Meta-Analysis Reveals Both the Promises and the Challenges of Clinical Metabolomics

Clinical metabolomics is a rapidly expanding field focused on identifying molecular biomarkers to aid in the efficient diagnosis and treatment of human diseases. Variations in study design, metabolomics methodologies, and investigator protocols raise serious concerns about the accuracy and reproducibility of these potential biomarkers. The explosive growth of the field has led to the recent availability of numerous replicate clinical studies, which permits an evaluation of the consistency of biomarkers identified across multiple metabolomics projects. Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death and has the lowest five-year survival rate primarily due to the lack of an early diagnosis and the limited treatment options. Accordingly, PDAC has been a popular target of clinical metabolomics studies. We compiled 24 PDAC metabolomics studies from the scientific literature for a detailed meta-analysis. A consistent identification across these multiple studies allowed for the validation of potential clinical biomarkers of PDAC while also highlighting variations in study protocols that may explain poor reproducibility. Our meta-analysis identified 10 metabolites that may serve as PDAC biomarkers and warrant further investigation. However, 87% of the 655 metabolites identified as potential biomarkers were identified in single studies. Differences in cohort size and demographics, p-value choice, fold-change significance, sample type, handling and storage, data collection, and analysis were all factors that likely contributed to this apparently large false positive rate. Our meta-analysis demonstrated the need for consistent experimental design and normalized practices to accurately leverage clinical metabolomics data for reliable and reproducible biomarker discovery

Constraints and Negotiations to Student Recreational Sport Center Usage

Studies have shown that student recreational sports centers offer a variety of benefits to emerging adult students. Previous research has identified the presence of constraints to physical activity and student recreational sports center (SRSC) usage (Flood & Parker, 2014; Stankowski, Trauntvein, & Hall, 2017; Young, Ross, & Barcelona, 2003). However, little research has examined the negotiation strategies which students use to increase their physical activity levels through use of the SRSC. This study focused on the constraints and negotiation strategies which affected both users and infrequent users of the SRSC at a mid-size, public university in the northeastern United States. Results suggested that constraints and negotiation strategies differed significantly between user groups and that several constraints and negotiations predict frequency of visitation to the SRSC. Findings suggest the importance of management actions which help students to create social networks, improve their planning and prioritization, and develop self-efficacy with regard to physical activity at the SRSC

Size Dependence in Non-sperm Ejaculate Production is Reflected in Daily Energy Expenditure and Resting Metabolic Rate

The non-sperm components of an ejaculate, such as copulatory plugs, can be essential to male reproductive success. But the costs of these ejaculate components are often considered trivial. In polyandrous species, males are predicted to increase energy allocation to the production of non-sperm components, but this allocation is often condition dependent and the energetic costs of their production have never been quantified. Red-sided garter snakes (Thamnophis sirtalis parietalis) are an excellent model with which to quantify the energetic costs of non-sperm components of the ejaculate as they exhibit a dissociated reproductive pattern in which sperm production is temporally disjunct from copulatory plug production, mating and plug deposition. We estimated the daily energy expenditure and resting metabolic rate of males after courtship and mating, and used bomb calorimetry to estimate the energy content of copulatory plugs. We found that both daily energy expenditure and resting metabolic rate were significantly higher in small mating males than in courting males, and a single copulatory plug without sperm constitutes 5–18% of daily energy expenditure. To our knowledge, this is the first study to quantify the energetic expense of size-dependent ejaculate strategies in any species

A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15

Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca2+-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca2+-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca2+-binding linker regions: canonical EC4-5 linker binding three Ca2+ ions, and non-canonical EC3-4 linker binding only two Ca2+ ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca2+-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.Molecular and Cellular Biolog

NMR Analysis of a Stress Response Metabolic Signaling Network

We previously hypothesized that Staphylococcus epidermidis senses a diverse set of environmental and nutritional factors associated with biofilm formation through a modulation in the activity of the tricarboxylic acid (TCA) cycle. Herein, we report our further investigation of the impact of additional environmental stress factors on TCA cycle activity and provide a detailed description of our NMR methodology. S. epidermidis wild-type strain 1457 was treated with stressors that are associated with biofilm formation, a sub-lethal dose of tetracycline, 5% NaCl, 2% glucose and autoinducer-2 (AI-2). As controls and to integrate our current data with our previous study, 4% ethanol stress and iron-limitation were also used. Consistent with our prior observations, the effect of many environmental stress factors on the S. epidermidis metabolome was essentially identical to the effect of TCA cycle inactivation in the aconitase mutant strain 1457-acnA::tetM. A detailed quantitative analysis of metabolite concentration changes using 2D 1H-13C HSQC and 1H-1H TOCSY spectra identified a network of 37 metabolites uniformly affected by the stressors and TCA cycle inactivation. We postulate that the TCA cycle acts as the central pathway in a metabolic signaling network

Accumulation of Microbial Biomass within Particulate Organic Matter of Aging Golf Greens

Microbial biomass (MB) is a key variable controlling soil organic matter dynamics in soil. Currently, there is little information on the amount and significance of MB in highly managed golf greens. Our objective was to determine the amount and distribution of MB within soil structural components of golf greens and its relationship to the location of organic substrates. During 1996, 47 greens were sampled from 12 golf courses within Nebraska (USA). Microbial biomass, determined as extractable lipid phosphate on field-moist soils, increased linearly with age of green (Y = 19.39 + 3.54x; r2 = 0.87, P = 0.001). In 1997 and 1999, selected greens were resampled and separated into mineral fraction (MF) and particulate organic matter (POM) fraction using a sodium metatungstate (NMT; r = 2.3 g cm-3). Then, POM was separated into light (L-POM) and heavy (H-POM) fractions using NMT (r = 2.0 g cm-3). Amount of MB of whole soil and POM was linearly related to green age (r2 = 0.76 and 0.68, respectively). Amount of MB in MF was not related to green age. The portion of total soil MB associated with POM increased significantly from 25.6% for an 8-yr-old green to 77.8% for a 28-yr-old green. Carbon in fulvic acid and humic acid increased with green age from 0.5 to 1.7 and 0.6 to 2.6 g kg-1 soil, respectively. As humus is a relatively stable form of soil organic matter, we hypothesized that humus accumulation within POM renders both POM and associated MB more resistant to degradation; thus, they accumulate

Structure and function of \u3ci\u3ePseudomonas aeruginosa\u3ci\u3e protein PA1324 (21–170)

Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human pathogen. P. aeruginosa is causatively associated with nosocomial infections and with cystic fibrosis. Antibiotic resistance in some strains adds to the inherent difficulties that result from biofilm formation when treating P. aeruginosa infections. Transcriptional profiling studies suggest widespread changes in the proteome during quorum sensing and biofilm development. Many of the proteins found to be upregulated during these processes are poorly characterized from a functional standpoint. Here, we report the solution NMR structure of PA1324, a protein of unknown function identified in these studies, and provide a putative biological functional assignment based on the observed prealbumin-like fold and FAST-NMR ligand screening studies. PA1324 is postulated to be involved in the binding and transport of sugars or polysaccharides associated with the peptidoglycan matrix during biofilm formation

Reconstitution of the tubular endoplasmic reticulum network with purified components

Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum (ER) is particularly intriguing, as it is comprised of morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation1–5, but how exactly they mediate network formation and whether they are all required is unclear. Here, we have reconstituted a dynamic tubular membrane network with purified ER proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p6,7 and the curvature-stabilizing protein Yop1p8,9 from Saccharomyces cerevisiae form a tubular network upon GTP addition. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favors the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon8 and REEP10 proteins from different species. Atlastin, the vertebrate ortholog of Sey1p6,11, forms a GTP-hydrolysis dependent network on its own, serving as both a fusion and curvature-stabilizing protein. Our results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly