Ethanol blocks leukocyte recruitment and endothelial cell activation in vivo and in vitro

Immune system impairment and increased susceptibility to infection among alcohol abusers is a significant but not well-understood problem. We hypothesized that acute ethanol administration would inhibit leukocyte recruitment and endothelial cell activation during inflammation and infection. Using LPS and carrageenan air pouch models in mice, we found that physiological concentrations of ethanol (1-5 g/kg) significantly blocked leukocyte recruitment (50-90%). Because endothelial cell activation and immune cell-endothelial cell interactions are critical regulators of leukocyte recruitment, we analyzed the effect of acute ethanol exposure on endothelial cell activation in vivo using the localized Shwartzman reaction model. In this model, ethanol markedly suppressed leukocyte accumulation and endothelial cell adhesion molecule expression in a dose-dependent manner. Finally, we examined the direct effects of ethanol on endothelial cell activation and leukocyte-endothelial cell interactions in vitro. Ethanol, at concentrations within the range found in human blood after acute exposure and below the levels that induce cytotoxicity (0.1-0.5%), did not induce endothelial cell activation, but significantly inhibited TNF-mediated endothelial cell activation, as measured by adhesion molecule (E-selectin, ICAM-1, VCAM-1) expression and chemokine (IL-8, MCP-1, RANTES) production and leukocyte adhesion in vitro. Studies exploring the potential mechanism by which ethanol suppresses endothelial cell activation revealed that ethanol blocked NF-kappaB nuclear entry in an IkappaBalpha-dependent manner. These findings support the hypothesis that acute ethanol overexposure may increase the risk of infection and inhibit the host inflammatory response, in part, by blocking endothelial cell activation and subsequent immune cell-endothelial cell interactions required for efficient immune cell recruitment.     

The application of ultrasound as a rapid method to provide DNA fragments suitable for detection by DNA biosensors

Contamination of food and water supplies by microorganisms such as Escherichia coli, the need for point-of-care bedside analysis of biological samples, and concerns about terrorist attacks using biological organisms, have made the development of fast, reliable, and sensitive analytical methodologies for use in monitoring of pathogens very important. With a variety of biosensors being developed for extremely sensitive and rapid nucleic acid diagnostics, it has become even more important to shift focus towards creation of methods to decrease the amount of time and effort necessary for sample preparation. The application of ultrasound has the potential to create DNA fragments from genomic material with lengths that are suitable for determination using biosensors and microarrays. For example, application of 85 W power at a frequency of 20 kHz can produce a preponderance of fragments of 100-400 base pairs (bp) within several seconds, and sample processing can lead to over 75% conversion from genomic material to fragments in times of 20-30 s. A proportion of these fragments are in a single-stranded state and are suitable for hydridization with immobilized single-stranded DNA probe oligonucleotides using a fiber optic biosensor. Control of factors such as salt concentration, exposure time, ultrasound power, and the initial temperature of the solution, can affect the length and form (single- or double-stranded) of DNA fragments that are generated by ultrasound, and average fragment length can be adjusted by selection of these operating parameters.     

A test of the social cohesion hypothesis: interactive female marmots remain at home

Individuals frequently leave home before reaching reproductive age, but the proximate causes of natal dispersal remain relatively unknown. The social cohesion hypothesis predicts that individuals who engage in more (affiliative) interactions are less likely to disperse. Despite the intuitive nature of this hypothesis, support is both limited and equivocal. We used formal social network analyses to quantify precisely both direct and indirect measures of social cohesion in yellow-bellied marmots. Because approximately 50 per cent of female yearlings disperse, we expected that social relationships and network measures of cohesion would predict dispersal. By contrast, because most male yearlings disperse, we expected that social relationships and cohesion would play a less important role. We found that female yearlings that interacted with more individuals, and those that were more socially embedded in their groups, were less likely to disperse. For males, social interactions were relatively unimportant determinants of dispersal. This is the first strong support for the social cohesion hypothesis and suggests that the specific nature of social relationships, not simply the number of affiliative relationships, may influence the propensity to disperse.     

Fulfilling the Promise of Personalized Medicine? Systematic Review and Field Synopsis of Pharmacogenetic Studies

Background

Studies of the genetic basis of drug response could help clarify mechanisms of drug action/metabolism, and facilitate development of genotype-based predictive tests of efficacy or toxicity (pharmacogenetics).

Objectives

We conducted a systematic review and field synopsis of pharmacogenetic studies to quantify the scope and quality of available evidence in this field in order to inform future research.

Data Sources

Original research articles were identified in Medline, reference lists from 24 meta-analyses/systematic reviews/review articles and U.S. Food and Drug Administration website of approved pharmacogenetic tests.

Study Eligibility Criteria, Participants, and Intervention Criteria

We included any study in which either intended or adverse response to drug therapy was examined in relation to genetic variation in the germline or cancer cells in humans.

Study Appraisal and Synthesis Methods

Study characteristics and data reported in abstracts were recorded. We further analysed full text from a random 10% subset of articles spanning the different subclasses of study.

Results

From 102,264 Medline hits and 1,641 articles from other sources, we identified 1,668 primary research articles (1987 to 2007, inclusive). A high proportion of remaining articles were reviews/commentaries (ratio of reviews to primary research approximately 25∶1). The majority of studies (81.8%) were set in Europe and North America focussing on cancer, cardiovascular disease and neurology/psychiatry. There was predominantly a candidate gene approach using common alleles, which despite small sample sizes (median 93 [IQR 40–222]) with no trend to an increase over time, generated a high proportion (74.5%) of nominally significant (p<0.05) reported associations suggesting the possibility of significance-chasing bias. Despite 136 examples of gene/drug interventions being the subject of ≥4 studies, only 31 meta-analyses were identified. The majority (69.4%) of end-points were continuous and likely surrogate rather than hard (binary) clinical end-points.

Conclusions and Implications of Key Findings

The high expectation but limited translation of pharmacogenetic research thus far may be explained by the preponderance of reviews over primary research, small sample sizes, a mainly candidate gene approach, surrogate markers, an excess of nominally positive to truly positive associations and paucity of meta-analyses. Recommendations based on these findings should inform future study design to help realise the goal of personalised medicines.

Systematic Review Registration Number

Not Registered     

The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.     

Unexpected Diversity of Feral Genetically Modified Oilseed Rape (Brassica napus L.) Despite a Cultivation and Import Ban in Switzerland

Despite cultivation and seed import bans of genetically modified (GM) oilseed rape (Brassica napus L.), feral GM plants were found growing along railway lines and in port areas at four sites in Switzerland in 2011 and 2012. All GM plants were identified as glyphosate-resistant GM event GT73 (Roundup Ready, Monsanto). The most affected sites were the Rhine port of Basel and the St. Johann freight railway station in Basel. To assess the distribution and intra- and interspecific outcrossing of GM oilseed rape in more detail, we monitored these two sites in 2013. Leaves and seed pods of feral oilseed rape plants, their possible hybridization partners and putative hybrid plants were sampled in monthly intervals and analysed for the presence of transgenes by real-time PCR. Using flow cytometry, we measured DNA contents of cell nuclei to confirm putative hybrids. In total, 2787 plants were sampled. The presence of GT73 oilseed rape could be confirmed at all previously documented sampling locations and was additionally detected at one new sampling location within the Rhine port. Furthermore, we found the glufosinate-resistant GM events MS8xRF3, MS8 and RF3 (all traded as InVigor, Bayer) at five sampling locations in the Rhine port. To our knowledge, this is the first time that feral MS8xRF3, MS8 or RF3 plants were detected in Europe. Real-time PCR analyses of seeds showed outcrossing of GT73 into two non-GM oilseed rape plants, but no outcrossing of transgenes into related wild species was observed. We found no hybrids between oilseed rape and related species. GM plants most frequently occurred at unloading sites for ships, indicating that ship cargo traffic is the main entry pathway for GM oilseed rape. In the future, it will be of major interest to determine the source of GM oilseed rape seeds.     

Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.     

Human liver epigenetic alterations in non-alcoholic steatohepatitis are related to insulin action

Both genetic and lifestyle factors contribute to the risk of non-alcoholic steatohepatitis (NASH). Additionally, epigenetic modifications may also play a key role in the pathogenesis of NASH. We therefore investigated liver DNA methylation, as a marker for epigenetic alterations, in individuals with simple steatosis and NASH, and further tested if these alterations were associated with clinical phenotypes. Liver biopsies obtained from 95 obese individuals (age: 49.5 ± 7.7 years, BMI: 43 ± 5.7 kg/m², type 2 diabetes [T2D]: 35) as a wedge biopsy during a Roux-en-Y gastric bypass operation were investigated. Thirty-four individuals had a normal liver phenotype, 35 had simple steatosis, and 26 had NASH. Genome-wide DNA methylation pattern was analyzed using the Infinium HumanMethylation450 BeadChip. mRNA expression was analyzed from 42 individuals using the HumanHT-12 Expression BeadChip. We identified 1,292 CpG sites representing 677 unique genes differentially methylated in liver of individuals with NASH (q < 0.001), independently of T2D, age, sex, and BMI. Focusing on the top-ranking 30 and another 37 CpG sites mapped to genes enriched in pathways of metabolism (q = 0.0036) and cancer (q = 0.0001) all together, 59 NASH-associated CpG sites correlated with fasting insulin levels independently of age, fasting glucose, or T2D. From these, we identified 30 correlations between DNA methylation and mRNA expression, for example LDHB (r = ?0.45, P = 0.003). We demonstrated that NASH, more than simple steatosis, associates with differential DNA methylation in the human liver. These epigenetic alterations in NASH are linked with insulin metabolism.     

Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.