Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour

Aims: This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development. Methods and Results: Extracellular DNA was purified and characterized in a 2‐day‐old mature biofilm developed by the reference strain H. pylori ATCC 43629, the clinical isolate H. pylori SDB60 and the environmental strain H. pylori MDC1. Subsequently, the role of eDNA in the H. pylori biofilm was evaluated by adding DNase I during biofilm formation and on mature biofilms. Extracellular DNA was detected in the 2‐day‐old EPS biofilm matrix of all analysed H. pylori strains. The DNA fingerprintings, performed by RAPD analysis, on eDNA and intracellular DNA (iDNA), showed some remarkable differences. The data obtained by microtitre biofilm assay as well as colony forming unit count and CLSM (confocal laser scanning microscopy) qualitative analysis did not show any significant differences between the DNase I‐treated biofilms and the corresponding not treated controls both in formation and on mature biofilms. Conclusions: In this study, we provide evidence that eDNA is a component of the EPS matrix of H. pylori biofilm. The different profiles of eDNA and iDNA indicate that lysed cells are not the primary source of eDNA release, suggesting that other active mechanisms might be involved in this process. Moreover, the biomass assay suggests that eDNA may not be the main component of biofilm matrix, suggesting that it could be primarily involved in other mechanisms such as recombination processes, via transformation, contributing to the wide genomic variability of this micro‐organism defined as a ‘quasi‐species’. Significance and Impact of the Study: The presence of eDNA in H. pylori biofilm can contribute to the active dynamic exchange of information aimed to reach the best condition for the bacterial survival in the host and in the environment.     

Detailed Dimethylacetal and Fatty Acid Composition of Rumen Content from Lambs Fed Lucerne or Concentrate Supplemented with Soybean Oil

Lipid metabolism in the rumen is responsible for the complex fatty acid profile of rumen outflow compared with the dietary fatty acid composition, contributing to the lipid profile of ruminant products. A method for the detailed dimethylacetal and fatty acid analysis of rumen contents was developed and applied to rumen content collected from lambs fed lucerne or concentrate based diets supplemented with soybean oil. The methodological approach developed consisted on a basic/acid direct transesterification followed by thin-layer chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo- fatty acid and fatty acid dimethylesters. The dimethylacetal composition was quite similar to the fatty acid composition, presenting even-, odd- and branched-chain structures. Total and individual odd- and branched-chain dimethylacetals were mostly affected by basal diet. The presence of 18∶1 dimethylacetals indicates that biohydrogenation intermediates might be incorporated in structural microbial lipids. Moreover, medium-chain fatty acid dimethylesters were identified for the first time in the rumen content despite their concentration being relatively low. The fatty acids containing 18 carbon-chain lengths comprise the majority of the fatty acids present in the rumen content, most of them being biohydrogenation intermediates of 18∶2n−6 and 18∶3n−3. Additionally, three oxo- fatty acids were identified in rumen samples, and 16-O-18∶0 might be produced during biohydrogenation of the 18∶3n−3.     

Bioaugmentation of a rotating biological contactor for degradation of 2-fluorophenol

The performance of a laboratory scale rotating biological contactor (RBC) towards shock loadings of 2-fluorophenol (2-FP) was investigated. During a period of ca. 2 months organic shock loadings of 25 mg L?1 of 2-FP were applied to the RBC. As no biodegradation of 2-FP was observed, bioaugmentation of the RBC with a 2-FP degrading strain was carried out and, along ca. 6 months, organic shock loadings within a range of 25-200 mg L?1 of 2-FP were applied. Complete biodegradation of 50 mg L?1 of 2-FP was observed during operation of the reactor. The RBC showed to be robust towards starvation periods, as after ca. 1month of non-supply of the target compound, the reactor resumed 2-FP degradation. The inoculated strain was retained within the biofilm in the disks, as the 2-FP degrading strain was recovered from the biofilm by the end of the experiment, thus bioaugmentation was successfully achieved.     

The fitness of the Brazilian damsel <Emphasis Type="Italic">Stegastes fuscus</Emphasis> is increased by sharing the territory with the dusky grouper <Emphasis Type="Italic">Epinephelus marginatus</Emphasis>

Stegastes fuscus and Epinephelus marginatus are known for co-habiting shelters. The damselfish S. fuscus uses the territory for nesting and must protect its eggs from grazers; the grouper E. marginatus is an omnivorous sit-and-wait predator. This study aims to evaluate the effect of juvenile groupers on the reproductive success of the Brazilian damsel. Twenty-five hours of underwater observations were done in São Sebastião and Ilhabela, Northern shore of São Paulo, Brazil. Fitness increase was measured by the egg-clutch area and number of contributing females in 130 nests shared by groupers and another 130 where damselfishes stood alone. An egg predator crab was placed into the damselfish territory, and behavioural responses during 2 min were recorded for nests with or without E. marginatus, 80 replicates each. Nests shared by the dusky groupers had more eggs and received eggs from more females too. While fathers who were alone in the territory had to deal with the egg predator crab, in shared nests, the grouper would take care of the intruder, sometimes feeding on it. Therefore, the Brazilian damsel may benefit from the presence of the dusky grouper by increasing the fitness and diminishing the costs of parental care.     

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM—which exhibited a significant diversity in their TN-C content—in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74–97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.     

Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background

Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B₁ receptor knockout mice (B₁ ^−/−) are leaner and exhibit improved insulin sensitivity.

Methodology/Principal Findings

Here we show that kinin B₁ receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B₁ receptors. In these cells, treatment with the B₁ receptor agonist des-Arg⁹-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B₁ ^−/− mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B₁ receptor was limited to cells of the adipose tissue (aP2-B₁/B₁ ^−/−). Similarly to B₁ ^−/− mice, aP2-B₁/B₁ ^−/− mice were leaner than wild type controls. However, exclusive expression of the kinin B₁ receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B₁ ^−/− mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B₁ receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B₁/B₁ ^−/− when compared to B₁ ^−/− mice. When subjected to high fat diet, aP2-B₁/B₁ ^−/− mice gained more weight than B₁ ^−/− littermates, becoming as obese as the wild types.

Conclusions/Significance

Thus, kinin B₁ receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.     

Role of IFN-γ +874 T/A single nucleotide polymorphism in the tuberculosis outcome among Brazilians subjects

Several genetic cytokine gene variants have been associated with host susceptibility to infectious diseases, including tuberculosis. Based upon the importance of IFN-γ in protective immunity against Mycobacterium tuberculosis, and the functional role of the IFN-γ + 874T/A single nucleotide polymorphism in IFN-γ production, we genotyped 93 Brazilian tuberculosis patients and 266 asymptomatic health care workers, including 150 individuals with a positive tuberculin skin test, and analyzed the possible association of the +874A low IFN-γ producer allele with tuberculosis occurrence. Using multivariable logistic regression models, genotype and allele frequencies of the mutant + 874A (low IFN-γ producer) allele were significantly associated with tuberculosis disease. Heterozygous carriers had a 25% increased chance, while individuals presenting the A/A homozygous genotype had an over two-fold risk of having active tuberculosis (95% CI, 1.16–5.91, P = 0.03). Despite the mixed ethnicity observed in Brazilian populations, the present data agree with observations reported in other populations and thus demonstrate that the functional +874T/A IFN-γ gene polymorphism is associated with tuberculosis in different populations.     

The role of urinary KIM-1, NGAL,CA19-9 and β2-microglobulin in the assessment of ureteropelvic junction obstruction in adults

Purpose: The objective of this study is to evaluate the diagnostic properties of urinary biomarkers in adults with ureteropelvic junction obstruction: KIM-1, NGAL, CA19-9, and β2-microglobulin. We also assessed urinary biomarker concentrations following pyeloplasty.

Material and methods: We prospectively studied adults from December 2013 to February 2015. We included 47 patients with a mean age of 38.6?±?12.7 years. Each patient provided four samples of voided urine for biomarker measurement, one at pre-operative consultation and the others at 1, 3, and 6 months of post-operative follow-up. The control group consisted of 40 healthy individuals with no hydronephrosis on ultrasound evaluation.

Results: KIM-1 had an area under the curve of 0.79 (95% CI 0.70–0.89), NGAL 0.71 (95% CI 0.61–0.83), CA19-9 0.70 (95% CI 0.60–0.81), and β2-microgloblin 0.61 (95% CI 0.50–0.73). KIM-1 was the most sensitive marker with a cut-off of 170.4?pg/mg creatinine (sensitivity 91.4%, specificity 59.1%), whereas CA19-9 was the most specific with a cut-off of 51.3?U/mg creatinine (sensitivity 48.9%, specificity 88.0%). Urinary concentrations of biomarkers decreased after pyeloplasty.

Conclusions: The evaluation of urinary biomarkers is useful in adults undergoing pyeloplasty. KIM-1, NGAL, and CA19-9 were elevated and significantly decreased after surgery.     

Using C. elegans to Decipher the Cellular and Molecular Mechanisms Underlying Neurodevelopmental Disorders

Neurodevelopmental disorders such as epilepsy, intellectual disability (ID), and autism spectrum disorders (ASDs) occur in over 2 % of the population, as the result of genetic mutations, environmental factors, or combination of both. In the last years, use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. Nevertheless, following association of novel genes with a given disease, interpretation of findings is often difficult due to lack of information on gene function and effect of a given mutation in the corresponding protein. This brings the need to validate genetic associations from a functional perspective in model systems in a relatively fast but effective manner. In this context, the small nematode, Caenorhabditis elegans, presents a good compromise between the simplicity of cell models and the complexity of rodent nervous systems. In this article, we review the features that make C. elegans a good model for the study of neurodevelopmental diseases. We discuss its nervous system architecture and function as well as the molecular basis of behaviors that seem important in the context of different neurodevelopmental disorders. We review methodologies used to assess memory, learning, and social behavior as well as susceptibility to seizures in this organism. We will also discuss technological progresses applied in C. elegans neurobiology research, such as use of microfluidics and optogenetic tools. Finally, we will present some interesting examples of the functional analysis of genes associated with human neurodevelopmental disorders and how we can move from genes to therapies using this simple model organism.