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

Case report:Thirty-year progression of an EMPF1 encephalopathy due to defective mitochondrial and peroxisomal fission caused by a novel de novo heterozygous DNM1L variant

Mutations in DNM1L (DRP1), which encode a key player of mitochondrial and peroxisomal fission, have been reported in patients with the variable phenotypic spectrum, ranging from non-syndromic optic atrophy to lethal infantile encephalopathy. Here, we report a case of an adult female patient presenting with a complex neurological phenotype that associates axonal sensory neuropathy, spasticity, optic atrophy, dysarthria, dysphasia, dystonia, and ataxia, worsening with aging. Whole-exome sequencing revealed a heterozygous de novo variant in the GTPase domain of DNM1L [NM_001278464.1: c.176C>A p.(Thr59Asn)] making her the oldest patient suffering from encephalopathy due to defective mitochondrial and peroxisomal fission-1. In silico analysis suggested a protein destabilization effect of the variant Thr59Asn. Unexpectedly, Western blotting disclosed profound decrease of DNM1L expression, probably related to the degradation of DNM1L complexes. A detailed description of mitochondrial and peroxisomal anomalies in transmission electron and 3D fluorescence microscopy studies confirmed the exceptional phenotype of this patient

Results from the CERN pilot CLOUD experiment

During a 4-week run in October–November 2006, a pilot experiment was performed at the CERN Proton Synchrotron in preparation for the Cosmics Leaving OUtdoor Droplets (CLOUD) experiment, whose aim is to study the possible influence of cosmic rays on clouds. The purpose of the pilot experiment was firstly to carry out exploratory measurements of the effect of ionising particle radiation on aerosol formation from trace H2SO4 vapour and secondly to provide technical input for the CLOUD design. A total of 44 nucleation bursts were produced and recorded, with formation rates of particles above the 3 nm detection threshold of between 0.1 and 100 cm -3 s -1, and growth rates between 2 and 37 nm h -1. The corresponding H2O concentrations were typically around 106 cm -3 or less. The experimentally-measured formation rates and htwosofour concentrations are comparable to those found in the atmosphere, supporting the idea that sulphuric acid is involved in the nucleation of atmospheric aerosols. However, sulphuric acid alone is not able to explain the observed rapid growth rates, which suggests the presence of additional trace vapours in the aerosol chamber, whose identity is unknown. By analysing the charged fraction, a few of the aerosol bursts appear to have a contribution from ion-induced nucleation and ion-ion recombination to form neutral clusters. Some indications were also found for the accelerator beam timing and intensity to influence the aerosol particle formation rate at the highest experimental SO2 concentrations of 6 ppb, although none was found at lower concentrations. Overall, the exploratory measurements provide suggestive evidence for ion-induced nucleation or ion-ion recombination as sources of aerosol particles. However in order to quantify the conditions under which ion processes become significant, improvements are needed in controlling the experimental variables and in the reproducibility of the experiments. Finally, concerning technical aspects, the most important lessons for the CLOUD design include the stringent requirement of internal cleanliness of the aerosol chamber, as well as maintenance of extremely stable temperatures (variations below 0.1 °C

Comparison of TaqMan PCR assays for detection of the melioidosis agent Burkholderia pseudomallei in clinical specimens

Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin

Comparison of TaqMan PCR assays for detection of the melioidosis agent Burkholderia pseudomallei in clinical specimens

Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin

Altering an Artificial Gagpolnef Polyprotein and Mode of ENV Co-Administration Affects the Immunogenicity of a Clade C HIV DNA Vaccine

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ+ CD8+ T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2d T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials

Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour, Burkholderia ubonensis, Using Real-Time PCR

Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei

Safety and Immunogenicity of a Recombinant Adenovirus Serotype 35-Vectored HIV-1 Vaccine in Adenovirus Serotype 5 Seronegative and Seropositive Individuals.

BACKGROUND: Recombinant adenovirus serotype 5 (rAd5)-vectored HIV-1 vaccines have not prevented HIV-1 infection or disease and pre-existing Ad5 neutralizing antibodies may limit the clinical utility of Ad5 vectors globally. Using a rare Ad serotype vector, such as Ad35, may circumvent these issues, but there are few data on the safety and immunogenicity of rAd35 directly compared to rAd5 following human vaccination. METHODS: HVTN 077 randomized 192 healthy, HIV-uninfected participants into one of four HIV-1 vaccine/placebo groups: rAd35/rAd5, DNA/rAd5, and DNA/rAd35 in Ad5-seronegative persons; and DNA/rAd35 in Ad5-seropositive persons. All vaccines encoded the HIV-1 EnvA antigen. Antibody and T-cell responses were measured 4 weeks post boost immunization. RESULTS: All vaccines were generally well tolerated and similarly immunogenic. As compared to rAd5, rAd35 was equally potent in boosting HIV-1-specific humoral and cellular immunity and responses were not significantly attenuated in those with baseline Ad5 seropositivity. Like DNA, rAd35 efficiently primed rAd5 boosting. All vaccine regimens tested elicited cross-clade antibody responses, including Env V1/V2-specific IgG responses. CONCLUSIONS: Vaccine antigen delivery by rAd35 is well-tolerated and immunogenic as a prime to rAd5 immunization and as a boost to prior DNA immunization with the homologous insert. Further development of rAd35-vectored prime-boost vaccine regimens is warranted

P4HB recurrent missense mutation causing Cole-Carpenter syndrome

BACKGROUND: Cole-Carpenter syndrome (CCS) is commonly classified as a rare Osteogenesis Imperfecta (OI) disorder. This was following the description of two unrelated patients with very similar phenotypes who were subsequently shown to have a heterozygous missense mutation in P4HB. OBJECTIVES: Here, we report a 3-year old female patient with severe OI who on exome sequencing was found to carry the same missense mutation in P4HB as reported in the original cohort. We discuss the genetic heterogeneity of CCS and underlying mechanism of P4HB in collagen production. METHODS: We undertook detailed clinical, radiological and molecular phenotyping in addition, to analysis of collagen in cultured fibroblasts and electron microscopic examination in the patient reported here. RESULTS: The clinical phenotype appears consistent in patients reported so far but interestingly, there also appears to be a definitive phenotypic clue (crumpling metadiaphyseal fractures of the long tubular bones with metaphyseal sclerosis which are findings that are uncommon in OI) to the underlying genotype (P4HB variant). DISCUSSION: P4HB (Prolyl 4-hydroxylase, betasubunit) encodes for PDI (Protein Disulfide isomerase) and in cells, in its tetrameric form, catalyses formation of 4-hydroxyproline in collagen. The recurrent variant in P4HB, c.1178A>G, p.Tyr393Cys, sits in the C-terminal reactive centre and is said to interfere with disulphide isomerase function of the C-terminal reactive centre. P4HB catalyses the hydroxylation of proline residues within the X-Pro-Gly repeats in the procollagen helical domain. Given the inter-dependence of extracellular matrix (ECM) components in assembly of a functional matrix, our data suggest that it is the organisation and assembly of the functional ECM that is perturbed rather than the secretion of collagen type I per se. CONCLUSIONS: We provide additional evidence of P4HB as a cause of a specific form of OI-CCS and expand on response to treatment with bisphosphonates in this rare disorder