Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication

Rare Variants in Ischemic Stroke: An Exome Pilot Study

The genetic architecture of ischemic stroke is complex and is likely to include rare or low frequency variants with high penetrance and large effect sizes. Such variants are likely to provide important insights into disease pathogenesis compared to common variants with small effect sizes. Because a significant portion of human functional variation may derive from the protein-coding portion of genes we undertook a pilot study to identify variation across the human exome (i.e., the coding exons across the entire human genome) in 10 ischemic stroke cases. Our efforts focused on evaluating the feasibility and identifying the difficulties in this type of research as it applies to ischemic stroke. The cases included 8 African-Americans and 2 Caucasians selected on the basis of similar stroke subtypes and by implementing a case selection algorithm that emphasized the genetic contribution of stroke risk. Following construction of paired-end sequencing libraries, all predicted human exons in each sample were captured and sequenced. Sequencing generated an average of 25.5 million read pairs (75 bp×2) and 3.8 Gbp per sample. After passing quality filters, screening the exomes against dbSNP demonstrated an average of 2839 novel SNPs among African-Americans and 1105 among Caucasians. In an aggregate analysis, 48 genes were identified to have at least one rare variant across all stroke cases. One gene, CSN3, identified by screening our prior GWAS results in conjunction with our exome results, was found to contain an interesting coding polymorphism as well as containing excess rare variation as compared with the other genes evaluated. In conclusion, while rare coding variants may predispose to the risk of ischemic stroke, this fact has yet to be definitively proven. Our study demonstrates the complexities of such research and highlights that while exome data can be obtained, the optimal analytical methods have yet to be determined

Diffusion of Innovation: State Factors that Influence the Spread of School Based Mental Health Policies and Programs

A number of trends in adolescent health have increased the importance of mental health services. In 1999, there were about 21 percent of children and adolescents between the ages of 9 and 17 that had a diagnosable mental disorder, causing some functional impairment (US DHHS, 1999). School based mental health (SBMH) has been reported to increase access to services (Armbruster, Gerstein & Fallon, 1997, Weist & Albus, 2004). Seventy-five percent of children who have mental health problems do not receive care (Kataoka, Zhang & Wells, 2002). Of those that receive care, 75 percent receive care from within the school (Burns, et al, 1995). ""SBMH services offer promise in the effort to bridge the gap between service need and service utilization by providing access to underserved populations""(Armbruster & Lichtman, 1999, p.493). However, states vary in their ability to provide services to children in school based mental health settings. This study conducted interviews with participants in nine states that are establishing connections between schools and mental health systems. Information gathered from these states was then used to seek opinions and perceptions of other State Mental Health Directors for Children. The model for this study combined the works of Rogers (1995), Berry and Berry (1990) and Mintrom and Vergari (1998) to examine the factors that influence the diffusion of innovation. The qualitative piece of this research identifies prominent necessities in an innovative SBMH program, factors that facilitate SBMH programs and barriers to innovative SBMH programs. The questionnaires that were sent to each of the state Directors also provided data on perceptions of SBMH innovations. The multivariate analyses included twenty programmatic measures and two per capita spending measures of readiness for innovation in SBMH. The models that were estimated revealed that state resources matter. The professionalism measure also showed a positive association. Bivariate analyses between measures of innovation and Census Divisions revealed some positive associations. The political variables did not show any associations with measures of readiness for innovation in the models

Diffusion of Innovation: State Factors that Influence the Spread of School Based Mental Health Policies and Programs

A number of trends in adolescent health have increased the importance of mental health services. In 1999, there were about 21 percent of children and adolescents between the ages of 9 and 17 that had a diagnosable mental disorder, causing some functional impairment (US DHHS, 1999). School based mental health (SBMH) has been reported to increase access to services (Armbruster, Gerstein & Fallon, 1997, Weist & Albus, 2004). Seventy-five percent of children who have mental health problems do not receive care (Kataoka, Zhang & Wells, 2002). Of those that receive care, 75 percent receive care from within the school (Burns, et al, 1995). ""SBMH services offer promise in the effort to bridge the gap between service need and service utilization by providing access to underserved populations""(Armbruster & Lichtman, 1999, p.493). However, states vary in their ability to provide services to children in school based mental health settings. This study conducted interviews with participants in nine states that are establishing connections between schools and mental health systems. Information gathered from these states was then used to seek opinions and perceptions of other State Mental Health Directors for Children. The model for this study combined the works of Rogers (1995), Berry and Berry (1990) and Mintrom and Vergari (1998) to examine the factors that influence the diffusion of innovation. The qualitative piece of this research identifies prominent necessities in an innovative SBMH program, factors that facilitate SBMH programs and barriers to innovative SBMH programs. The questionnaires that were sent to each of the state Directors also provided data on perceptions of SBMH innovations. The multivariate analyses included twenty programmatic measures and two per capita spending measures of readiness for innovation in SBMH. The models that were estimated revealed that state resources matter. The professionalism measure also showed a positive association. Bivariate analyses between measures of innovation and Census Divisions revealed some positive associations. The political variables did not show any associations with measures of readiness for innovation in the models

A comparative analysis of library prep approaches for sequencing low input translatome samples

Abstract Background Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. Results We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a ‘gold standard’ control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit. Conclusion RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts

Whole-Genome Sequences of Burkholderia pseudomallei Isolates Exhibiting Decreased Meropenem Susceptibility.

We report here paired isogenic Burkholderia pseudomallei genomes obtained from three patients receiving intravenous meropenem for melioidosis treatment, with post-meropenem isolates developing decreased susceptibility. Two genomes were finished, and four were drafted to improved high-quality standard. These genomes will be used to identify meropenem resistance mechanisms in B.?pseudomallei

Peripheral blood transcriptomic profiling of molecular mechanisms commonly regulated by binge drinking and placebo effects

Abstract Molecular responses to alcohol consumption are dynamic, context-dependent, and arise from a complex interplay of biological and external factors. While many have studied genetic risk associated with drinking patterns, comprehensive studies identifying dynamic responses to pharmacologic and psychological/placebo effects underlying binge drinking are lacking. We investigated transcriptome-wide response to binge, medium, and placebo alcohol consumption by 17 healthy heavy social drinkers enrolled in a controlled, in-house, longitudinal study of up to 12 days. Using RNA-seq, we identified 251 and 13 differentially expressed genes (DEGs) in response to binge drinking and placebo, respectively. Eleven protein-coding DEGs had very large effect sizes in response to binge drinking (Cohen’s d > 1). Furthermore, binge dose significantly impacted the Cytokine-cytokine receptor interaction pathway (KEGG: hsa04060) across all experimental sequences. Placebo also impacted hsa04060, but only when administered following regular alcohol drinking sessions. Similarly, medium-dose and placebo commonly impacted KEGG pathways of Systemic lupus erythematosus, Neutrophil extracellular trap formation, and Alcoholism based on the sequence of drinking sessions. These findings together indicate the “dose-extending effects” of placebo at a molecular level. Furthermore, besides supporting alcohol dose-specific molecular changes, results suggest that the placebo effects may induce molecular responses within the same pathways regulated by alcohol

Serum Antibody Repertoire Profiling Using In Silico Antigen Screen

<div><p>Serum antibodies are valuable source of information on the health state of an organism. The profiles of serum antibody reactivity can be generated by using a high throughput sequencing of peptide-coding DNA from combinatorial random peptide phage display libraries selected for binding to serum antibodies. Here we demonstrate that the targets of immune response, which are recognized by serum antibodies directed against sequential epitopes, can be identified using the serum antibody repertoire profiles generated by high throughput sequencing. We developed an algorithm to filter the results of the protein database BLAST search for selected peptides to distinguish real antigens recognized by serum antibodies from irrelevant proteins retrieved randomly. When we used this algorithm to analyze serum antibodies from mice immunized with human protein, we were able to identify the protein used for immunizations among the top candidate antigens. When we analyzed human serum sample from the metastatic melanoma patient, the recombinant protein, corresponding to the top candidate from the list generated using the algorithm, was recognized by antibodies from metastatic melanoma serum on the western blot, thus confirming that the method can identify autoantigens recognized by serum antibodies. We demonstrated also that our unbiased method of looking at the repertoire of serum antibodies reveals quantitative information on the epitope composition of the targets of immune response. A method for deciphering information contained in the serum antibody repertoire profiles may help to identify autoantibodies that can be used for diagnosing and monitoring autoimmune diseases or malignancies.</p></div