Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.     

Design,synthesis, and docking of highly hypolipidemic agents: Schizosaccharomyces pombe as a new model for evaluating α-asarone-based HMG-CoA reductase inhibitors

A series of α-asarone-based analogues was designed by conducting docking experiments with published crystal structures of human HMG-CoA reductase. Indeed, synthesis and evaluation of this series showed a highly hypocholesterolemic in vivo activity in a murine model, as predicted by previous docking studies. In agreement with this model, the polar groups attached to the benzene ring could play a key role in the enzyme binding and probably also in its biological activity, mimicking the HMG-moiety of the natural substrate. The hypolipidemic action mechanism of these compounds was investigated by developing a simple, efficient, and novel model for determining HMG-CoA reductase inhibition. The partial purification of the enzyme from Schizosaccharomyces pombe allowed for testing of α-asarone- and fibrate-based analogues, resulting in positive and significant inhibitory activity.     

Seroprevalence of Angiostrongylus cantonensis in wild rodents from the Canary Islands

Background

Angiostrongylus cantonensis is a lungworm of rats (Muridae) that is the causative agent of human cerebral angiostrongyliasis. The life cycle of A. cantonensis involves rats and mollusks as the definitive and intermediate hosts, respectively. This study was designed to increase the knowledge about the occurrence and distribution of A. cantonensis in its definitive host in the Canary Islands, using parasitological and serological analysis in different areas and age groups.

Methodology/Principal Findings

Between 2009 and 2010, 54 black rats (Rattus rattus) from Tenerife were captured from six human-inhabited areas and sera samples were obtained. The lung nematodes were identified by morphological and molecular tools as A. cantonensis. The 31-kDa glycoprotein antigen was purified from A. cantonensis adult worms by electrophoresis and electroelution. Of the 54 tested rodents, 30 showed IgG antibodies against A. cantonensis 31-kDa antigen by ELISA. Therefore, the overall seroprevalence was 55.6% (95% CI: 42.4–68). Seroprevalent rodents were found in all the 6 areas. This 31-kDa antigen was not recognized by some sera of rats infected by other helminth species (but not A. cantonensis). Seroprevalence of IgG antibodies against A. cantonensis and prevalence based on the presence of adult worms showed significant correlation (R² = 0.954, p<0.05).

Conclusions/Significance

The present results could indicate a high prevalence of A. cantonensis in Tenerife and suggest the inclusion of two new zones in the distribution area of the parasite. The commonness and wide distribution of A. cantonensis in rats implies the presence of intermediate hosts, indicating that humans may be at risk of getting infected.     

Aerobic biodegradation of a mixture of sulfonated azo dyes by a bacterial consortium immobilized in a two‐stage sparged packed‐bed biofilm reactor

The biodegradation of the sulfonated azo dyes, Acid Orange 7 (AO7) and Acid Red 88 (AR88), by a bacterial consortium isolated from water and soil samples obtained from sites receiving discharges from textile industries, was evaluated. For a better removal of azo dyes and their biodegradation byproducts, an aerobically operated two‐stage rectangular packed‐bed biofilm reactor (2S‐RPBR) was constructed. Because the consortium's metabolic activity is affected by oxygen, the effect of the interstitial air flow rate Q_GI on 2S‐RPBR's zonal values of the oxygen mass transfer coefficient k_La was estimated. In the operational conditions probed in the bioreactor, the k_La values varied from 3 to 60 h^?1, which roughly correspond to volumetric oxygen transfer rates, dc_L/dt, ranging from 20 to 375 mg O₂ L^?1h^?1. Complete biodegradation of azo dyes was attained at loading rates B_V,AZ up to 40 mg L^?1d^?1. At higher B_V,AZ values (80 mg L^?1 d^?1), dye decolorization and biodegradation of the intermediaries 4‐amino‐naphthalenesulphonic acid (4‐ANS) and 1‐amino‐2‐naphthol (1‐A2N) was almost complete. However, a diminution in COD and TOC removal efficiencies was observed in correspondence to the 4‐aminobenzenesulfonic acid (4‐ABS) accumulation in the bioreactor. Although the oxygen transport rate improved the azo dye mineralization, the results suggest that the removal efficiency of azo dyes was affected by biofilm detachment at relatively high Q_GI and B_V,AZ values. After 225 days of continuous operation of the 2S‐RFBR, eight bacterial strains were isolated from the biofilm attached to the porous support. The identified genera were: Arthrobacter, Variovorax, Agrococcus, Sphingomonas, Sphingopyxis, Methylobacterium, Mesorhizobium, and Microbacterium.     

The FTO gene is associated with adulthood obesity in the Mexican population

Common polymorphisms in the fat mass and obesity-associated gene (FTO) have shown strong association with obesity in several populations. In the present study, we explored the association of FTO gene polymorphisms with obesity and other biochemical parameters in the Mexican population. We also assessed FTO gene expression levels in adipose tissue of obese and nonobese individuals. The study comprised 788 unrelated Mexican-Mestizo individuals and 31 subcutaneous fat tissue biopsies from lean and obese women. FTO single-nucleotide polymorphisms (SNPs) rs9939609, rs1421085, and rs17817449 were associated with obesity, particularly with class III obesity, under both additive and dominant models (P = 0.0000004 and 0.000008, respectively). These associations remained significant after adjusting for admixture (P = 0.000003 and 0.00009, respectively). Moreover, risk alleles showed a nominal association with lower insulin levels and homeostasis model assessment of B-cell function (HOMA-B), and with higher homeostasis model assessment of insulin sensitivity (HOMA-S) only in nonobese individuals (P (dom) = 0.031, 0.023, and 0.049, respectively). FTO mRNA levels were significantly higher in subcutaneous fat tissue of class III obese individuals than in lean individuals (P = 0.043). Risk alleles were significantly associated with higher FTO expression in the class III obesity group (P = 0.047). In conclusion, FTO is a major risk factor for obesity (particularly class III) in the Mexican-Mestizo population, and is upregulated in subcutaneous fat tissue of obese individuals.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.     

Impact of the Non-Contributory Social Pension Program 70 y más on Older Adults’ Mental Well-Being

Background

In 2007, a non-contributory pension program was launched in rural areas of Mexico. The program consisted in a non-conditional cash transfer of US$40 monthly to all older adults (OA) aged 70 and over. We evaluate the effect of the program on mental well-being of its beneficiaries.

Methods and Findings

Quantitative and qualitative methods were used. For the quantitative component, we used the selection criteria established by the program (age and locality size) to form the Intervention (OA aged 70–74 residing in rural localities, <2500 inhabitants) and Control groups (OA aged 70–74, in localities with 2501–2700 inhabitants). Baseline data collection was conducted in 2007 where 5,465 OA were interviewed. The follow-up survey was conducted in 2008, and it was possible to interview 5,270 OA, with a response rate of 96%. A difference-in-difference linear probability model with individual fixed effect was used to estimate the impact of the program on mental well-being indicators. In 2009 a qualitative component was designed to explore possible causal pathways of such effect.

Results

After a year of exposure, the program had a significant effect on reduction of depressive symptoms (β = −0.06, CI_95% −0.12; −0.01) and an increase in empowerment indicators: OA participated in important household decisions (β = 0.09, CI_95% 0.03;0.15); and OA participated in household decisions pertaining to expenses (β = 0.11, CI_95% 0.05;0.18). Qualitative analysis found a strong trend showing a reduction of sadness, and feeling of increasing empowerment.

Conclusions

These results suggest that a non-conditional transfer in older ages have an impact beyond the economic sphere, impacting even the mental well-being. This effect could be explained because the pension produces feelings of safety and welfare. It is recommendable that governments should invest efforts towards universalizing the non-contributory pension programs in order to ensure a basic income for the elderly.     

Study of the design and analytical properties of the lethality neutralization assay used to estimate antivenom potency against Bothrops asper snake venom

The lethality neutralization assay performed in mice is the standard recommended by the World Health Organization to estimate antivenom potency. The interpretation of its results without considering its analytical capacity may lead to erroneous conclusions. Therefore, laboratories that manufacture or control antivenoms must demonstrate the appropriateness of their models. A study of the method used at Instituto Clodomiro Picado, Costa Rica, to estimate the potency of antivenoms against Bothrops asper snake venom was performed. Results show that venom doses ranging from 2 to 6 Median Lethal Doses (LD₅₀) are appropriate to be used as challenge in this test. Variables such as the injection route, number of mice used per venom/antivenom level, and weight of the animals are critical in the estimation of the Median Effective Dose (ED₅₀), whereas incubation time is not. The assay has an acceptable selectivity, linearity, and limits of detection and quantification. Accuracy of the lethality neutralization assay, expressed as percentage recovery, was between 71% and 127%. Intermediate precision, expressed as relative standard deviation, was ≤17%. It is concluded that the analytical characteristics of this assay are adequate enough to prove product compliance and to have statistical control over an industrial line of antivenom serial production.