Lower Fetuin-A,Retinol Binding Protein 4 and Several Metabolites after Gastric Bypass Compared to Sleeve Gastrectomy in Patients with Type 2 Diabetes

Background

Bypass of foregut secreted factors promoting insulin resistance is hypothesized to be one of the mechanisms by which resolution of type 2 diabetes (T2D) follows roux-en-y gastric bypass (GBP) surgery.

Aim

To identify insulin resistance-associated proteins and metabolites which decrease more after GBP than after sleeve gastrectomy (SG) prior to diabetes remission.

Methods

Fasting plasma from 15 subjects with T2D undergoing GBP or SG was analyzed by proteomic and metabolomic methods 3 days before and 3 days after surgery. Subjects were matched for age, BMI, metformin therapy and glycemic control. Insulin resistance was calculated using homeostasis model assessment (HOMA-IR). For proteomics, samples were depleted of abundant plasma proteins, digested with trypsin and labeled with iTRAQ isobaric tags prior to liquid chromatography-tandem mass spectrometry analysis. Metabolomic analysis was performed using gas chromatography-mass spectrometry. The effect of the respective bariatric surgery on identified proteins and metabolites was evaluated using two-way analysis of variance and appropriate post-hoc tests.

Results

HOMA-IR improved, albeit not significantly, in both groups after surgery. Proteomic analysis yielded seven proteins which decreased significantly after GBP only, including Fetuin-A and Retinol binding protein 4, both previously linked to insulin resistance. Significant decrease in Fetuin-A and Retinol binding protein 4 after GBP was confirmed using ELISA and immunoassay. Metabolomic analysis identified significant decrease of citrate, proline, histidine and decanoic acid specifically after GBP.

Conclusion

Greater early decrease was seen for Fetuin-A, Retinol binding protein 4, and several metabolites after GBP compared to SG, preceding significant weight loss. This may contribute to enhanced T2D remission observed following foregut bypass procedures.     

Abundance-based Classifier for the Prediction of Mass Spectrometric Peptide Detectability Upon Enrichment (PPA)

The function of a large percentage of proteins is modulated by post-translational modifications (PTMs). Currently, mass spectrometry (MS) is the only proteome-wide technology that can identify PTMs. Unfortunately, the inability to detect a PTM by MS is not proof that the modification is not present. The detectability of peptides varies significantly making MS potentially blind to a large fraction of peptides. Learning from published algorithms that generally focus on predicting the most detectable peptides we developed a tool that incorporates protein abundance into the peptide prediction algorithm with the aim to determine the detectability of every peptide within a protein. We tested our tool, “Peptide Prediction with Abundance” (PPA), on in-house acquired as well as published data sets from other groups acquired on different instrument platforms. Incorporation of protein abundance into the prediction allows us to assess not only the detectability of all peptides but also whether a peptide of interest is likely to become detectable upon enrichment. We validated the ability of our tool to predict changes in protein detectability with a dilution series of 31 purified proteins at several different concentrations. PPA predicted the concentration dependent peptide detectability in 78% of the cases correctly, demonstrating its utility for predicting the protein enrichment needed to observe a peptide of interest in targeted experiments. This is especially important in the analysis of PTMs. PPA is available as a web-based or executable package that can work with generally applicable defaults or retrained from a pilot MS data set.Post-translational modification (PTM)¹ of proteins is a key regulatory mechanism in the vast majority of biological processes. Historically, to follow PTMs, site-specific antibodies had to be generated in a time-consuming and laborious process associated with high failure rates. Mass spectrometry (MS) holds enormous promise in PTM analysis as it is currently the only technique that has the ability to both discover, localize, and quantify proteome-wide modifications (1). Recent advances in instrumentation and method optimization makes it possible to detect the complete yeast proteome within one hour (2), an ever increasing proportion of the human proteome (3–6), and more than 10,000 phosphorylation sites in a single MS experiment (7, 8). As a result one of the major publicly available databases (www.phosphosite.org (9)) has curated >200,000 phosphorylation sites.Although the number of proteins and PTMs that can be identified is impressive, many modifications have still not been identified in any MS-based experiment. The identification and quantification of biologically relevant modifications is challenging for three reasons: (1) many proteins of interest are of very low abundance rendering them difficult to detect and quantify; (2) many modifications sites are present at substoichiometric quantities, further reducing their detectability; and (3) as large scale proteomics is based on the detection of peptides after a proteolytic digest, and the detectability of a peptide is determined by its physiochemical properties (10), many peptides from highly abundant proteins are never detected. This is particularly important, as there is a shift in the use of MS-based proteomics from large scale, unbiased, discovery-focused experiments toward directed experiments for accurate and precise quantification of biologically relevant PTMs. Protein and peptide enrichment strategies and/or targeted MS experiments like single reaction monitoring (SRM) (11) have increased the number of detectable peptides; however, both of these methods are laborious, and often not successful, that is, the peptide carrying the modification of interest is still not observed as it is fundamentally very difficult to detect.Protein enrichment is the method choice for most experimentalists, but there is no current way to determine whether this is likely to succeed prior to engaging in lengthy biochemical and/or analytical experiments. In an effort to gauge the chances of success for detecting a particular peptide we sought to develop an algorithm that can predict both the chances of detecting a particular peptide and, more importantly, what enrichment it would take to detect a particular peptide that is not easily detected. Here we present such a tool that predicts the detectability and estimates an enrichment factor, i.e. an increase in signal over the background that is necessary to actually detect a particular peptide. Our algorithm development was motivated by two premises: (1) In silico methods have been developed that focus on the prediction of easily detectable “proteotypic” peptides (peptides that are likely to provide the best detection sensitivity) with good accuracy (12–15). (2) Comprehensive proteome studies have shown that the number of detected peptides per protein, and thus the sequence coverage, varies with protein abundance (which is the basis for spectral counting-based protein quantification (16, 17)). We find that incorporation of protein abundance in a peptide classification tool improves the accuracy of the prediction of peptide detectability allowing us to predict the detectability of all peptides within a protein as well as the amount of enrichment needed to detect a peptide of interest.We used a set of 120 purified in vitro expressed proteins as a training set to develop a prediction tool. We deliver this in the form of a web-based interface that provides information about: (1) the probability of detecting the different tryptic peptides of a protein, and (2) the fold enrichment that would be required to bring a peptide of interest into the detectable range. This tool will help guide researchers in their efforts to monitor particular peptides and their modified cognates by MS, specifically, in prioritizing their efforts toward enriching proteins where they would be likely to be able to detect a peptide or modification of interest.     

Practical method of germination for a key jarrah forest species: Snottygobble (Persoonia longifolia)

Summary Snottygobble ( Persoonia longifolia ) is an ecologically and economically important species in the jarrah forest in Western Australia but is not well represented in jarrah forest restoration projects because it is difficult to germinate. In restored bauxite mines the establishment density of Snottygobble from the soil seed bank is variable and often inadequate. Alcoa considers it a priority to re-establish such key species at adequate densities in its restored mine areas The aims of the present research were: (i) to determine if the species could be cued to germinate; and (ii) to develop a practical method to re-establish the species in restored bauxite mines. We found that fresh Snottygobble seed has high viability (> 90%) but seed stored at 4°C rapidly loses viability over the first year after seed fall. We obtained up to 40% germination using fresh seed that had been treated with gibberellic acid (GA3) after having part of the endocarp chipped away, sown on the soil surface and watered twice daily in an ambient temperature glasshouse in winter/spring. We found that the key to successful germination was combining surface sowing, endocarp chipping and GA3 treatment. Germination involved the breakdown of mechanical and, probably, chemical dormancy. There also appears to be a cool temperature requirement for germination. Practical recommendations to germinate Snottygobble are made. This germination method will have application to land managers, restoration practitioners and the horticultural industry. Alcoa will continue work to translate this success into an adequate stocking of Snottygobble in restored bauxite mines.
Key words bauxite mine, dormancy, endocarp chipping, endogenous, germination, gibberellic acid , Persoonia longifolia , restoration, Snottygobble.     

Synthesis and initial structure-activity relationships of a novel series of imidazolo[1,2-a]pyrimid-5-ones as potent GnRH receptor antagonists

SAR studies of 2-arylimidazolo[1,2-a]pyrimid-5-ones 10a-m, which were derived from initial lead 3a, resulted in the discovery of a series of potent nonpeptide human GnRH receptor antagonists. Compounds with good potency (e.g., 10e, K(i)=7.5 nM) were prepared by introduction of a 2-(2-pyridyl)ethyl at the basic nitrogen and a 3-pentyl ester at the 6-position of the bicyclic core.     

Empathy,social media,and directed altruistic living organ donation

In this article we explore some of the ethical dimensions of using social media to increase the number of living kidney donors. Social media provides a platform for changing non‐identifiable ‘statistical victims’ into ‘real people’ with whom we can identify and feel empathy: the so‐called ‘identifiable victim effect’, which prompts charitable action. We examine three approaches to promoting kidney donation using social media which could take advantages of the identifiable victim effect: (a) institutionally organized campaigns based on historical cases aimed at promoting non‐directed altruistic donation; (b) personal case‐based campaigns organized by individuals aimed at promoting themselves/or someone with whom they are in a relationship as a recipient of directed donation; (c) institutionally organized personal case‐based campaigns aimed at promoting specific recipients for directed donation. We will highlight the key ethical issues raised by these approaches, and will argue that the third option, despite raising ethical concerns, is preferable to the other two.     

Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence

Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L. donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L. donovani compared to typical visceral disease associated L. donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L. donovani introduced into the virulent L. donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L. donovani including RagC complexing proteins, RagA and Raptor. L. donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L. donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L. donovani in Sri Lanka.     

Engineering vitamin E content: from Arabidopsis mutant to soy oil

We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of delta-tocopherol and decreased gamma-tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed delta-tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (gamma-tocopherol methyltransferase) in soybean, the seed accumulated to >95% alpha-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of alpha-tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.     

Perspective: Evolution and detection of genetic robustness

Abstract Robustness is the invariance of phenotypes in the face of perturbation. The robustness of phenotypes appears at various levels of biological organization, including gene expression, protein folding, metabolic flux, physiological homeostasis, development, and even organismal fitness. The mechanisms underlying robustness are diverse, ranging from thermodynamic stability at the RNA and protein level to behavior at the organismal level. Phenotypes can be robust either against heritable perturbations (e.g., mutations) or nonheritable perturbations (e.g., the weather). Here we primarily focus on the first kind of robustness–genetic robustness–and survey three growing avenues of research: (1) measuring genetic robustness in nature and in the laboratory; (2) understanding the evolution of genetic robustness; and (3) exploring the implications of genetic robustness for future evolution.     

Decorticate, decerebrate and opisthotonic posturing and seizures in Kenyan children with cerebral malaria

Background

Intermittent preventive treatment of malaria during pregnancy (IPTp) is a key intervention in the national strategy for malaria control in Tanzania. SP, the current drug of choice, is recommended to be administered in the second and third trimesters of pregnancy during antenatal care (ANC) visits. To allow for a proper design of planned scaling up of IPT services in Tanzania it is useful to understand the IPTp strategy's acceptability to health managers, ANC service providers and pregnant women. This study assesses the knowledge, attitudes and practices of these groups in relation to malaria control with emphasis on IPTp services.

Methods

The study was conducted in February 2004, in Korogwe District, Tanzania. It involved in-depth interviews with the district medical officer (DMO), district hospital medical officer in charge and relevant health service staff at two peripheral dispensaries, and separate focus group discussions (FGDs) with district Council Health Management Team members at district level and pregnant women at dispensary and community levels.

Results

Knowledge of malaria risks during pregnancy was high among pregnant women although some women did not associate coma and convulsions with malaria. Contacting traditional healers and self-medication with local herbs for malaria management was reported to be common. Pregnant women and ANC staff were generally aware of SP as the drug recommended for IPTp, albeit some nurses and the majority of pregnant women expressed concern about the use of SP during pregnancy. Some pregnant women testified that sometimes ANC staff allow the women to swallow SP tablets at home which gives a room for some women to throw away SP tablets after leaving the clinic. The DMO was sceptical about health workers' compliance with the direct observed therapy in administering SP for IPTp due to a shortage of clean water and cups at ANC clinics. Intensified sensitization of pregnant women about the benefits of IPTp was suggested by the study participants as an important approach for improving IPTp compliance.

Conclusion

The successful implementation of the IPTp strategy in Tanzania depends on the proper planning of, and support to, the training of health staff and sustained sensitization of pregnant women at health facility and community levels about the benefits of IPTp for the women and their unborn babies.