Proteomic identification of posttranslational modifications: cAMP-induced changes of phosphorylation and investigation of novel approaches detecting posttranslational modifications at lysine and arginine residues

Cyclic adenosine monophosphate (cAMP) is an important second messenger which can be generated in response to signals binding to stimulatory G-protein coupled receptors. Inside the cell, protein kinase A is a well-known cAMP sensor. Within the present study, cAMP-dependent changes in the phosphorylation (phospho) pattern of lysosomal membrane and lysosome-associated proteins were investigated. The serine phospho-site at position 133 (S133) of the lysosome-associated SNARE-associated protein (SNAPIN) was found to be significantly downregulated in the phosphoproteomic data set of the lysosomal membrane fraction after elevating the intracellular cAMP level by a Forskolin/dibutyryl-cAMP (FSK/db-cAMP) treatment. In addition to that, a tendency of SNAPIN to dissociate from the lysosomes upon elevated cAMP levels was observed. Therefore, the cAMP-dependent downregulation of the S133 phospho-site detected in the MS-based approach could have also been caused by a translocation of SNAPIN after FSK/db-cAMP treatment. Nevertheless, a dephosphorylation of SNAPIN was observed in 2D-gelelectrophoresis of whole cell protein lysate after elevating the intracellular cAMP level. This observation confirmed the findings of the phosphoproteomic study although the dephosphorylation site could not be mapped to a specific amino acid position by 2D-gelelectrophoresis. Pull-down assays with a phosphomimetic proteoform of the S133 phospho-site (S133E) indicate an interaction of the biogenesis of lysosome-related organelles complex-1 subunit 6 (BLOC1S6) with the S133E but not the S133A SNAPIN isoform (serine to alanine substitution mimics unphosphorylated SNAPIN at position 133). Furthermore, SNAPIN knock-out (KO) was associated with decreased endocytosis and increased exocytosis of lysosomal hydrolases compared to wildtype cells. Elevation of intracellular cAMP levels neither influenced these processes in wildtype nor in SNAPIN KO cells. This indicates that the dephosphorylation of SNAPIN detected after FSK/db-cAMP treatment might not be involved in regulating these secretion or uptake events. Posttranslational modifications (PTMs) of lysine and arginine residues are important regulators in different cellular processes. In bottom-up mass spectrometry-based approaches, these PTMs are most commonly enriched with pan-specific antibodies. Here, the investigations are limited to one specific PTM and depend on the quality and availability of proper antibodies in order to obtain an efficient enrichment of modified peptides. In addition to that, huge amounts of samples are usually required in these immunoprecipitation approaches which might be problematic when dealing with limited amounts of starting material. The present study aimed at developing and testing of two novel antibody-free techniques for improving the detection of specific PTMs from small quantities of starting sample. For example, such techniques would facilitate PTM analysis of enriched lysosomes or other purified subcellular compartments. The first technique, called the serial digestion workflow, was assumed to enhance the identification of lysine modifications. Compared to that, the second method, called the Nα-selective derivatization workflow (derivatization of primary amine group at amino-terminus), was hypothesized to improve the detection of PTMs occurring on both, lysine and arginine residues. Applying the two workflows to proteins from human embryonic kidney (HEK 293) cell lysate was not associated with an increased identification of lysine modifications. In contrast, sample treatment according to the Nα-selective derivatization method yielded an increase in the detection rate of arginine methylation. However, a similar rise was observed in both workflows after strong cation exchange chromatography which was originally performed as a sample clean-up procedure. Here, depending on the sample type, either the identification of arginine or of lysine and arginine methylation was increased. Comparison of the methylated peptides detected in the different samples showed only a partial overlap. This indicates the necessity of analyzing different samples coming from both workflows in order to get the most comprehensive protein methylation data set. Based on these findings, a new hybrid workflow is suggested

Prolonged Antibiotic Treatment does not Prevent Intra-Abdominal Abscesses in Perforated Appendicitis

Contains fulltext : 89619.pdf (publisher's version ) (Open Access)BACKGROUND: Children with perforated appendicitis have a relatively high risk of intra-abdominal abscesses. There is no evidence that prolonged antibiotic treatment after surgery reduces intra-abdominal abscess formation. We compared two patient groups with perforated appendicitis with different postoperative antibiotic treatment protocols. METHODS: We retrospectively reviewed patients younger than age 18 years who underwent appendectomy for perforated appendicitis at two academic hospitals between January 1992 and December 2006. Perforation was diagnosed during surgery and confirmed during histopathological evaluation. Patients in hospital A received 5 days of antibiotics postoperatively, unless decided otherwise on clinical grounds. Patients in hospital B received antibiotics for 5 days, continued until serum C-reactive protein (CRP) was <20 mg/l. Univariate logistic regression analysis was performed on intention-to-treat basis. p < 0.05 was considered significant. RESULTS: A total of 149 children underwent appendectomy for perforated appendicitis: 68 in hospital A, and 81 in hospital B. As expected, the median (range) use of antibiotics was significantly different: 5 (range, 1-16) and 7 (range, 2-32) days, respectively (p < 0.0001). However, the incidence of postoperative intra-abdominal abscesses was similar (p = 0.95). Regression analysis demonstrated that sex (female) was a risk factor for abscess formation, whereas surgical technique and young age were not. CONCLUSIONS: Prolonged use of antibiotics after surgery for perforated appendicitis in children based on serum CRP does not reduce postoperative abscess formation.1 december 201

Functional classification of protein toxins as a basis for bioinformatic screening

Proteins are fundamental to life and exhibit a wide diversity of activities, some of which are toxic. Therefore, assessing whether a specific protein is safe for consumption in foods and feeds is critical. Simple BLAST searches may reveal homology to a known toxin, when in fact the protein may pose no real danger. Another challenge to answer this question is the lack of curated databases with a representative set of experimentally validated toxins. Here we have systematically analyzed over 10,000 manually curated toxin sequences using sequence clustering, network analysis, and protein domain classification. We also developed a functional sequence signature method to distinguish toxic from non-toxic proteins. The current database, combined with motif analysis, can be used by researchers and regulators in a hazard screening capacity to assess the potential of a protein to be toxic at early stages of development. Identifying key signatures of toxicity can also aid in redesigning proteins, so as to maintain their desirable functions while reducing the risk of potential health hazards

The human nuclear exosome targeting complex is loaded onto newly synthesized RNA to direct early ribonucleolysis

SummaryThe RNA exosome complex constitutes the major nuclear eukaryotic 3′-5′ exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT) complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and 3′-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3′ ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3′-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3′ ends

Non-perturbative dynamics of hot non-Abelian gauge fields: beyond leading log approximation

Many aspects of high-temperature gauge theories, such as the electroweak baryon number violation rate, color conductivity, and the hard gluon damping rate, have previously been understood only at leading logarithmic order (that is, neglecting effects suppressed only by an inverse logarithm of the gauge coupling). We discuss how to systematically go beyond leading logarithmic order in the analysis of physical quantities. Specifically, we extend to next-to-leading-log order (NLLO) the simple leading-log effective theory due to Bodeker that describes non-perturbative color physics in hot non-Abelian plasmas. A suitable scaling analysis is used to show that no new operators enter the effective theory at next-to-leading-log order. However, a NLLO calculation of the color conductivity is required, and we report the resulting value. Our NLLO result for the color conductivity can be trivially combined with previous numerical work by G. Moore to yield a NLLO result for the hot electroweak baryon number violation rate.Comment: 20 pages, 1 figur

A Learning Approach to Monitoring and Evaluation

This article draws on literature from both monitoring and evaluation (M&amp;E) and organisational learning to explore synergies between these two fields in support of organisational performance. Two insights from the organisational learning literature are that organisations learn through ‘double?loop’ learning: reflecting on experience and using this to question critically underlying assumptions; and that power relations within an organisation will influence what and whose learning is valued and shared. This article identifies four incentives that can help link M&amp;E with organisational learning: the incentive to learn why; the incentive to learn from below; the incentive to learn collaboratively; and the incentive to take risks. Two key elements are required to support these incentives: (1) establishing and promoting an ‘evaluative culture’ within an organisation; and (2) having accountability relationships where value is placed on learning ‘why’, as well as on learning from mistakes, which requires trust

The psychometric properties of the 'Hospital Survey on Patient Safety Culture' in Dutch hospitals

BACKGROUND: In many different countries the Hospital Survey on Patient Safety Culture (HSOPS) is used to assess the safety culture in hospitals. Accordingly, the questionnaire has been translated into Dutch for application in the Netherlands. The aim of this study was to examine the underlying dimensions and psychometric properties of the questionnaire in Dutch hospital settings, and to compare these results with the original questionnaire used in USA hospital settings. METHODS: The HSOPS was completed by 583 staff members of four general hospitals, three teaching hospitals, and one university hospital in the Netherlands. Confirmatory factor analyses were performed to examine the applicability of the factor structure of the American questionnaire to the Dutch data. Explorative factor analyses were performed to examine whether another composition of items and factors would fit the data better. Supplementary psychometric analyses were performed, including internal consistency and construct validity. RESULTS: The confirmatory factor analyses were based on the 12-factor model of the original questionnaire and resulted in a few low reliability scores. 11 Factors were drawn with explorative factor analyses, with acceptable reliability scores and a good construct validity. Two items were removed from the questionnaire. The composition of the factors was very similar to that of the original questionnaire. A few items moved to another factor and two factors turned out to combine into a six-item dimension. All other dimensions consisted of two to five items. CONCLUSION: The Dutch translation of the HSOPS consists of 11 factors with acceptable reliability and good construct validity. and is similar to the original HSOPS factor structure. (aut. ref.

Space micropropulsion systems for Cubesats and small satellites: From proximate targets to furthermost frontiers

Rapid evolution of miniaturized, automatic, robotized, function-centered devices has redefined space technology, bringing closer the realization of most ambitious interplanetary missions and intense near-Earth space exploration. Small unmanned satellites and probes are now being launched in hundreds at a time, resurrecting a dream of satellite constellations, i.e., wide, all-covering networks of small satellites capable of forming universal multifunctional, intelligent platforms for global communication, navigation, ubiquitous data mining, Earth observation, and many other functions, which was once doomed by the extraordinary cost of such systems. The ingression of novel nanostructured materials provided a solid base that enabled the advancement of these affordable systems in aspects of power, instrumentation, and communication. However, absence of efficient and reliable thrust systems with the capacity to support precise maneuvering of small satellites and CubeSats over long periods of deployment remains a real stumbling block both for the deployment of large satellite systems and for further exploration of deep space using a new generation of spacecraft. The last few years have seen tremendous global efforts to develop various miniaturized space thrusters, with great success stories. Yet, there are critical challenges that still face the space technology. These have been outlined at an inaugural International Workshop on Micropropulsion and Cubesats, MPCS-2017, a joint effort between Plasma Sources and Application Centre/Space Propulsion Centre (Singapore) and the Micropropulsion and Nanotechnology Lab, the G. Washington University (USA) devoted to miniaturized space propulsion systems, and hosted by CNR-Nanotec - P.Las.M.I. lab in Bari, Italy. This focused review aims to highlight the most promising developments reported at MPCS-2017 by leading world-reputed experts in miniaturized space propulsion systems. Recent advances in several major types of small thrusters including Hall thrusters, ion engines, helicon, and vacuum arc devices are presented, and trends and perspectives are outlined.This work was supported in part by the following funds and organizations: OSTIn-SRP/EDB through National Research Foundation and in part by MoE AcRF (Rp6/16 Xs), Singapore; National Natural Science Foundation of China (Grant Nos. 51777045 and 51477035); National Technical Basic Scientific Research of China, Grant No. JSZL2016203c006; NASA DC Space Grant Consortium; Grant-in-Aid for Scientific Research under Grant S: 21226019 and Grant B: 17H02295 through the Japan Society for the Promotion of Science, and by NIFS budget code NIFS17KLER063, and KAKENHI grant: Grant-in-Aid for Scientific Research (S), No. JP16H06370; S.S. thanks late Professor K. Toki, late Dr. K. P. Shamrai, Dr. Kuwahara, and the HEAT project members for their contribution Y.R. acknowledges the support from the US DOE under Contract No. DE-AC02-09CH11466; I.L. acknowledges the support from the School of Chemistry, Physics and Mechanical Engineering, Science and Engineering Faculty, Queensland University of Technology; special thanks to L. Xu, M. Lim, S. Huang, and the entire PSAC/SPCS for their help

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN