA</i> were not significant. Data are presented as the mean and standard error of two biological replicates, and analyzed by two-way ANOVA followed by Bonferroni posttest. (B) SrbA binding to the promoter of <i>srbA</i>, <i>srbB</i>, and <i>erg11A</i> in Δ<i>srbB</i> was examined by ChIP-qPCR. Compared to wild type, SrbA enrichment on the gene promoters was not altered by disruption of SrbB. Data are presented as the mean and standard error of two biological replicates and analyzed by two-way ANOVA followed by Bonferroni posttests.</p

A sub-set of SrbA ChIP Target Genes are co-regulated by SrbB.

<p>The RNA-seq data for annotated genes corresponding to SrbA ChIP-seq peaks are shown as ratios of 30 and 120-minute wild-type hypoxia vs. wild-type normoxia, and gene deletion strains are shown as the deletion strain vs. the equivalent wild type hypoxia time point. Genes discussed and/or examined in detail in this manuscript are noted with asterisks. MeV analysis was performed using hierarchical clustering. Optimized gene and leaf ordering groups the wild type 30- and 120-minute hypoxic conditions together, with the 30-minute Δ<i>srbA</i> sample more similar to wild type for the SrbA targets, using Pearson correlation with complete linkage clustering.</p

RNA-seq and nCounter Analyses of Δ<i>srbA</i> Confirms SrbA Regulation of ChIP-seq Target Genes <i>in vitro</i> and <i>in vivo</i> during Invasive Pulmonary Aspergillosis.

<p>(A). Enrichment of RNA-seq differentially expressed genes in GO/Funcat categories of up- and down-regulated genes in srbA cells under hypoxia 120 minutes versus WT (B). Analysis of transcript levels of 12 of the ChiP-seq target genes <i>in vivo</i> in a murine model of invasive pulmonary aspergillosis for wild type (CEA10) and <i>in vitro</i> under normoxic/hypoxic conditions for Δ<i>srbA</i> and wild type <i>in vivo</i> samples were at 48–96 hours post-infection (grey, n = 16). <i>in vitro</i> samples were wild type normoxia (red, n = 2) and hypoxia (blue, n = 6) followed by Δ<i>srbA</i> under normoxia (red, n = 2) and hypoxia (blue, n = 6). Time under hypoxia for both wild type and Δ<i>srbA</i> ranged from 30 to 120 minutes. Expression values are represented as total number of normalized counts per transcript. Quantitation and normalization was as follows: Digital counts for 60 genes (ChIP targets, housekeeping genes and other genes of interest) were adjusted for binding efficiency with background subtraction using the included positive and negative controls from the manufacturer as per NanoString nCounter data analysis guidelines. Data sets were normalized to facilitate across sample comparisons using the geometric mean of 20 stably expressed genes.</p