Exploring the Microbiome-Wide Lysine Acetylation, Succinylation, and Propionylation in Human Gut Microbiota.

Lysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes and regulate diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. Here, we show that the serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation, and succinylation from human gut microbiome samples. A new bioinformatic workflow consisting of an unrestricted database search confidently identified >60,000 acetylated, and ∼20,000 propionylated and succinylated gut microbial peptides. The characterization of these identified modification-specific metaproteomes, i.e., meta-PTMomes, demonstrates that lysine acylations are differentially distributed in microbial species with different metabolic capabilities. This study provides an analytical framework for the study of lysine acylations in the microbiome, which enables functional microbiome studies at the post-translational level.

Targeted Quantification of Peptides Using Miniature Mass Spectrometry.

Proteomics by mass spectrometry (MS) allows for the identification of amino acid/peptide sequences in complex mixtures. Peptide analysis and quantitation enables screening of protein biomarkers and targeted protein biomarker analysis for clinical applications. Whereas miniature mass spectrometers have primarily demonstrated point-of-care analyses with simple procedures aiming at drugs and lipids, it would be interesting to explore their potential in analyzing proteins and peptides. In this work, we adapted a miniature MS instrument for peptide analysis. A mass range as wide as 100-2000 m/z was achieved for obtaining peptide spectra using this instrument with dual linear ion traps. MS2 and MS3 can be performed to analyze a wide range of peptides. The parameters of pressure, electric potentials, and solution conditions were optimized to analyze peptides with molecular weights between 900 and 1800 Da. The amino acid sequences were identified using both beam-type and in-trap collision-induced dissociation, and the results were comparable to those obtained by a commercial quadrupole time-of-flight mass spectrometer. With product ion monitoring scan mode, peptide quantitation was performed with a limit of detection of 20 nM achieved for the Met peptide. The method developed has also been applied to the analysis of the trypsin-digested cell lysate of SKBR3 cells with a low expression level of the Met gene.

PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.

Widespread protein lysine acetylation in gut microbiome and its alterations in patients with Crohn's disease.

Lysine acetylation (Kac), an abundant post-translational modification (PTM) in prokaryotes, regulates various microbial metabolic pathways. However, no studies have examined protein Kac at the microbiome level, and it remains unknown whether Kac level is altered in patient microbiomes. Herein, we use a peptide immuno-affinity enrichment strategy coupled with mass spectrometry to characterize protein Kac in the microbiome, which successfully identifies 35,200 Kac peptides from microbial or human proteins in gut microbiome samples. We demonstrate that Kac is widely distributed in gut microbial metabolic pathways, including anaerobic fermentation to generate short-chain fatty acids. Applying to the analyses of microbiomes of patients with Crohn's disease identifies 52 host and 136 microbial protein Kac sites that are differentially abundant in disease versus controls. This microbiome-wide acetylomic approach aids in advancing functional microbiome research.

Faculty and students' self-assessment of client communication skills and professional ethics in three veterinary medical schools.

Client communication skills and professional ethics are areas that have received much attention in veterinary education in recent years. The objectives of this study were to: i) establish the confidence level of faculty teaching in three veterinary schools with regard to their client communication skills, ii) establish a baseline of professional ethics indicators in the same faculty, and iii) compare veterinary students of all levels to faculty in both areas. Students and faculty received identical questionnaires, including statements addressing client communication skills and professional ethics. The results indicate that students are generally comfortable with their communication skills, except in the areas of visual and/or audio aid use, handling emotional clients, and discussing costs of care and payment. Faculty were more comfortable than students in all areas of client communication, although they also had low confidence when dealing with costs of care and payment. Ethically, students and faculty answered similarly. Faculty showed a stronger belief that people are basically honest and ethical, but both cohorts responded similarly when asked about reporting an ethical violation admitted to them by their best friend. Further research is needed to determine whether students are communicating as effectively as they believe they are, with particular attention paid to improving communications with emotional clients and the business aspects of veterinary medicine. Additional work is needed to ensure that veterinary students are learning how to cope with ethical issues objectively. This may begin by ensuring that faculty are teaching and, more importantly, modeling these behaviors during the clinical year(s).

Synthetic PreImplantation Factor (sPIF) induces posttranslational protein modification and reverses paralysis in EAE mice.

An autoimmune response against myelin protein is considered one of the key pathogenic processes that initiates multiple sclerosis (MS). The currently available MS disease modifying therapies have demonstrated to reduce the frequency of inflammatory attacks. However, they appear limited in preventing disease progression and neurodegeneration. Hence, novel therapeutic approaches targeting both inflammation and neuroregeneration are urgently needed. A new pregnancy derived synthetic peptide, synthetic PreImplantation Factor (sPIF), crosses the blood-brain barrier and prevents neuro-inflammation. We report that sPIF reduces paralysis and de-myelination of the brain in a clinically-relevant experimental autoimmune encephalomyelitis mice model. These effects, at least in part, are due to post-translational modifications, which involve cyclic AMP dependent protein kinase (PKA), calcium-dependent protein kinase (PKC), and immune regulation. In terms of potential MS treatment, sPIF was successfully tested in neurodegenerative animal models of perinatal brain injury and experimental autoimmune encephalitis. Importantly, sPIF received a FDA Fast Track Approval for first in human trial in autommuninty (completed).

Assessment of clinical reasoning skills in veterinary students prior to and after the clinical year of training.

OBJECTIVE: To examine the effect of various clinical tracks within the veterinary medical clinical curriculum at Texas A&M University on clinical diagnostic proficiency as determined by pre- and post-training assessment. We expected that the clinical track chosen by the student would impact their measured outcome with bias toward higher scores in their chosen field. DESIGN: Prospective cohort study. STUDY POPULATION: 32 students from the College of Veterinary Medicine and Biomedical Sciences at Texas A&M University. PROCEDURES: By use of standardized, written case scenarios, clinical reasoning was assessed twice: once prior to the clinical (fourth) year of the curriculum and again at completion of the clinical year. Students demonstrated their abilities to collect and organize appropriate clinical data (history, physical examination, and laboratory findings), determine clinical diagnoses, and formulate and implement acceptable treatment modalities. Data from clinical assessments were compared for a given cohort and correlated with other measures (eg, grades, standardized test scores, and species-specific curricular track). RESULTS: Differences were detected in clinical diagnostic proficiency among students in different clinical tracks and for different species groups in the case scenarios. Tracking by species group in the clinical veterinary curriculum appeared to affect development of clinical reasoning and resulted in differential proficiency among cases for differing species groups. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in clinical experiences between small animal tracks and all other track opportunities (large animal, mixed animal, and alternative) influenced the development of clinical proficiency in fourth-year veterinary students during their clinical training period.

Mass spectrometry-based proteomic analysis of the DNA damage response.

In response to DNA damage, cells have evolved mechanisms to halt cell cycle progression, activate repair, or initiate apoptosis. These DNA damage response (DDR) pathways are critical for cellular survival in response to genomic insult, and play important roles in growth, development, and disease. Historically, mediators of DNA damage response signaling have been studied one or a few proteins at a time. Advances in mass spectrometry instrumentation and enrichment methods now allow for more global analysis of the DDR in cells and tissues. In this review we will discuss current methods in liquid chromatography tandem mass spectrometry (LC-MS/ MS), enrichment strategies, and targeted analyses for the study of damage signaling. These methods have allowed a greater understanding of the DNA damage response and have highlighted the far-reaching effects of activation of damage-induced pathways.

Outcomes assessment of an alternative career choice program.

OBJECTIVE: To assess the outcomes of an alternative track program designed to address shortages of veterinarians in nonpractice areas of the veterinary profession. DESIGN: Telephone survey. STUDY POPULATION: Recent veterinary graduates. PROCEDURES: A telephone survey of recent graduates from the Texas A&M University College of Veterinary Medicine and Biomedical Sciences Alternative Career Choice program was conducted. Respondents were asked to give open-ended responses to a list of 9 interview questions. RESULTS: Of the 21 alternative career choice students who graduated between the years 2000 and 2005, 17 were interviewed. CONCLUSION: Analysis of the data suggested that it may be possible to decrease the total number of weeks allotted to the alternate career choice program without impairing the ability of graduates to find employment in their chosen career fields.

Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy.

Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis.

Gaining insight into normal cellular signaling and disease biology is a critical goal of proteomic analyses. The ability to perform these studies successfully to extract the maximum value and discovery of biologically relevant candidate biomarkers is therefore of primary importance. Many successful studies in the past have focused on total proteome analysis (changes at the protein level) combined with phosphorylation analysis by metal affinity enrichment (changes at the PTM level). Here, we use the gastric carcinoma cell line MKN-45 treated with the c-Met inhibitor SU11274 and PKC inhibitor staurosporine to investigate the most efficient and most comprehensive strategies for both total protein and PTM analysis. Under the conditions used, total protein analysis yielded few changes in response to either compound, while analysis of phosphorylation identified thousands of sites that changed differentially between the two treatments. Both metal affinity and antibody-based enrichments were used to assess phosphopeptide changes, and the data generated by the two methods was largely complementary (non-overlapping). Label-free quantitation of peptide peak abundances was used to accurately determine fold-changes between control and treated samples. Protein interaction network analysis allowed the data to be placed in a biologically relevant context, and follow-up validation of selected findings confirmed the accuracy of the proteomic data. Together, this study provides a framework for start-to-finish proteomic analysis of any experimental system under investigation to maximize the value of the proteomic study and yield the best chance for uncovering actionable target candidates.

Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system.

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.

Assessing the activity of cediranib, a VEGFR-2/3 tyrosine kinase inhibitor, against VEGFR-1 and members of the structurally related PDGFR family.

Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1 tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-ß. Cediranib inhibited VEGF-A-stimulated VEGFR-1 activation in AG1-G1-Flt1 cells (IC(50) = 1.2 nmol/L). VEGF-A induced greatest phosphorylation of VEGFR-1 at tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against VEGFR-1 was comparable with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC(50) = 1-3 nmol/L) and in a stem cell factor-induced proliferation assay (IC(50) = 13 nmol/L). Furthermore, phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (≥1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-ß and PDGFR-α was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both receptor phosphorylation (IC(50) = 12-32 nmol/L) and PDGF-BB-stimulated cellular proliferation (IC(50) = 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. In vivo, ligand-induced PDGFR-ß phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-α and PDGFR-ß was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, EGFR, FGFR1, and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR kinase inhibitor with similar activity against c-Kit but is significantly less potent than PDGFR-α and PDGFR-ß.

Conditional expression of the mutant Ki-rasG12C allele results in formation of benign lung adenomas: development of a novel mouse lung tumor model.

To determine the effects of expression of mutant Ki-ras on lung tumorigenesis, we developed a bitransgenic mouse model that expresses the human Ki-ras(G12C) allele in alveolar type II and/or Clara cells in a tetracycline-inducible, lung-specific manner. Expression of Ki-ras(G12C) caused multiple, small lung tumors over a 12-month time period. Although tumor multiplicity increased upon continued Ki-ras expression, most lung lesions were hyperplasias or well-differentiated adenomas. This is in contrast to the more severe phenotypes observed in other transgenic mouse models in which different mutant Ki-ras alleles were expressed in the lung. Expression of Ki-ras(G12C) was associated with a 2-fold increase in the activation of the Ras and Ral signaling pathways and increased phosphorylation of Ras downstream effectors, including Erk, p90 ribosomal S6 kinase, ribosomal S6 protein, p38 and MAPKAPK-2. In contrast, expression of the transgene had no effect on the activation of the JNK and Akt signaling pathways. Withdrawal of doxycycline for 1 month resulted in almost a complete absence of proliferative pulmonary lesions, suggesting tumor regression in the absence of Ki-ras expression. Mutant Ki-ras(G12C) expression was sufficient for initial lung tumor transformation, required for maintenance of tumor phenotype, and induced transformation of lung epithelial cells by the activation of multiple effector pathways. These results describe a novel mouse lung tumor model demonstrating benign tumor development in the absence of tumor progression, which will provide a new tool for understanding the early stages of lung tumor pathogenesis.