Global Metabolic Responses to Salt Stress in Fifteen Species

Cells constantly adapt to unpredictably changing extracellular solute concentrations. A cornerstone of the cellular osmotic stress response is the metabolic supply of energy and building blocks to mount appropriate defenses. Yet, the extent to which osmotic stress impinges on the metabolic network remains largely unknown. Moreover, it is mostly unclear which, if any, of the metabolic responses to osmotic stress are conserved among diverse organisms or confined to particular groups of species. Here we investigate the global metabolic responses of twelve bacteria, two yeasts and two human cell lines exposed to sustained hyperosmotic salt stress by measuring semiquantitative levels of hundreds of cellular metabolites using nontargeted metabolomics. Beyond the accumulation of osmoprotectants, we observed significant changes of numerous metabolites in all species. Global metabolic responses were predominantly species-specific, yet individual metabolites were characteristically affected depending on species’ taxonomy, natural habitat, envelope structure or salt tolerance. Exploiting the breadth of our dataset, the correlation of individual metabolite response magnitudes across all species implicated lower glycolysis, tricarboxylic acid cycle, branched-chain amino acid metabolism and heme biosynthesis to be generally important for salt tolerance. Thus, our findings place the global metabolic salt stress response into a phylogenetic context and provide insights into the cellular phenotype associated with salt tolerance.     

Q Fever Knowledge,Attitudes and Vaccination Status of Australia’s Veterinary Workforce in 2014

Q fever, caused by Coxiella burnetii, is a serious zoonotic disease in humans with a worldwide distribution. Many species of animals are capable of transmitting C. burnetii, and consequently all veterinary workers are at risk for this disease. An effective Q fever vaccine has been readily available and used in Australia for many years in at-risk groups, and the European Centre for Disease Prevention and Control has recently also called for the use of this vaccine among at-risk groups in Europe. Little is known about attitudes towards this vaccine and vaccine uptake in veterinary workers. This study aimed to determine the Q fever vaccination status of veterinarians and veterinary nurses in Australia and to assess and compare the knowledge and attitudes towards Q fever disease and vaccination of each cohort. An online cross-sectional survey performed in 2014 targeted all veterinarians and veterinary nurses in Australia. Responses from 890 veterinarians and 852 veterinary nurses were obtained. Binary, ordinal and multinomial logistic regression were used to make comparisons between the two cohorts. The results showed that 74% of veterinarians had sought vaccination compared to only 29% of veterinary nurses. Barriers to vaccination among those not vaccinated did not differ between cohorts, and included a lack of perceived risk, financial expense, time constraints, and difficulty in finding a vaccine provider. Poor knowledge and awareness of Q fever disease and vaccination were additional and notable barriers for the veterinary nursing cohort, suggesting veterinary clinics and veterinarians may not be meeting their legal responsibility to educate staff about risks and risk prevention. Further evaluation is needed to identify the drivers behind seeking and recommending vaccination so that recommendations can be made to improve vaccine uptake.     

A Meta-Analysis of Retinoblastoma Copy Numbers Refines the List of Possible Driver Genes Involved in Tumor Progression

BackgroundWhile RB1 loss initiates retinoblastoma development, additional somatic copy number alterations (SCNAs) can drive tumor progression. Although SCNAs have been identified with good concordance between studies at a cytoband resolution, accurate identification of single genes for all recurrent SCNAs is still challenging. This study presents a comprehensive meta-analysis of genome-wide SCNAs integrated with gene expression profiling data, narrowing down the list of plausible retinoblastoma driver genes.MethodsWe performed SCNA profiling of 45 primary retinoblastoma samples and eight retinoblastoma cell lines by high-resolution microarrays. We combined our data with genomic, clinical and histopathological data of ten published genome-wide SCNA studies, which strongly enhanced the power of our analyses (N = 310).ResultsComprehensive recurrence analysis of SCNAs in all studies integrated with gene expression data allowed us to reduce candidate gene lists for 1q, 2p, 6p, 7q and 13q to a limited gene set. Besides the well-established driver genes RB1 (13q-loss) and MYCN (2p-gain) we identified CRB1 and NEK7 (1q-gain), SOX4 (6p-gain) and NUP205 (7q-gain) as novel retinoblastoma driver candidates. Depending on the sample subset and algorithms used, alternative candidates were identified including MIR181 (1q-gain) and DEK (6p gain). Remarkably, our study showed that copy number gains rarely exceeded change of one copy, even in pure tumor samples with 100% homozygosity at the RB1 locus (N = 34), which is indicative for intra-tumor heterogeneity. In addition, profound between-tumor variability was observed that was associated with age at diagnosis and differentiation grades.InterpretationSince focal alterations at commonly altered chromosome regions were rare except for 2p24.3 (MYCN), further functional validation of the oncogenic potential of the described candidate genes is now required. For further investigations, our study provides a refined and revised set of candidate retinoblastoma driver genes.     

Antifungal Penetration into the Nail and New Topicals for Onychomycosis

Onychomycosis is a common and difficult to treat infection, owing predominately to the limited penetration of topical drugs to the site of infection. Systemic drugs are not an option for all patients due to adverse events and drug-drug interactions. In this article, we review the nail penetration and clinical efficacy data of topical drugs, including older agents such as ciclopirox and amorolfine, as well as the newer agents, efinaconazole and tavaborole. Additionally, we describe some unresolved questions in the management of onychomycosis.     

The European antibody network's practical guide to finding and validating suitable antibodies for research

Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.     

Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer

The role of telomeres and telomerase in colorectal cancer (CRC) is well established as the major driving force in generating chromosomal instability. However, their potential as prognostic markers remains unclear. We investigated the outcome implications of telomeres and telomerase in this tumour type. We considered telomere length (TL), ratio of telomere length in cancer to non-cancer tissue (T/N ratio), telomerase activity and TERT levels; their relation with clinical variables and their role as prognostic markers. We analyzed 132 CRCs and paired non-cancer tissues. Kaplan-Meier curves for disease-free survival were calculated for TL, T/N ratio, telomerase activity and TERT levels. Overall, tumours had shorter telomeres than non-tumour tissues (P < 0.001) and more than 80% of CRCs displayed telomerase activity. Telomere lengths of non-tumour tissues and CRCs were positively correlated (P < 0.001). Considering telomere status and clinical variables, the lowest degree of telomere shortening was shown by tumours located in the rectum (P = 0.021). Regarding prognosis studies, patients with tumours showing a mean TL < 6.35 Kb experienced a significantly better clinical evolution (P < 0.001) and none of them with the highest degree of tumour telomere shortening relapsed during the follow-up period (P = 0.043). The mean TL in CRCs emerged as an independent prognostic factor in the Cox analysis (P = 0.017). Telomerase-positive activity was identified as a marker that confers a trend toward a poor prognosis. In CRC, our results support the use of telomere status as an independent prognostic factor. Telomere status may contribute to explaining the different molecular identities of this tumour type.     

Internal Jugular Vein Cross-Sectional Area Enlargement Is Associated with Aging in Healthy Individuals

Background

Internal jugular vein (IJV) narrowing has been implicated in central nervous system pathologies, however normal physiological age- and gender-related IJV variance in healthy individuals (HIs) has not been adequately assessed.

Objectives

We assessed the relationship between IJV cross-sectional area (CSA) and aging.

Materials and Methods

This study involved 193 HIs (63 males and 130 females) who received 2-dimensional magnetic resonance venography at 3T. The minimum CSA of the IJVs at cervical levels C2/C3, C4, C5/C6, and C7/T1 was obtained using a semi-automated contouring-thresholding technique. Subjects were grouped by decade. Pearson and partial correlation (controlled for cardiovascular risk factors, including hypertension, heart disease, smoking and body mass index) and analysis of variance analyses were used, with paired t-tests comparing side differences.

Results

Mean right IJV CSA ranges were: in males, 41.6 mm² (C2/C3) to 82.0 mm² (C7/T1); in females, 38.0 mm² (C2/C3) to 62.3 mm² (C7/T1), while the equivalent left side ranges were: in males, 28.0 mm² (C2/C3) to 52.2 mm² (C7/T1); in females, 27.2 mm² (C2/C3) to 47.8 mm² (C7/T1). The CSA of the right IJVs was significantly larger (p<0.001) than the left at all cervical levels. Controlling for cardiovascular risk factors, the correlation between age and IJV CSA was more robust in males than in the females for all cervical levels.

Conclusions

In HIs age, gender, hand side and cervical location all affect IJV CSA. These findings suggest that any definition of IJV stenosis needs to account for these factors.     

Evolution of E. coli on [U-13C]Glucose Reveals a Negligible Isotopic Influence on Metabolism and Physiology

¹³C-Metabolic flux analysis (¹³C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled ¹³C-glucose, a commonly used isotope for ¹³C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (¹³C) or unlabeled (¹²C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between ¹²C and ¹³C-glucose as a carbon source for E. coli growth.     

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid–base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na⁺]_i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na⁺]_i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye–ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na⁺]_i, whereas no significant differences in branchial [Na⁺]_i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na⁺]_i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.