Denaturing and non-denaturing microsolution isoelectric focussing to mine the metalloproteome

Metals bound to proteins play essential roles in living systems. Elements such as phosphorus, selenium and iodine are commonly covalently linked to proteins while others are non-covalently complexed. Thus, the identification and characterization of the metal-protein complexes require a careful hyphenation of techniques able to separate and detect the intact binding complexes with both high resolution and high sensitivity. This study has investigated for the first time the potential of microsolution isoelectric focussing to separate a mixture of metal-binding protein standards under well-established denaturing conditions and a novel non-denaturing separation protocol has also been developed. SEC-ICP-MS analysis was used to evaluate the ability of the two separation procedures to separate and maintain the integrity of standard metal-protein complexes. Microsolution isoelectric focussing under denaturing conditions separates the metalloprotein mixtures with high resolution, although the stability of the complexes is affected. Microsolution isoelectric focussing under our newly developed non-denaturing conditions shows a lower degree of resolution, although the stability of the metal-protein complexes is preserved. The applicability of the two procedures to a biological metalloproteome has also been evaluated.     

Diversity and endemism of Murinae rodents in Thai limestone karsts

This study aims to investigate the species diversity of rodents living in karst ecosystems of Thailand. A survey has been conducted throughout Thailand, 122 karsts sampled and 477 Murinae rodents live-trapped. Phylogenetic reconstructions were carried out using two mitochondrial markers (cytb, COI). A sequence-based species delimitation method completed by the analysis of the level of genetic divergence was then applied to define species boundaries within our dataset. The phylogenetic position of Niviventer hinpoon was also investigated and sequences obtained from the holotype specimen of this species were used to reliably identify samples of N. hinpoon. A total of 12 described Murinae species, corresponding to 17 deeply divergent genetic lineages, were encountered in limestone karsts of Thailand. Our study revealed an important genetic diversity within the traditionally recognized species Maxomys surifer (four highly divergent genetic lineages), Leopoldamys neilli (two highly divergent genetic lineages) and Berylmys bowersi (two highly divergent genetic lineages). These species could be considered as species complex and require further taxonomic work. This study also provides valuable information on the distribution of the two rodent species endemic to limestone karsts of Thailand, L. neilli and N. hinpoon. Leopoldamys neilli was the most abundant species encountered in Thai karsts during our survey. However, L. neilli specimens from western Thailand are genetically highly divergent from the remaining populations of L. neilli and could represent a separate species. Niviventer hinpoon, phylogenetically closely related to N. fulvescens, is much rarer and its distribution limited to central Thailand. Most of the other captured species are typically associated with forest ecosystems. This study suggests that limestone karsts play a key role in the preservation of the rodent species endemic to such habitat, but they would also provide refuges for the forest-dwelling Murinae rodents in deforested regions.     

Habitat differentiation of Lauraceae species in a tropical lower montane forest in northern Thailand

Dependency on topographical habitat was examined for Lauraceae tree species in a lower montane forest using a large-scale research plot established at Doi Inthanon National Park, northern Thailand. Twenty species of 10 genera of Lauraceae were recorded in a 7.5-ha part of the plot; Lauraceae accounted for 18% of the total basal area. Lauraceae was the most species-rich and most abundant family in the plot. In a cluster analysis based on the matrix of spatial associations between species, two clusters were recognized. Members of one cluster seemed to associate with lower-elevation habitats, and members of the other associated with habitats on ridges. By subdividing the study plot into 20m×20m squares, a discriminant analysis could be applied to the presence–absence data for the 17 species that had sufficient population density. The predictor variables used were the relative elevation, slope inclination, slope direction (transformed to deviation from SSW) and slope convexity for each of the squares. The discriminant models were tested statistically by applying the random shift technique. The models were significant for 11 of the species (65% of the species examined) and were associated with the topographical condition of the habitat. Stepwise selection of the predictor variables for these 11 species revealed that relative elevation and slope convexity were the most important factors for predicting the presence or absence of the Lauraceae species. Both these variables were considered to indicate the hydrological condition of the habitat.     

Neutralizing Dengue Antibody in Pregnant Thai Women and Cord Blood

Background

The WHO ‘Global Strategy for Dengue Prevention and Control, 2012–2020’ addresses the growing need for the treatment of dengue, and targets a 25% reduction in morbidity and 50% in mortality (using 2010 estimates as baseline). Achieving these goals requires future dengue prevention strategies that will employ both potential vaccines and sustainable vector-control measures. Maternally transferred dengue antibody is an important factor in determining the optimal age for dengue vaccination.

Objectives

To estimate the seroprevalence of dengue antibodies among mothers living in an area of high endemicity – Ban Pong, Ratchaburi Province – and to assess maternal dengue antibodies transferred to cord blood.

Materials & Methods

A cross-sectional study was conducted with 141 pregnant women who delivered at Ban Pong Hospital, Ratchaburi, Thailand. Maternal-cord paired sera were tested for dengue neutralizing (NT) antibody by PRNT₅₀ assay. A ratio of ≥ 1:10 NT titer to dengue serotype was considered seropositive.

Results

Most mothers (137/141, 97.2%) had NT antibodies to at least one dengue serotype in their sera. At birth, the proportion of cord sera with NT antibodies to DEN-1, DEN-2, DEN-3, and DEN-4, were high and similar to the sera of their mothers, at 93.6%, 97.2%, 97.9%, and 92.2%, respectively. The dengue geometric mean titers (GMT) in cord blood were significantly higher than the maternal antibodies (p<0.001): highest in DEN-2, followed by DEN-3, and then DEN-1. The GMT of DEN-4 was the lowest among all four serotypes.

Conclusions

Dengue infection is highly prevalent among pregnant women in this dengue-endemic area. Most of the cord blood had transferred dengue antibodies, which may have an impact on the disease burden in this population.     

A Code of Conduct for Human and Regional Security Around the South China Sea

In the past decade, there have been calls for a regional code of conduct for the South China Sea to ensure peace and stability in a region replete with conflicting territorial claims over offshore geographical features. This paper will distinguish between the process of codification of rules and principles in one document, on the one hand, and the process of implementing existing rules and principles, on the other hand. It will be argued that rules and principles governing conduct for human and regional security around the South China Sea already exist in many forms and that the South China Sea states are obligated by international law to implement them without waiting for their consolidation in one document.     

Strategies for Diagnosis and Treatment of Suspected Leptospirosis: A Cost-Benefit Analysis

Background

Symptoms and signs of leptospirosis are non-specific. Several diagnostic tests for leptospirosis are available and in some instances are being used prior to treatment of leptospirosis-suspected patients. There is therefore a need to evaluate the cost-effectiveness of the different treatment strategies in order to avoid misuse of scarce resources and ensure best possible health outcomes for patients.

Methods

The study population was adult patients, presented with uncomplicated acute febrile illness, without an obvious focus of infection or malaria or typical dengue infection. We compared the cost and effectiveness of 5 management strategies: 1) no patients tested or given antibiotic treatment; 2) all patients given empirical doxycycline treatment; patients given doxycycline when a patient is tested positive for leptospirosis using: 3) lateral flow; 4) MCAT; 5) latex test. The framework used is a cost-benefit analysis, accounting for all direct medical costs in diagnosing and treating patients suspected of leptospirosis. Outcomes are measured in length of fever after treatment which is then converted to productivity losses to capture the full economic costs.

Findings

Empirical doxycycline treatment was the most efficient strategy, being both the least costly alternative and the one that resulted in the shortest duration of fever. The limited sensitivity of all three diagnostic tests implied that their use to guide treatment was not cost-effective. The most influential parameter driving these results was the cost of treating patients with complications for patients who did not receive adequate treatment as a result of incorrect diagnosis or a strategy of no-antibiotic-treatment.

Conclusions

Clinicians should continue treating suspected cases of leptospirosis on an empirical basis. This conclusion holds true as long as policy makers are not prioritizing the reduction of use of antibiotics, in which case the use of the latex test would be the most efficient strategy.     

Novel endotoxin-sequestering compounds with terephthalaldehyde-bis-guanylhydrazone scaffolds

We have shown that lipopolyamines bind to the lipid A moiety of lipopolysaccharide, a constituent of Gram-negative bacterial membranes, and neutralize its toxicity in animal models of endotoxic shock. In an effort to identify non-polyamine scaffolds with similar endotoxin-recognizing features, we had observed an unusually high frequency of hits containing guanylhydrazone scaffolds in high-throughput screens. We now describe the syntheses and preliminary structure-activity relationships in a homologous series of bis-guanylhydrazone compounds decorated with hydrophobic functionalities. These first-generation compounds bind and neutralize lipopolysaccharide with a potency comparable to that of polymyxin B, a peptide antibiotic known to sequester LPS.     

Development and application of mass spectrometric methods for the analysis of progranulin N-glycosylation

PGRN is a modular protein with 7 1/2 repeats of the granulin domain separated by short spacer sequences. Elevated expression of PGRN is associated with cancer growth, while mutations of PGRN cause frontotemporal lobar degeneration (FTLD), an early onset form of dementia. PGRN is a glycoprotein, containing five N-glycosylation consensus sequons, three of which fall within granulin domains. A method tailored to enable detailed analysis of the PGRN oligosaccharides and glycopeptides has been developed. The approach involves in-gel deglycosylation using peptide-N-glycosidase F (PNGase F) followed by permethylation of the released oligosaccharides. Permethylation was applied for rapid sample clean-up and to improve sensitivity of MS detection and mass spectrometric fragmentation. Reversed-phase monolithic LC–ESI–MS/MS was used for analysis of permethylated oligosaccharides, enabling structural characterization of released N-linked glycans in one chromatographic run. In-gel tryptic digestion was further applied to the gel pieces containing deglycosylated protein, for N-glycosylation site determination. In addition, glycopeptides were produced using in-solution pronase digestion to identify species of N-glycan attached at particular sites. The method developed was applied to progranulin (PGRN) to characterize the structures of the released glycans and to identify the sites of glycosylation. Glycosylation of four out of five potential PGRN N-glycosylation consensus sites was demonstrated (the final one remains undetermined), with one of the four observed to be partially occupied. Two of the observed glycosylation sites occur within granulin domains, which may have important implications for understanding the structural basis of PGRN action.