Fast DNA Serotyping and Antimicrobial Resistance Gene Determination of Salmonella enterica with an Oligonucleotide Microarray-Based Assay

Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4–5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping.     

Dimethyl fumarate treatment after traumatic brain injury prevents depletion of antioxidative brain glutathione and confers neuroprotection

Dimethyl fumarate (DMF) is an immunomodulatory compound to treat multiple sclerosis and psoriasis with neuroprotective potential. Its mechanism of action involves activation of the antioxidant pathway regulator Nuclear factor erythroid 2‐related factor 2 thereby increasing synthesis of the cellular antioxidant glutathione (GSH). The objective of this study was to investigate whether post‐traumatic DMF treatment is beneficial after experimental traumatic brain injury (TBI). Adult C57Bl/6 mice were subjected to controlled cortical impact followed by oral administration of DMF (80 mg/kg body weight) or vehicle at 3, 24, 48, and 72 h after the inflicted TBI. At 4 days after lesion (dal), DMF‐treated mice displayed less neurological deficits than vehicle‐treated mice and reduced histopathological brain damage. At the same time, the TBI‐evoked depletion of brain GSH was prevented by DMF treatment. However, nuclear factor erythroid 2‐related factor 2 target gene mRNA expression involved in antioxidant and detoxifying pathways was increased in both treatment groups at 4 dal. Blood brain barrier leakage, as assessed by immunoglobulin G extravasation, inflammatory marker mRNA expression, and CD45⁺ leukocyte infiltration into the perilesional brain tissue was induced by TBI but not significantly altered by DMF treatment. Collectively, our data demonstrate that post‐traumatic DMF treatment improves neurological outcome and reduces brain tissue loss in a clinically relevant model of TBI. Our findings suggest that DMF treatment confers neuroprotection after TBI via preservation of brain GSH levels rather than by modulating neuroinflammation.

     

Zoonotic agents in small ruminants kept on city farms in southern Germany

Sheep and goats are popular examples of livestock kept on city farms. In these settings, close contacts between humans and animals frequently occur. Although it is widely accepted that small ruminants can carry numerous zoonotic agents, it is unknown which of these agents actually occur in sheep and goats on city farms in Germany. We sampled feces and nasal liquid of 48 animals (28 goats, 20 sheep) distributed in 7 city farms and on one activity playground in southern Germany. We found that 100% of the sampled sheep and 89.3% of the goats carried Shiga toxin-producing Escherichia coli (STEC). The presence of Staphylococcus spp. in 75% of both sheep and goats could be demonstrated. Campylobacter spp. were detected in 25% and 14.3% of the sheep and goats, respectively. Neither Salmonella spp. nor Coxiella burnetii was found. On the basis of these data, we propose a reasonable hygiene scheme to prevent transmission of zoonotic agents during city farm visits.     

Membrane cholesterol modulates {beta}-amyloid-dependent tau cleavage by inducing changes in the membrane content and localization of N-methyl-D-aspartic acid receptors

We have previously shown that β-amyloid (Aβ) treatment resulted in an age-dependent calpain activation leading to Tau cleavage into a neurotoxic 17-kDa fragment in a cellular model of Alzheimer disease. This detrimental cellular response was mediated by a developmentally regulated increase in membrane cholesterol levels. In this study, we assessed the molecular mechanisms by which cholesterol modulated Aβ-induced Tau cleavage in cultured hippocampal neurons. Our results indicated that these mechanisms did not involve the regulation of the binding of Aβ aggregates to the plasma membrane. On the other hand, experiments using N-methyl-d-aspartic acid receptor inhibitors suggested that these receptors played an essential role in cholesterol-mediated Aβ-dependent calpain activity and 17-kDa Tau production. Biochemical and immunocytochemical analyses demonstrated that decreasing membrane cholesterol levels in mature neurons resulted in a significant reduction of the NR1 subunit at the membrane as well as an increase in the number of large NR1, NR2A, and NR2B subunit clusters. Moreover, the majority of these larger N-methyl-d-aspartic acid receptor subunit immunoreactive spots was not juxtaposed to presynaptic sites in cholesterol-reduced neurons. These data suggested that changes at the synaptic level underlie the mechanism by which membrane cholesterol modulates developmental changes in the susceptibility of hippocampal neurons to Aβ-induced toxicity.     

Complex I Deficiency Due to Selective Loss of Ndufs4 in the Mouse Heart Results in Severe Hypertrophic Cardiomyopathy

Mitochondrial complex I, the primary entry point for electrons into the mitochondrial respiratory chain, is both critical for aerobic respiration and a major source of reactive oxygen species. In the heart, chronic dysfunction driving cardiomyopathy is frequently associated with decreased complex I activity, from both genetic and environmental causes. To examine the functional relationship between complex I disruption and cardiac dysfunction we used an established mouse model of mild and chronic complex I inhibition through heart-specific Ndufs4 gene ablation. Heart-specific Ndufs4-null mice had a decrease of ∼50% in complex I activity within the heart, and developed severe hypertrophic cardiomyopathy as assessed by magnetic resonance imaging. The decrease in complex I activity, and associated cardiac dysfunction, occurred absent an increase in mitochondrial hydrogen peroxide levels in vivo, accumulation of markers of oxidative damage, induction of apoptosis, or tissue fibrosis. Taken together, these results indicate that diminished complex I activity in the heart alone is sufficient to drive hypertrophic cardiomyopathy independently of alterations in levels of mitochondrial hydrogen peroxide or oxidative damage.     

The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.     

Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality

The measurement of yeast''s intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r² = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P = 0.433), ICP via flow cytometry did (P = 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population''s vitality via a short measurement, which is suitable for routine analysis.Yeast plays an important role in the food industry. It is primarily used for making bread, beer, and wine, and the flavor and aromatic compounds it produces are characteristic of these fermented products. The physiological state of the yeast biomass influences the fermentation performance and thus the quality of the resulting product, e.g., of beer (1, 32). Food processors who depend on yeast health for consistent fermentations demand a method to measure yeast''s physiological condition during yeast growth, that is, its vitality. The term “vitality” refers to the health of living biomass where high vitality results in a fast fermentation with minimal undesired by-products, while low vitality results in sluggish or poorly attenuating fermentation. In contrast, the term “viability” only distinguishes between dead and alive cells.Several methods exist to measure yeast vitality such as carbon dioxide production (10), vicinal diketone reduction (3), glycogen and trehalose staining (16-18), vital titration (30), measurement of the specific oxygen uptake rate (27, 38), and the acidification power test (12, 22). One important method for vitality measurement is the detection of intracellular pH (ICP) (9, 19, 20, 31).Intracellular metabolic reactions in yeast are catalyzed by enzymes that have their optimum working pH in a neutral range (23). During fermentation or propagation, yeast produces carbon dioxide and organic acids and releases protons to the medium. To maintain a desired ICP, yeast continuously pumps protons against a gradient from the cytosol to the extracellular medium. The regulating proton pump is plasma-membrane-ATPase (4). The transmembrane proton gradient has to be sustained since the transport of important nutrients such as maltose or amino acids depends on it. The efficiency of glycolysis and gluconeogenesis is affected by ICP. The higher the proton extrusion rate, the more active is the metabolism. Thus, a high ICP indicates high yeast vitality. Yeast with the highest ICP ferment the best (1).Techniques using fluorescence photometry and the pH-dependent dye, 5,6-carboxyfluorescein (CF), for measuring yeast ICP have been published (1, 20, 21, 33). These proved reliable but only yield an average value of ICP of a yeast population. Based on the method of Imai (21), a shorter method for measuring ICP was developed by Thiele and Back (33) using photometry whereby the sample preparation time was reduced from approximately 3.5 to 1.25 h. This reduction made it suitable for routine analysis.In the present study and according to Thiele and Back (33), the nonfluorescent, esterified form of CF, 5,6-carboxyfluorescein-diacetate (CF-DA), was used for cell loading because it is more capable of passing through cell membranes than its nonesterified counterpart (6). After the cells were infused with CF-DA, yeast esterase enzymes split the molecule into acetate residues and the actual fluorescent probe CF. Excitation with a certain wavelength results in different fluorescent intensities that depend on both the pH of the cell cytoplasm and the dye concentration. In order to receive results independent on probe concentration, a ratio between pH-dependent and pH-independent fluorescence intensities of the sample can be used to produce data depending on pH only (20, 35).In addition to measuring ICP via fluorescence photometry, flow cytometry can also serve as detection system of CF fluorescent intensities corresponding to ICP. Flow cytometry is becoming a standard tool in biotechnology research and has been successfully applied to analyze and optimize yeast processes (13, 14, 15, 24, 36). Cytometric measurements provide the opportunity to detect single cells. With this method it is possible to describe a whole yeast population in detail by illustrating the distribution of ICP (35, 39). In contrast, photometry only reflects an average value of the population.We compare here the application of the short ICP method executed via fluorescence photometry to flow cytometry. During the propagation of brewer''s yeast, three different growth phases (the lag, exponential, and stationary phases) were compared to determine whether the vitality varied during growth and whether this variation was detectable by both methods. Since flow cytometry offers the opportunity to identify subpopulations (35, 36), we hypothesized that it was possible to detect yeast subpopulations with differing ICP during propagation.