Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

Intramitochondrial pyruvate attenuates hydrogen peroxide-induced apoptosis in bovine pulmonary artery endothelium

In the hydrogen peroxide (H₂O₂) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H₂O₂ in the absence and presence of 0.5 mM -cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H₂O₂ decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1–20 mM) enhanced cell viability in a dose-dependent manner, with 85% viable cells at 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. -Cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85 ± 3 to 71 ± 4% (p = 0.023) in presence of 3 mM pyruvate plus H₂O₂; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since -cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H₂O₂ apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm.     

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.     

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson''s disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser⁶⁵) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser¹¹¹) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser¹¹¹ of each of the Rabs, we demonstrate that Rab Ser¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser⁶⁵. We further show mechanistically that phosphorylation at Ser¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser¹¹¹ may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson''s disease.     

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.