Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the mu2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of mu2 phosphorylation demonstrated that clathrin is a specific activator of the mu2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of m2 phosphorylation. Furthermore, phosphorylated mu2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-mu2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.     

Expansion of the BioCyc collection of pathway/genome databases to 160 genomes

The BioCyc database collection is a set of 160 pathway/genome databases (PGDBs) for most eukaryotic and prokaryotic species whose genomes have been completely sequenced to date. Each PGDB in the BioCyc collection describes the genome and predicted metabolic network of a single organism, inferred from the MetaCyc database, which is a reference source on metabolic pathways from multiple organisms. In addition, each bacterial PGDB includes predicted operons for the corresponding species. The BioCyc collection provides a unique resource for computational systems biology, namely global and comparative analyses of genomes and metabolic networks, and a supplement to the BioCyc resource of curated PGDBs. The Omics viewer available through the BioCyc website allows scientists to visualize combinations of gene expression, proteomics and metabolomics data on the metabolic maps of these organisms. This paper discusses the computational methodology by which the BioCyc collection has been expanded, and presents an aggregate analysis of the collection that includes the range of number of pathways present in these organisms, and the most frequently observed pathways. We seek scientists to adopt and curate individual PGDBs within the BioCyc collection. Only by harnessing the expertise of many scientists we can hope to produce biological databases, which accurately reflect the depth and breadth of knowledge that the biomedical research community is producing.     

Translational database selection and multiplexed sequence capture for up front filtering of reliable breast cancer biomarker candidates

Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.     

SSRIs Increase Risk of Blood Transfusion in Patients Admitted for Hip Surgery

Background

Recent studies have shown that an increased bleeding tendency can be caused by Selective Serotonin Reuptake Inhibitors (SSRI) use. We aimed to investigate the occurrence and risk of blood transfusion in SSRI users compared to non-SSRI users in a cohort of patients admitted for hip-surgery.

Methods

We conducted a retrospective cohort study of patients who underwent planned or emergency hip surgery from 1996 to 2011 in the Academic Medical Center in Amsterdam. Primary outcome measure was risk of blood transfusion. Secondary outcome measures were pre- and postoperative hemoglobin level. Multivariate logistic regression was used to adjust for potential confounders.

Results

One-hundred and fourteen SSRI users were compared to 1773 non-SSRI users. Risk of blood transfusion during admission was increased for SSRI users in multivariate analyses (OR 1.7 [95% CI 1.1–2.5]). Also, pre-operative hemoglobin levels were lower in SSRI users (7.8±1.0 mmol/L) compared to non-SSRI users (8.0±1.0 mmol/L) (p = 0.042)), as were postoperative hemoglobin levels (6.2±1.0 mmol/L vs. 6.4±1.0 mmol/L respectively) (p = 0.017)).

Conclusions

SSRI users undergoing hip surgery have an increased risk for blood transfusion during admission, potentially explained by a lower hemoglobin level before surgery. SSRI use should be considered as a potential risk indicator for increased blood loss in patients admitted for hip surgery. These results need to be confirmed in a prospective study.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Oscillations of phospholipase C activity triggered by depolarization and Ca2+ influx in insulin-secreting cells

Phospholipase C (PLC) is a ubiquitous enzyme involved in the regulation of a variety of cellular processes. Its dependence on Ca2+ is well recognized, but it is not known how PLC activity is affected by physiological variations of the cytoplasmic Ca2+ concentration ([Ca2+](i)). Here, we applied evanescent wave microscopy to monitor PLC activity in parallel with [Ca2+](i) in individual insulin-secreting INS-1 cells using the phosphatidylinositol 4,5-bisphosphate- and inositol 1,4,5-trisphosphate-binding pleckstrin homology domain from PLCdelta(1) fused to green fluorescent protein (PH(PLCdelta1)-GFP) and the Ca2+ indicator fura red. In resting cells, PH(PLCdelta1)-GFP was located predominantly at the plasma membrane. Activation of PLC by muscarinic or purinergic receptor stimulation resulted in PH(PLCdelta1)-GFP translocation from the plasma membrane to the cytoplasm, detected as a decrease in evanescent wave-excited PH(PLCdelta1)-GFP fluorescence. Using this translocation as a measure of PLC activity, we found that depolarization by raising extracellular [K+] triggered activation of the enzyme. This effect could be attributed both to a rise of [Ca2+](i) and to depolarization per se, because some translocation persisted during depolarization in a Ca2+-deficient medium containing the Ca2+ chelator EGTA. Moreover, oscillations of [Ca2+](i) resulting from depolarization with Ca2+ influx evoked concentration-dependent periodic activation of PLC. We conclude that PLC activity is under tight dynamic control of [Ca2+](i). In insulin-secreting beta-cells, this mechanism provides a link between Ca2+ influx and release from intracellular stores that may be important in the regulation of insulin secretion.     

Historic Titles and Historic Rights in the Law of the Sea in the Light of the South China Sea Arbitration

Historic titles and historic rights have been a complicated issue in the law of the sea both conceptually and practically. The South China Sea Arbitration between the Philippines and China raised important issues regarding the contemporary relevance and validity of historic claims, and the relationship between the Law of the Sea Convention and historic rights. This articles examines historic rights and historic titles in the law of the sea in the light of the South China Sea Arbitration and evaluates the contribution of the Tribunal's Awards to the clarification of these concepts.     

In Vivo Evidence for Serine Biosynthesis-Defined Sensitivity of Lung Metastasis,but Not of Primary Breast Tumors,to mTORC1 Inhibition

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Interspecific hybridization causes long‐term phylogenetic discordance between nuclear and mitochondrial genomes in freshwater fishes

Classification, phylogeography and the testing of evolutionary hypotheses rely on correct estimation of species phylogeny. Early molecular phylogenies often relied on mtDNA alone, which acts as a single linkage group with one history. Over the last decade, the use of multiple nuclear sequences has often revealed conflict among gene trees. This observation can be attributed to hybridization, lineage sorting, paralogy or selection. Here, we use 54 groups of fishes from 48 studies to estimate the degree of concordance between mitochondrial and nuclear gene trees in two ecological grades of fishes: marine and freshwater. We test the hypothesis that freshwater fish phylogenies should, on average, show more discordance because of their higher propensity for hybridization in the past. In keeping with this idea, concordance between mitochondrial and nuclear gene trees (as measured by proportion of components shared) is on average 50% higher in marine fishes. We discuss why this difference almost certainly results from introgression caused by greater historical hybridization among lineages in freshwater groups, and further emphasize the need to use multiple nuclear genes, and identify conflict among them, in estimation of species phylogeny.