Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.     

Change in HbA1c Levels between the Age of 8 Years and the Age of 12 Years in Dutch Children without Diabetes: The PIAMA Birth Cohort Study

Objective

HbA1c is associated with cardiovascular risk in persons without diabetes and cardiovascular risk accumulates over the life course. Therefore, insight in factors determining HbA1c from childhood onwards is important. We investigated (lifestyle) determinants of HbA1c at age 12 years and the effects of growth on change in HbA1c and the tracking of HbA1c between the age of 8 and 12 years.

Study Design and Methods

Anthropometric measurements were taken and HbA1c levels were assessed in 955 children without diabetes aged around 12 years participating in the PIAMA birth cohort study. In 363 of these children HbA1c was also measured at age 8 years. Data on parents and children were collected prospectively by questionnaires.

Results

We found no significant association between known risk factors for diabetes and HbA1c at age 12 years. Mean(SD) change in HbA1c between ages 8 and 12 years was 0.6(0.7) mmol/mol per year (or 0.1(0.1) %/yr). Anthropometric measures at age 8 and their change between age 8 and 12 years were not associated with the change in HbA1c. 68.9% of the children remained in the same quintile or had an HbA1c one quintile higher or lower at age 8 years compared to age 12 years.

Conclusion

The lack of association between known risk factors for diabetes and HbA1c suggest that HbA1c in children without diabetes is relatively unaffected by factors associated with glycaemia. HbA1c at age 8 years is by far the most important predictor of HbA1c at age 12. Therefore, the ranking of HbA1c levels appear to be fairly stable over time.     

Pediatric Drug Safety Surveillance in FDA-AERS: A Description of Adverse Events from GRiP Project

Individual case safety reports (ICSRs) are a cornerstone in drug safety surveillance. The knowledge on using these data specifically for children is limited. We studied characteristics of pediatric ICSRs reported to the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS). Public available ICSRs reported in children (0–18 years) to FAERS were downloaded from the FDA-website for the period Jan 2004-Dec 2011. Characteristics of these ICSRs, including the reported drugs and events, were described and stratified by age-groups. We included 106,122 pediatric ICSRs (55% boys and 58% from United States) with a median of 1 drug [range 1–3] and 1 event [1–2] per ICSR. Mean age was 9.1 years. 90% was submitted through expedited (15-days) (65%) or periodic reporting (25%) and 10% by non-manufacturers. The proportion and type of pediatric ICSRs reported were relatively stable over time. Most commonly reported drug classes by decreasing frequency were ‘nervous system drugs’ (58%), ‘antineoplastics’ (32%) and ‘anti-infectives’ (25%). Most commonly reported system organ classes were ‘general’ (13%), ‘nervous system’ (12%) and ‘psychiatric’ (11%) disorders. Duration of use could be calculated for 19.7% of the reported drugs, of which 14.5% concerned drugs being used long-term (>6 months). Knowledge on the distribution of the drug classes and events within FAERS is a key first step in developing pediatric specific methods for drug safety surveillance. Because of several differences in terms of drugs and events among age-categories, drug safety signal detection analysis in children needs to be stratified by each age group.     

Mapping Soil Properties of Africa at 250 m Resolution: Random Forests Significantly Improve Current Predictions

80% of arable land in Africa has low soil fertility and suffers from physical soil problems. Additionally, significant amounts of nutrients are lost every year due to unsustainable soil management practices. This is partially the result of insufficient use of soil management knowledge. To help bridge the soil information gap in Africa, the Africa Soil Information Service (AfSIS) project was established in 2008. Over the period 2008–2014, the AfSIS project compiled two point data sets: the Africa Soil Profiles (legacy) database and the AfSIS Sentinel Site database. These data sets contain over 28 thousand sampling locations and represent the most comprehensive soil sample data sets of the African continent to date. Utilizing these point data sets in combination with a large number of covariates, we have generated a series of spatial predictions of soil properties relevant to the agricultural management—organic carbon, pH, sand, silt and clay fractions, bulk density, cation-exchange capacity, total nitrogen, exchangeable acidity, Al content and exchangeable bases (Ca, K, Mg, Na). We specifically investigate differences between two predictive approaches: random forests and linear regression. Results of 5-fold cross-validation demonstrate that the random forests algorithm consistently outperforms the linear regression algorithm, with average decreases of 15–75% in Root Mean Squared Error (RMSE) across soil properties and depths. Fitting and running random forests models takes an order of magnitude more time and the modelling success is sensitive to artifacts in the input data, but as long as quality-controlled point data are provided, an increase in soil mapping accuracy can be expected. Results also indicate that globally predicted soil classes (USDA Soil Taxonomy, especially Alfisols and Mollisols) help improve continental scale soil property mapping, and are among the most important predictors. This indicates a promising potential for transferring pedological knowledge from data rich countries to countries with limited soil data.     

Serum Level of Soluble Receptor for Advanced Glycation End Products Is Associated with A Disintegrin And Metalloproteinase 10 in Type 1 Diabetes

Background

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of diabetic complications, and soluble forms of the receptor (sRAGE) can counteract the detrimental action of the full-length receptor by acting as decoy. Soluble RAGE is produced by alternative splicing [endogenous secretory RAGE (esRAGE)] and/or by proteolytic cleavage of the membrane-bound receptor. We have investigated the role of A Disintegrin And Metalloproteinase 10 (ADAM10) in the ectodomain shedding of RAGE.

Methods

Constitutive and insulin-induced shedding of RAGE in THP-1 macrophages by ADAM10 was evaluated using an ADAM10-specific metalloproteinase inhibitor. Serum ADAM10 level was measured in type 1 diabetes and control subjects, and the association with serum soluble RAGE was determined. Serum total sRAGE and esRAGE were assayed by ELISA and the difference between total sRAGE and esRAGE gave an estimated measure of soluble RAGE formed by cleavage (cRAGE).

Results

RAGE shedding (constitutive and insulin-induced) was significantly reduced after inhibition of ADAM10 in macrophages, and insulin stimulated ADAM10 expression and activity. Diabetic subjects have higher serum total sRAGE and esRAGE (p<0.01) than controls, and serum ADAM10 was also increased (p<0.01). Serum ADAM10 correlated with serum cRAGE in type 1 diabetes (r = 0.40, p<0.01) and in controls (r = 0.31. p<0.01) but no correlations were seen with esRAGE. The association remained significant after adjusting for age, gender, BMI, smoking status and HbA1c.

Conclusion

Our data suggested that ADAM10 contributed to the shedding of RAGE. Serum ADAM10 level was increased in type 1 diabetes and was a significant determinant of circulating cRAGE.     

Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.     

Ist2 recruits the lipid transporters Osh6/7 to ER–PM contacts to maintain phospholipid metabolism

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER–PM contacts through a binding site located in Ist2’s disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.     

Clinal variation in the morph ratio of Black Sparrowhawks Accipiter melanoleucus in South Africa and its correlation with environmental variables

The morph ratio distribution in polymorphic species often varies clinally, with a gradual change in morph ratios across the distributional range of the species. In polymorphic bird populations, clinal variation is rarely quantified. We describe a cline in the morph ratios of Black Sparrowhawks across South Africa, which is principally driven by a higher ratio of dark morph birds in the newly colonized southwest of the country. Across the 1400 km of our cline, the probability of a bird being a dark morph declined from over 80% close to the Cape Peninsula to under 20% in the northeast. Higher frequencies of dark morphs were associated with a higher proportion of rainfall falling during the winter breeding months. Further investigation revealed relationships between the proportion of dark morphs and altitude, amount of rainfall during the breeding months, and an interaction between this variable and temperature. These results provide some support for the suggestion that the higher frequency of dark morphs in the southwest is an adaptive response, rather than the result of a founder effect or genetic drift. These findings also suggest that, in theory, polymorphic species may be better adapted to cope with the challenges of climate change or may be able to expand their ranges more quickly into novel climatic areas, since selection pressure can act on a pre‐existing trait that may be beneficial in new conditions.