Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses*

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.The broad-reaching use and application of mass spectrometry-based proteomics in the international research community continues to exponentially grow and expand. As the technology has developed and practitioners have become skilled in performing complex workflows, the community has not only gained interest in assessing data across laboratories but also in maintaining consistent quality control within a laboratory. Koecher et al. raised the issue of quality control measures and how this aspect of mass spectrometry-based proteomics is generally neglected in scientific publications (1). Fortunately, studies characterizing the stability of liquid chromatography-tandem MS (LC-MSMS)¹ quality control performance among numerous laboratories are emerging. The relationship between sample preparation schemes, data acquisition and reduction strategies, and bioinformatic analyses have been comprehensively reviewed by Tabb (2).Several studies exist where intra- and interlaboratory reproducibility between multiple sites has been assessed under different settings. Perhaps the most systematic and detailed of these investigations are from the Human Proteome Organization (HuPO) test sample working group (3); the National Cancer Institute Clinical Proteomic Tumor Analysis Consortium (NCI CPTAC) (4); and the ProteoRed Consortium (5, 6). The HuPO group utilized an equimolar mixture of 20 highly purified recombinant human proteins (5 pmol per protein) distributed to 27 different laboratories and analyzed without constraint according to optimized LCMS and database search protocols from each of the laboratories (3). The study was not an assessment of instrument performance for highly sensitive detection of proteins, as all participating laboratories had acquired raw data of sufficient quality to identify all 20 proteins (and a specific subset of tryptic peptides). The study revealed, however, that discrepancies in peptide identification and protein assignment were the result of differences in data analysis strategies rather than data collection.The NCI CPTAC group used a standardized Saccharomyces cerevisiae proteome digest that was analyzed on ion-trap-based LCMS platforms in five independent laboratories according to both an established standard operating procedure (SOP) and with no SOP constraint (4). All data analysis was centralized, and thus, any observed variations were entirely because of the LCMS platform. By applying the performance metrics developed by Rudnick et al. (7), several key points emerged: (1) as expected, intralaboratory variation was less than interlaboratory variation; and (2) overall, the interlaboratory variation in peptide identifications and some of the other performance metrics were comparable between instruments, although there were large differences in the average values for some metrics (e.g. MS1 signal intensity, dynamic sampling).The ProteoRed Consortium initiated the ProteoRed Multicenter Experiment for Quality Control (PMEQC) (5, 6). This longitudinal QC multicenter study involved 12 institutes, and was designed to assess: (1) intralaboratory repeatability of LC-MSMS proteomic data; (2) interlaboratory reproducibility; and (3) reproducibility across multiple instrument platforms. Participants received samples of undigested or tryptically digested yeast proteins and were requested to follow strict analytical guidelines. Data analysis was centralized and performed under standard procedures using a common workflow. The study revealed that the overall performance with respect to metrics such as reproducibility, sensitivity, dynamic range etc. was directly related to the degree of operator expertise, and less dependent on instrumentation.Several studies not specifically focused on quality control have also yielded insight into proteomic reproducibility. The HuPO plasma proteome project (HuPO PPP) distributed 20 human samples (five serum plus 3 × 5 plasma samples treated with three different anticoagulants) to 35 laboratories spanning 13 countries (8). The purpose of this large-scale study was not to assess reproducibility per se, but rather to generate the largest and most comprehensive data set on the protein composition of human plasma/serum. On a smaller scale, the ISB standard 18 protein mixture (purified proteins from cow, horse, rabbit, chicken, E. coli, and B. licheniformus) was also assessed between laboratories on eight different LCMS platforms (9). These data reside in a comprehensive, multiplatform database as a resource for the proteomic community. Additional interlaboratory assessments have consisted of multiple reaction monitoring-based measurements of peptides/proteins in plasma (10, 11) and protein–protein interactions at both the biochemical and proteomic level (12).For team leaders/directors of proteomic laboratories and any researcher collaborating with such groups, major questions that may arise concerning data consistency are: how well are quality controls being implemented in the daily operations? Do the quality control measures effectively support data reproducibility? To address this, the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) designed a study whereby LC-MSMS data obtained from the analysis of a commercially available bovine protein mixture predigested with trypsin were collected at routine intervals over a period of 9 months. Raw MS data files from a total of 64 participating laboratories were accumulated, and HPLC and MS performance were evaluated through QC metrics (13). The main impetus of the study was to recognize key sources of variability in HPLC and MS analyses under extended and routine operating conditions for each laboratory and to catalog the state of quality control in a diverse set of proteomic laboratories.No standard operating protocol was imposed on the participants; instead, contributors were encouraged to employ the methods that were typically applied in individual laboratories. Optimization of instrument methods on the provided sample was discouraged. A survey was conducted with each sample submission to catalog individual laboratory practices, instrument configurations, acquisition settings, including routine and nonroutine maintenance procedures. Unlike previous investigations where emphasis was placed on the preparation, distribution, and evaluation of protein standards to appraise and/or standardize LCMS platforms between laboratories, the key interest in this study was purely to determine the intralaboratory performance, reproducibility, and consistency of participating laboratories over an extended period of time.The rapidly expanding number of proteomic laboratories have incorporated divergent HPLC systems, mass spectrometers, solvent systems, columns etc. As a result, analyzing data from a large number of laboratories necessitates tools that can accommodate data from a broad range of platforms. For example, to expect a small laboratory with a decade-old three-dimensional ion trap mass spectrometer to achieve the same sensitivity as a laboratory with a high-resolution hybrid instrument would be unfair. Correspondingly, the data analysis needs to include axes beyond simple peptide-level sensitivity. Nevertheless, the laboratory with the older instrumentation may be consistently better at maximizing performance from the chosen instrument platform compared with a laboratory with the latest high-end equipment.The focus of this study was to estimate the degree of variability in intralaboratory performance over a 9-month period. This goal was achieved using quality metrics that are applicable to most LC-MSMS workflows. The inclusion of data from many laboratories will enable the proteomic community to determine the current state of quality control within a typical laboratory. The survey data enabled the mapping of some alterations in instrument performance to documented laboratory events, e.g. mass spectrometer calibration. The study was designed neither to compare one laboratory with another, nor to discriminate between classes of instrumentation.Questions of data quality and performance in the proteomic community are appropriately aligned with the heightened awareness of a perceived lack of reproducibility of scientific findings in general (1). This community has endeavored to provide tools to assess proteomic data quality, and this study provides additional insight into the application of such tools and the quality of data within respective laboratories.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type="Italic">in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Molecular biology of insect olfaction:recent progress and conceptual models

Insects have an enormous impact on global public health as disease vectors and as agricultural enablers as well as pests and olfaction is an important sensory input to their behavior. As such it is of great value to understand the interplay of the molecular components of the olfactory system which, in addition to fostering a better understanding of insect neurobiology, may ultimately aid in devising novel intervention strategies to reduce disease transmission or crop damage. Since the first discovery of odorant receptors in vertebrates over a decade ago, much of our view on how the insect olfactory system might work has been derived from observations made in vertebrates and other invertebrates, such as lobsters or nematodes. Together with the advantages of a wide range of genetic tools, the identification of the first insect odorant receptors in Drosophila melanogaster in 1999 paved the way for rapid progress in unraveling the question of how olfactory signal transduction and processing occurs in the fruitfly. This review intends to summarize much of this progress and to point out some areas where advances can be expected in the near future.