Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia

Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area

Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species

Gene-Based Analysis of Regionally Enriched Cortical Genes in GWAS Data Sets of Cognitive Traits and Psychiatric Disorders

Background: Despite its estimated high heritability, the genetic architecture leading to differences in cognitive performance remains poorly understood. Different cortical regions play important roles in normal cognitive functioning and impairment. Recently, we reported on sets of regionally enriched genes in three different cortical areas (frontomedial, temporal and occipital cortices) of the adult rat brain. It has been suggested that genes preferentially, or specifically, expressed in one region or organ reflect functional specialisation. Employing a gene-based approach to the analysis, we used the regionally enriched cortical genes to mine a genome-wide association study (GWAS) of the Norwegian Cognitive NeuroGenetics (NCNG) sample of healthy adults for association to nine psychometric tests measures. In addition, we explored GWAS data sets for the serious psychiatric disorders schizophrenia (SCZ) (n = 3 samples) and bipolar affective disorder (BP) (n = 3 samples), to which cognitive impairment is linked. Principal Findings: At the single gene level, the temporal cortex enriched gene RAR-related orphan receptor B (RORB) showed the strongest overall association, namely to a test of verbal intelligence (Vocabulary, P = 7.7E-04). We also applied gene set enrichment analysis (GSEA) to test the candidate genes, as gene sets, for enrichment of association signal in the NCNG GWAS and in GWASs of BP and of SCZ. We found that genes differentially expressed in the temporal cortex showed a significant enrichment of association signal in a test measure of non-verbal intelligence (Reasoning) in the NCNG sample. Conclusion: Our gene-based approach suggests that RORB could be involved in verbal intelligence differences, while the genes enriched in the temporal cortex might be important to intellectual functions as measured by a test of reasoning in the healthy population. These findings warrant further replication in independent samples on cognitive traits

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Geographical distribution of isolates from this study.

<p>Circle size represents the number of isolates found at a specific sampling site and the color represents different species as determined by <i>recA</i> or 16s <i>rRNA</i> sequencing. Division of circles indicates that multiple species were found at a site. <i>B</i>. <i>ubonensis</i> is commonly found throughout Darwin and the surrounding areas. Several species were found in the same soil sample as <i>B</i>. <i>pseudomallei</i> including <i>B</i>. <i>ubonensis</i>, several <i>B</i>. <i>cepacia</i> complex species, proposed <i>B</i>. <i>humptydooensis</i>, <i>Pandoraea</i>, and <i>Cupriavidus</i>.</p

Colony morphology of MSMB isolates on Ashdown’s Agar (ASA) compared to <i>B</i>. <i>pseudomallei</i> MSHR305.

<p>ASA is considered a selective growth medium for <i>Burkholderia pseudomallei</i>. In this study, at least 20 different species demonstrated the ability to grow on ASA including multiple <i>Burkholderia</i> speices as well as <i>Ralstonia</i>, <i>Cupriavidus</i>, and <i>Panoraea</i>. Many different colony morphologies were observed during this study as evident in the pictures (a) proposed <i>B</i>. <i>humptydooensis</i>MSMB43, (b) unknown <i>Burkholderia spp</i>. MSMB 175, (c) <i>B</i>. <i>multivorans</i> MSMB105, (d) <i>B</i>. <i>thailandensis</i> MSMB60, (e) <i>B</i>. <i>ubonensis</i> MSMB153, (f) <i>B</i>. <i>pseudomallei</i> MSHR 305. Each strain was grown on ASA for 72 hours at 37°C aerobically.</p

Comparison of environmental species identified in the Darwin region of Australia.

<p>(-) indicates that no <i>B</i>. <i>pseudomallei</i> was found to occur with a species or that the molecular assay was negative for that target.</p><p>^a Site refers to the GPS location from which soil or water samples were taken.</p><p>^b<i>B</i>. <i>pseudomallei</i> were recovered from the same soil or water sample as the isolate.</p><p>Comparison of environmental species identified in the Darwin region of Australia.</p

Detection of <it>Burkholderia pseudomallei</it> O-antigen serotypes in near-neighbor species

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei</it> is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in <it>B</it>. <it>pseudomallei</it>. Complicating this strategy are the four different <it>B. pseudomallei</it> LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of <it>Burkholderia</it> species. Here, we identified the presence of multiple <it>B. pseudomallei</it> O-antigen types and sero-crossreactivity in its near-neighbor species.</p> <p>Results</p> <p>PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of <it>B. mallei</it> and <it>B. thailandensis</it> strains contained the typical O-antigen type A. In contrast, most of <it>B. ubonensis</it> and <it>B. thailandensis</it>-like strains expressed the atypical O-antigen types B and B2, respectively. Most <it>B</it>. <it>oklahomensis</it> strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in <it>B</it>. <it>thailandensis</it> 82172, <it>B</it>. <it>ubonensis</it> MSMB108, and <it>Burkholderia</it> sp. MSMB175. Interestingly, <it>B</it>. <it>thailandensis</it>-like MSMB43 contained a novel serotype B positive O-antigen.</p> <p>Conclusions</p> <p>This study expands the number of species which express <it>B. pseudomallei</it> O-antigen types. Further work is required to elucidate the full structures and how closely these are to the <it>B. pseudomallei</it> O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.</p