Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

Dealing with bad guys: actor- and process-level determinants of the “devil shift” in policy making

Policy actors tend to misinterpret and distrust opponents in policy processes. This phenomenon, known as the “devil shift”, consists of the following two dimensions: actors perceive opponents as more powerful and as more evil than they really are. Analysing nine policy processes in Switzerland, this article highlights the drivers of the devil shift at two levels. On the actor level, interest groups, political parties and powerful actors suffer more from the devil shift than state actors and powerless actors. On the process level, the devil shift is stronger in policy processes dealing with socio-economic issues as compared with other issues. Finally, and in line with previous studies, there is less empirical evidence of the power dimension of the devil shift phenomenon than of its evilness dimension.

Structural and Institutional Determinants of Influence Reputation: A Comparison of Collaborative and Adversarial Policy Networks in Decision Making and Implementation

The central assumption in the literature on collaborative networks and policy networks is that political outcomes are affected by a variety of state and nonstate actors. Some of these actors are more powerful than others and can therefore have a considerable effect on decision making. In this article, we seek to provide a structural and institutional explanation for these power differentials in policy networks and support the explanation with empirical evidence. We use a dyadic measure of influence reputation as a proxy for power, and posit that influence reputation over the political outcome is related to vertical integration into the political system by means of formal decision-making authority, and to horizontal integration by means of being well embedded into the policy network. Hence, we argue that actors are perceived as influential because of two complementary factors: (a) their institutional roles and (b) their structural positions in the policy network. Based on temporal and cross-sectional exponential random graph models, we compare five cases about climate, telecommunications, flood prevention, and toxic chemicals politics in Switzerland and Germany. The five networks cover national and local networks at different stages of the policy cycle. The results confirm that institutional and structural drivers seem to have a crucial impact on how an actor is perceived in decision making and implementation and, therefore, their ability to significantly shape outputs and service delivery.

IFPA Meeting 2013 Workshop Report III: maternal placental immunological interactions, novel determinants of trophoblast cell fate, dual ex vivo perfusion of the human placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2013 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of placental function, cell turnover and immunology: 1) immunology; 2) novel determinants of placental cell fate; 3) dual perfusion of human placental tissue.

Determinants of renal tissue oxygenation as measured with BOLD-MRI in chronic kidney disease and hypertension in humans.

Experimentally renal tissue hypoxia appears to play an important role in the pathogenesis of chronic kidney disease (CKD) and arterial hypertension (AHT). In this study we measured renal tissue oxygenation and its determinants in humans using blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) under standardized hydration conditions. Four coronal slices were selected, and a multi gradient echo sequence was used to acquire T2* weighted images. The mean cortical and medullary R2* values ( = 1/T2*) were calculated before and after administration of IV furosemide, a low R2* indicating a high tissue oxygenation. We studied 195 subjects (95 CKD, 58 treated AHT, and 42 healthy controls). Mean cortical R2 and medullary R2* were not significantly different between the groups at baseline. In stimulated conditions (furosemide injection), the decrease in R2* was significantly blunted in patients with CKD and AHT. In multivariate linear regression analyses, neither cortical nor medullary R2* were associated with eGFR or blood pressure, but cortical R2* correlated positively with male gender, blood glucose and uric acid levels. In conclusion, our data show that kidney oxygenation is tightly regulated in CKD and hypertensive patients at rest. However, the metabolic response to acute changes in sodium transport is altered in CKD and in AHT, despite preserved renal function in the latter group. This suggests the presence of early renal metabolic alterations in hypertension. The correlations between cortical R2* values, male gender, glycemia and uric acid levels suggest that these factors interfere with the regulation of renal tissue oxygenation.

Good intents but low impacts: Various determinants for different indicators of overall energy use

Energy consumption in industrialized countries by far exceeds a sustainable level. Previous research on determinants of overall consumption levels has yielded contradictory results as to what the main drivers are. While research on the relationship of environmental concerns and pro-environmental behavior emphasizes the importance of motivational aspects, more impact-oriented research challenges these findings and underlines the impacts of a person’s social standing. The aim of our research was to determine which amount of per-capita energy consumption can be explained by structural, socio-demographic, and pro-environmentally motivational variables. Data come from standardized interviews with a representative sample (N=1014) in Germany. Different indicators of per-capita use were collected and will provide the basis for calculating the overall consumption level. In addition, person variables, lifestyle milieus, self-reported energy use, and motivational variables were assessed. First regression analyses show various patterns of determinants for different indicators of overall energy use. While variance in self-reported use is mainly explained by environmental concern, more impact-oriented indicators, such as the size of personal living space and distances of vacation trips, predominantly correlate with status-relevant predictors. These preliminary results support the suspicion that although environmentally aware people intend to reduce their energy use, they rarely go beyond low-impact actions.

Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering

Polydnaviruses (genera Ichnovirus and Bracovirus) have a segmented genome of circular double-stranded DNA molecules, replicate in the ovary of parasitic wasps and are essential for successful parasitism of the host. Here we show the first detailed analysis of various segments of a bracovirus, the Chelonus inanitus virus (CiV). Four segments were sequenced and two of them, CiV12 and CiV14, were found to be closely related while CiV14.5 and CiV16.8 were unrelated. CiV12, CiV14.5 and CiV16.8 are unique while CiV14 occurs also nested in another larger segment. All four segments are predicted to contain genes and predictions could be substantiated in most cases. Comparison with databases revealed no significant similarities at either the nucleotide or amino acid level. Inverted repeats with identities between 77% and 92% and lengths between 26 bp and 100 bp were found on all segments outside of predicted genes. Hybridization experiments indicate that CiV12 and CiV14 are both flanked by other virus segments, suggesting that proviral CiV segments are clustered in the genome of the wasp. The integration/excision site of CiV14 was analysed and compared to that of CiV12. On both termini of proviral CiV12 and CiV14 as well as in the excised circular molecule and the rejoined DNA a very similar repeat of 14 bp was found. A model to illustrate where the terminal repeats might recombine to yield the circular molecule is presented. Excision of CiV12 and CiV14 is restricted to the female and sets in at a very specific time-point in pupal-adult development.

Genetic Resistance Determinants, In Vitro Time-Kill Curve Analysis and Pharmacodynamic Functions for the Novel Topoisomerase II Inhibitor ETX0914 (AZD0914) in Neisseria gonorrhoeae.

Resistance in Neisseria gonorrhoeae to all available therapeutic antimicrobials has emerged and new efficacious drugs for treatment of gonorrhea are essential. The topoisomerase II inhibitor ETX0914 (also known as AZD0914) is a new spiropyrimidinetrione antimicrobial that has different mechanisms of action from all previous and current gonorrhea treatment options. In this study, the N. gonorrhoeae resistance determinants for ETX0914 were further described and the effects of ETX0914 on the growth of N. gonorrhoeae (ETX0914 wild type, single step selected resistant mutants, and efflux pump mutants) were examined in a novel in vitro time-kill curve analysis to estimate pharmacodynamic parameters of the new antimicrobial. For comparison, ciprofloxacin, azithromycin, ceftriaxone, and tetracycline were also examined (separately and in combination with ETX0914). ETX0914 was rapidly bactericidal for all wild type strains and had similar pharmacodynamic properties to ciprofloxacin. All selected resistant mutants contained mutations in amino acid codons D429 or K450 of GyrB and inactivation of the MtrCDE efflux pump fully restored the susceptibility to ETX0914. ETX0914 alone and in combination with azithromycin and ceftriaxone was highly effective against N. gonorrhoeae and synergistic interaction with ciprofloxacin, particularly for ETX0914-resistant mutants, was found. ETX0914, monotherapy or in combination with azithromycin (to cover additional sexually transmitted infections), should be considered for phase III clinical trials and future gonorrhea treatment.

A retrospective study of 1- versus 2-cm excision margins for cutaneous malignant melanomas thicker than 2 mm.

BACKGROUND Most guidelines recommend at least 2-cm excision margin for melanomas thicker than 2 mm. OBJECTIVE We evaluated whether 1- or 2-cm excision margins for melanoma (>2 mm) result in different outcomes. METHODS This is a retrospective cohort study on patients with melanomas (>2 mm) who underwent tumor excision with 1-cm (228 patients) or 2-cm (97 patients) margins to investigate presence of local recurrences, locoregional and distant metastases, and disease-free and overall survival. RESULTS In all, 325 patients with mean age of 61.84 years and Breslow thickness of 4.36 mm were considered for the study with a median follow-up of 1852 days (1995-2012). There was no significant difference in the frequency of locoregional and distant metastasis between the 2 groups (P = .311 and .571). The survival analysis showed no differences for disease-free (P = .800; hazard ratio 0.948; 95% confidence interval 0.627-1.433) and overall (P = .951; hazard ratio 1.018; 95% confidence interval 0.575-1.803) survival. LIMITATIONS The study was not prospectively randomized. CONCLUSIONS Our study did not show any significant differences in important outcome parameters such as local or distant metastases and overall survival. A prospective study testing 1- versus 2-cm excision margin is warranted.

3CAPS – a structural AP-site analogue as a tool to investigate DNA base excision repair

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3′-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

Determinants of Weight Evolution Among HIV-Positive Patients Initiating Antiretroviral Treatment in Low-Resource Settings.

BACKGROUND In resource-limited settings, clinical parameters, including body weight changes, are used to monitor clinical response. Therefore, we studied body weight changes in patients on antiretroviral treatment (ART) in different regions of the world. METHODS Data were extracted from the "International Epidemiologic Databases to Evaluate AIDS," a network of ART programmes that prospectively collects routine clinical data. Adults on ART from the Southern, East, West, and Central African and the Asia-Pacific regions were selected from the database if baseline data on body weight, gender, ART regimen, and CD4 count were available. Body weight change over the first 2 years and the probability of body weight loss in the second year were modeled using linear mixed models and logistic regression, respectively. RESULTS Data from 205,571 patients were analyzed. Mean adjusted body weight change in the first 12 months was higher in patients started on tenofovir and/or efavirenz; in patients from Central, West, and East Africa, in men, and in patients with a poorer clinical status. In the second year of ART, it was greater in patients initiated on tenofovir and/or nevirapine, and for patients not on stavudine, in women, in Southern Africa and in patients with a better clinical status at initiation. Stavudine in the initial regimen was associated with a lower mean adjusted body weight change and with weight loss in the second treatment year. CONCLUSIONS Different ART regimens have different effects on body weight change. Body weight loss after 1 year of treatment in patients on stavudine might be associated with lipoatrophy.

Sociodemographic, behavioral and genetic determinants of allostatic load in a Swiss population-based study.

Allostatic load (AL) is a marker of physiological dysregulation which reflects exposure to chronic stress. High AL has been related to poorer health outcomes including mortality. We examine here the association of socioeconomic and lifestyle factors with AL. Additionally, we investigate the extent to which AL is genetically determined. We included 803 participants (52% women, mean age 48±16years) from a population and family-based Swiss study. We computed an AL index aggregating 14 markers from cardiovascular, metabolic, lipidic, oxidative, hypothalamus-pituitary-adrenal and inflammatory homeostatic axes. Education and occupational position were used as indicators of socioeconomic status. Marital status, stress, alcohol intake, smoking, dietary patterns and physical activity were considered as lifestyle factors. Heritability of AL was estimated by maximum likelihood. Women with a low occupational position had higher AL (low vs. high OR=3.99, 95%CI [1.22;13.05]), while the opposite was observed for men (middle vs. high OR=0.48, 95%CI [0.23;0.99]). Education tended to be inversely associated with AL in both sexes(low vs. high OR=3.54, 95%CI [1.69;7.4]/OR=1.59, 95%CI [0.88;2.90] in women/men). Heavy drinking men as well as women abstaining from alcohol had higher AL than moderate drinkers. Physical activity was protective against AL while high salt intake was related to increased AL risk. The heritability of AL was estimated to be 29.5% ±7.9%. Our results suggest that generalized physiological dysregulation, as measured by AL, is determined by both environmental and genetic factors. The genetic contribution to AL remains modest when compared to the environmental component, which explains approximately 70% of the phenotypic variance.

Determinants of stimulated salivary flow among haematopoietic stem cell transplantation recipients.

OBJECTIVES The aetiology of hyposalivation in haematopoietic stem cell transplantation (HSCT) recipients is not fully understood. This study examined the effects of treatment-related aetiological factors, particularly medications, on stimulated salivary flow in HSCT recipients. SUBJECTS AND METHODS Adult HSCT recipients (N = 118, 66 males, 27 autologous and 91 allogeneic transplants) were examined. Stimulated whole salivary flow rates (SWSFR) were measured before HSCT and at 6 and 12 months post-HSCT. Linear regression models were used to analyse the associations of medications and transplant-related factors with salivary flow rates, which were compared to salivary flow rates of generally healthy controls (N = 247). RESULTS The SWSFR of recipients were lower pre-HSCT (mean ± standard deviation, 0.88 ± 0.56 ml/min; P < 0.001), 6 months post-HSCT (0.84 ± 0.61; P < 0.001) and 12 months post-HSCT (1.08 ± 0.67; P = 0.005) than the SWSFR of controls (1.31 ± 0.65). In addition, hyposalivation (<0.7 ml/min) was more frequent among HSCT recipients pre-HSCT (P < 0.001), 6 months post-HSCT (P < 0.001) and 12 months post-HSCT (P = 0.01) than among controls. The SWSFR was observed to improve over time being significantly higher 12 months post-HSCT compared to pre-HSCT (P < 0.001). The observed decrease of salivary flow could not be explained by the examined transplant-related factors and medications. CONCLUSIONS Decreased stimulated salivary flow rates could not be explained by the examined factors alone; these findings indicate that hyposalivation in HSCT recipients exhibits a multifactorial aetiology. CLINICAL RELEVANCE All HSCT recipients should be considered to be at high risk of hyposalivation and consequent oral diseases, and they should be treated accordingly.