Morphological and genetic diversity of the thermophilic cyanobacterium,Mastigocladus laminosus (Stigonematales,Cyanobacteria) from Japan and Myanmar

The morphology and phylogeny of 13 strains of a thermophilic cyanobacterium, Mastigocladus laminosus Cohn, isolated from hot springs in Japan and Myanmar were analyzed to determine taxonomy and biogeography. From the morphological observations of cell size, there were significant differences among strains. Phylogenetic analyses based on 16S rRNA gene sequences revealed two lineages: Lineages I and II. Lineage I consisted of strains collected in Japan and reference strains from a previous study (CCMEE 5329 and 5331, Hakone, Japan); Lineage II included all of the Myanmar strains and one Japanese strain, and was a novel lineage in phylogeographic studies on M. laminosus. Since strains in the Lineage II tended to have larger cells than those in the Lineage I, the morphological and phylogenetic lineages corresponded well with each other.     

Changes in the Treatment Responses to Artesunate-Mefloquine on the Northwestern Border of Thailand during 13 Years of Continuous Deployment

Background

Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS₃), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border.

Methods and Findings

3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS₃. The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p<0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00–2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS₃ efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07–1.19, p<0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0–98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2–5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend).

Conclusion

Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage.     

Neuropathogenesis of Japanese Encephalitis in a Primate Model

Background

Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.

Methodology/Principal Findings

Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.

Conclusions/Significance

The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.     

Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues

Hypoxia-inducible factor-1 alpha (HIF-1α) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1α are being developed, but the ability to measure drug-induced changes in HIF-1α is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1α in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1α. We tested various strategies for HIF-1α stabilization in solid tumors, including nitrogen gas-purged lysis buffer, the addition of proteasome inhibitors or the prolyl hydroxylase inhibitor 2-hydroxyglutarate, and bead homogenization. Degassing and the addition of 2-hydroxyglutarate to the collection buffer significantly increased HIF-1α recovery, whereas bead homogenization in sealed tubes improved HIF-1α recovery and reduced sample variability. Validation of the ELISA demonstrated intra- and inter-assay variability of less than 15% and accuracy of 99.8 ± 8.3% as assessed by spike recovery. Inter-laboratory reproducibility was also demonstrated (R² = 0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5 μg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.     

Isolation and characterization of a new cellulosome-producing Clostridium thermocellum strain

The anaerobic thermophilic bacterium, Clostridium thermocellum, is a potent cellulolytic microorganism that produces large extracellular multienzyme complexes called cellulosomes. To isolate C. thermocellum organisms that possess effective cellulose-degrading ability, new thermophilic cellulolytic strains were screened from more than 800 samples obtained mainly from agriculture residues in Thailand using microcrystalline cellulose as a carbon source. A new strain, C. thermocellum S14, having high cellulose-degrading ability was isolated from bagasse paper sludge. Cellulosomes prepared from S14 demonstrated faster degradation of microcrystalline cellulose, and 3.4- and 5.6-fold greater Avicelase activity than those from C. thermocellum ATCC27405 and JW20 (ATCC31449), respectively. Scanning electron microscopic analysis showed that S14 had unique cell surface features with few protuberances in contrast to the type strains. In addition, the cellulosome of S14 was resistant to inhibition by cellobiose that is a major end product of cellulose hydrolysis. Saccharification tests conducted using rice straw soaked with sodium hydroxide indicated the cellulosome of S14 released approximately 1.5-fold more total sugars compared to that of ATCC27405. This newly isolated S14 strain has the potential as an enzyme resource for effective lignocellulose degradation.     

Global diversity and balancing selection of 23 leading Plasmodium falciparum candidate vaccine antigens

Investigation of the diversity of malaria parasite antigens can help prioritize and validate them as vaccine candidates and identify the most common variants for inclusion in vaccine formulations. Studies of vaccine candidates of the most virulent human malaria parasite, Plasmodium falciparum, have focused on a handful of well-known antigens, while several others have never been studied. Here we examine the global diversity and population structure of leading vaccine candidate antigens of P. falciparum using the MalariaGEN Pf3K (version 5.1) resource, comprising more than 2600 genomes from 15 malaria endemic countries. A stringent variant calling pipeline was used to extract high quality antigen gene ‘haplotypes’ from the global dataset and a new R-package named VaxPack was used to streamline population genetic analyses. In addition, a newly developed algorithm that enables spatial averaging of selection pressure on 3D protein structures was applied to the dataset. We analysed the genes encoding 23 leading and novel candidate malaria vaccine antigens including csp, trap, eba175, ama1, rh5, and CelTOS. Our analysis shows that current malaria vaccine formulations are based on rare haplotypes and thus may have limited efficacy against natural parasite populations. High levels of diversity with evidence of balancing selection was detected for most of the erythrocytic and pre-erythrocytic antigens. Measures of natural selection were then mapped to 3D protein structures to predict targets of functional antibodies. For some antigens, geographical variation in the intensity and distribution of these signals on the 3D structure suggests adaptation to different human host or mosquito vector populations. This study provides an essential framework for the diversity of P. falciparum antigens to be considered in the design of the next generation of malaria vaccines.     

Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1

An alkaliphilic bacterium, Bacillus sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, β-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60°C and 5.5, respectively. Metal ions such as Fe²⁺, Ca²⁺, and Mg²⁺ greatly increased the xylanase activity, whereas Mn²⁺ strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.     

Multi-Purpose Utility of Circulating Plasma DNA Testing in Patients with Advanced Cancers

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5–1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical ‘hot-spot’ mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.     

Proof of Concept,Randomized, Placebo-Controlled Study of the Effect of Simvastatin on the Course of Age-Related Macular Degeneration

Background

HMG Co-A reductase inhibitors are ubiquitous in our community yet their potential role in age-related macular degeneration (AMD) remains to be determined.

Methodology/Principal Findings

Objectives: To evaluate the effect of simvastatin on AMD progression and the effect modification by polymorphism in apolipoprotein E (ApoE) and complement factor H (CFH) genes. Design: A proof of concept double-masked randomized controlled study. Participants: 114 participants aged 53 to 91 years, with either bilateral intermediate AMD or unilateral non-advanced AMD (with advanced AMD in fellow eye), BCVA≥20/60 in at least one eye, and a normal lipid profile. Intervention: Simvastatin 40 mg/day or placebo, allocated 1∶1. Main outcome measures: Progression of AMD either to advanced AMD or in severity of non-advanced AMD. Results. The cumulative AMD progression rates were 70% in the placebo and 54% in the simvastatin group. Intent to treat multivariable logistic regression analysis, adjusted for age, sex, smoking and baseline AMD severity, showed a significant 2-fold decrease in the risk of progression in the simvastatin group: OR 0.43 (0.18–0.99), p = 0.047. Post-hoc analysis stratified by baseline AMD severity showed no benefit from treatment in those who had advanced AMD in the fellow eye before enrolment: OR 0.97 (0.27–3.52), p = 0.96, after adjusting for age, sex and smoking. However, there was a significant reduction in the risk of progression in the bilateral intermediate AMD group compared to placebo [adjusted OR 0.23 (0.07–0.75), p = 0.015]. The most prominent effect was observed amongst those who had the CC (Y402H) at risk genotype of the CFH gene [OR 0.08 (0.02–0.45), p = 0.004]. No evidence of harm from simvastatin intervention was detected.

Conclusion/Significance

Simvastatin may slow progression of non-advanced AMD, especially for those with the at risk CFH genotype CC (Y402H). Further exploration of the potential use of statins for AMD, with emphasis on genetic subgroups, is warranted.

Trial Registration

Australian New Zealand Clinical Trial Registry (ANZCTR) ACTRN1260500032065