Analysis of 16S rRNA Primer Systems for Profiling of Thermophilic Microbial Communities

Microbiology - Thermophilic microorganisms are of special interest for phylogenetics and research in prokaryotic evolution, since many of them belong to deep branches of the tree of life. For this...     

Chromatin-Based Classification of Genetically Heterogeneous AMLs into Two Distinct Subtypes with Diverse Stemness Phenotypes

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Fractionated breath condensate sampling: H2O2 concentrations of the alveolar fraction may be related to asthma control in children

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.     

Estradiol abolishes reduction in cell death by the opioid agonist Met5-enkephalin after oxygen glucose deprivation in isolated cardiomyocytes from both sexes

There is evidence for differences in the response to the treatment of cardiovascular disease in men and women. In addition, there are conflicting results regarding the effectiveness of pharmacologically induced protection or ischemic preconditioning in females. We investigated whether the ability of Met(5)-enkephalin (ME) to reduce cell death after oxygen-glucose deprivation (OGD) is influenced by the presence of 17beta-estradiol (E(2)) in a nitric oxide (NO)- and estrogen receptor-dependent manner. On postnatal day 7 to 8, murine cardiomyocytes from wild-type or inducible NO synthase (iNOS) knockout mice were separated by sex, isolated by collagenase digestion, cultured for 24 h, and subjected to 90 min OGD and 180 min reoxygenation at 37 degrees C (n = 4 to 5 replicates). Cell cultures were incubated in E(2) for 15 min or 24 h before OGD. ME was used to increase cell survival. Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. Data are presented as means +/- SE. As a result, in both sexes, ME-induced cell survival was lost in the presence of E(2), and the ability of ME to improve cell survival was restored after treatment with the estrogen receptor antagonist ICI-182780. Furthermore, iNOS was necessary for ME to increase cell survival following OGD in vitro. We conclude that ME-induced reduction in cell death is abolished by E(2) in a sex-independent manner via activation of estrogen receptors, and this interaction is dependent on iNOS.     

Sex differences in the mechanism of Met5-enkephalin-induced cardioprotection: role of PI3K/Akt

Met(5)-enkephalin (ME)-induced cardioprotection occurs via epidermal growth factor receptor (EGFR) transactivation with the subsequent activation of phosphatidylinositol 3-kinase (PI3K). In the present study, we investigated whether there is a sex difference in ME-elicited PI3K signaling. Neonatal murine cardiomyocytes were isolated by collagenase digestion and subjected to 90 min hypoxia and 180 min reoxygenation at 37 degrees C (n = 5 to 7 replicates). PI3K/Akt signaling was interrogated using pharmacological inhibitors and small interfering RNA (siRNA). Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. The data are presented as means +/- SE. There was not a sex difference in the basal content of total Akt. ME (100 microM) elicited comparable protection in both sexes. Wortmannin and the nonselective Akt inhibitor IV completely abolished ME-induced protection in male cardiomyocytes but only attenuated protection in female cardiomyocytes. Isoform-selective knockdown of Akt in males with siRNAs against Akt1/2 completely abolished ME-induced cardioprotection, whereas the siRNAs against Akt3 only attenuated protection of approximately 40%. In contrast, in females the siRNAs against Akt1/2 attenuated and against Akt3 eliminated ME-induced cardioprotection. There is not a sex difference in the degree of ME-induced protection, and there is a sex difference in the cardioprotective signaling pathways after the administration of ME; ME-induced cardioprotection in males primarily utilizes a PI3K/Akt1/2 pathway and in females primarily utilizes a PI3K/Akt3 pathway. The incomplete loss of protection in females following the blockade of PI3K suggests that additional factors may facilitate the maintenance or function of activated Akt.     

Low genetic diversity and lack of population structure in the endangered Galápagos penguin (<Emphasis Type="Italic">Spheniscus mendiculus</Emphasis>)

Long-term monitoring of the endangered Galápagos penguin (Spheniscus mendiculus) has indicated poor reproductive periods and severe population fluctuations in association with El Niño – Southern Oscillation events. An earlier mark and recapture study indicated that adults exhibit some degree of breeding-site and mate fidelity, and that juveniles potentially move more frequently than adults; however, the extent to which migrants and gene flow occur between islands within the Galápagos archipelago is largely unknown. This study tested the hypothesis that geographic isolation and adult breeding philopatry has led to a degree of genetic differentiation between island subpopulations within the archipelago. We examined the genetic diversity within and among different subpopulations and the extent to which gene flow occurs between island subpopulations. Estimates of allelic richness and gene diversity were not significantly different between subpopulations. Tests to detect genetic heterogeneity failed to reject the H ₀ of no difference in allele frequencies for chi-square (P = 0.28) and Fisher’s exact test (P = 0.19). All pairwise values of the F _ST variant θ were not significant, while a power analysis revealed a >99% probability of detecting a biologically true F _ST of 0.05. Migration estimates in BAYESASS+ suggest symmetrical gene flow throughout the species’ distribution. Our results indicate a low level of genetic diversity throughout the population and a seemingly high level of gene flow between subpopulations. We argue that the Galápagos penguin should be managed as one panmictic population and we discuss the risk of disease threats in the archipelago.     

Microbial diversity and autotrophic activity in Kamchatka hot springs

Microbial communities of Kamchatka Peninsula terrestrial hot springs were studied using molecular, radioisotopic and cultural approaches. Analysis of 16S rRNA gene fragments performed by means of high-throughput sequencing revealed that aerobic autotrophic sulfur-oxidizing bacteria of the genus Sulfurihydrogenibium (phylum Aquificae) dominated in a majority of streamers. Another widely distributed and abundant group was that of anaerobic bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). Archaea of the genus Vulcanisaeta were abundant in a high-temperature, slightly acidic hot spring, where they were accompanied by numerous Nanoarchaeota, while the domination of uncultured Thermoplasmataceae A10 was characteristic for moderately thermophilic acidic habitats. The highest rates of inorganic carbon assimilation determined by the in situ incubation of samples in the presence of ¹⁴C-labeled bicarbonate were found in oxygen-dependent streamers; in two sediment samples taken from the hottest springs this process, though much weaker, was found to be not dependent on oxygen. The isolation of anaerobic lithoautotrophic prokaryotes from Kamchatka hot springs revealed a wide distribution of the ability for sulfur disproportionation, a new lithoautotrophic process capable to fuel autonomous anaerobic ecosystems.

     

Circulating angiopoietin-2 as a biomarker in ANCA-associated vasculitis

The endothelial-specific Angiopoietin-Tie2 ligand-receptor system is an important regulator of endothelial activation. Binding of angiopoietin-2 (Ang-2) to Tie2 receptor renders the endothelial barrier responsive to pro-inflammatory cytokines. We previously showed that circulating Ang-2 correlated with disease severity in a small cohort of critically ill patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. The current study reassessed Ang-2 as a biomarker of disease activity and relapse in AAV. Circulating Ang-2 was measured in 162 patients with severe AAV (BVAS/WG≥3, with or without glomerulonephritis) in a clinical trial. Ang-2 levels during active AAV were compared to levels in the same patients during remission (BVAS/WG = 0). Levels in clinical subsets of AAV were compared, and association with future disease course was assessed. Ang-2 levels were elevated in severe disease (median 3.0 ng/ml, interquartile range 1.9–4.4) compared to healthy controls (1.2, 0.9–1.5). However, they did not reliably decline with successful treatment (median 2.6 ng/ml, interquartile range 1.9–3.8, median change −0.1). Ang-2 correlated weakly with BVAS/WG score (r = 0.17), moderately with markers of systemic inflammation (r = 0.25–0.41), and inversely with renal function (r = −0.36). Levels were higher in patients with glomerulonephritis, but levels adjusted for renal dysfunction were no different in patients with or without glomerulonephritis. Levels were higher in patients with newly diagnosed AAV and lower in patients in whom treatment had recently been started. Ang-2 levels during active disease did not predict response to treatment, and Ang-2 levels in remission did not predict time to flare. Thus, Ang-2 appears to have limited practical value in AAV as a biomarker of disease activity at time of measurement or for predicting future activity.     

The promising effects of BMP2 transfected mesenchymal stem cells on human osteosarcoma

Selective targeting of transfected mesenchymal stem cells (MSCs) carrying specific antioncogenes to the tumor was suggested as a treatment option. Bone morphogenetic protein-2 (BMP2) was shown to inhibit the proliferation and aggressiveness of osteosarcoma (OS) cells. Here, we aimed to assess the homing efficiency of intraperitoneally administered hMSCs transfected with BMP2 to the tumoral site and their effects on OS using an orthotopic xenograft murine model. Orthotopic xenograft murine model of OS in six-week-old female NOD/SCID mice using 143B cells was established. hMSCs transfected with BMP2 (BMP2⁺hMSC) were used. In vivo experiments performed on four groups of mice that received no treatment, or intraperitoneally administered BMP2, hMSCs, and BMP2⁺hMSCs. Histopathological and immunohistochemical studies were used to evaluate the pathological identification and to assess the dimensions and necrotic foci of the tumor, the features of lung metastases, and immunostaining against p27, Ki-67, and caspase-3 antibodies. The osteogenic differentiation markers BMP2, BMP4, COL1A1, OPN, OCN and PF4 evaluated using RT-PCR. The tumor dimensions in the hMSCs group were significantly higher than those of the remaining groups (p < 0.01). The number of metastatic foci in the BMP2⁺hMSCs group was significantly lower than those of the other groups (p < 0.01). The current results showed that the intraperitoneal route could be efficiently used for targeting hMSCs to the tumoral tissues for effective BMP2 delivery. In this study, the effects of BMP2 transfected hMSCs on human OS and metastasis were promising for achieving osteogenic differentiation and reduced metastatic process.