Three “hotspots” important for adenosine A2B receptor activation: a mutational analysis of transmembrane domains 4 and 5 and the second extracellular loop

G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Despite the progress made in the last decade in molecular and structural biology, their exact activation mechanism is still unknown. Here we describe new insights in specific regions essential in adenosine A2B receptor activation (A2BR), a typical class A GPCR. We applied unbiased random mutagenesis on the middle part of the human adenosine A2BR, consisting of transmembrane domains 4 and 5 (TM4 and TM5) linked by extracellular loop 2 (EL2), and subsequently screened in a medium-throughput manner for gain-of-function and constitutively active mutants. For that purpose, we used a genetically engineered yeast strain (Saccharomyces cerevisiae MMY24) with growth as a read-out parameter. From the random mutagenesis screen, 12 different mutant receptors were identified that form three distinct clusters; at the top of TM4, in a cysteine-rich region in EL2, and at the intracellular side of TM5. All mutant receptors show a vast increase in agonist potency and most also displayed a significant increase in constitutive activity. None of these residues are supposedly involved in ligand binding directly. As a consequence, it appears that disrupting the relatively “silent” configuration of the wild-type receptor in each of the three clusters readily causes spontaneous receptor activity

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio

Elemental composition of vegetables cultivated over coal-mining waste

ABSTRACT We assessed elemental composition of the liver in mice subjected to one-time or chronic consumption of the juice of vegetables cultivated in a vegetable garden built over deposits of coal waste. Lactuca sativa L. (lettuce), Beta vulgaris L. (beet), Brassica oleracea L. var. italica (broccoli) and Brassica oleracea L. var. acephala (kale) were collected from the coal-mining area and from a certified organic farm (control). Elemental composition was analyzed by particle-induced X-ray emission (PIXE) method. Concentrations of Mg, S, and Ca of mice subjected to one-time consumption of broccoli and concentrations of these same elements plus Si of mice receiving kale were higher in the coal-mining area. Concentrations of P, K, and Cu were increase after chronic consumption of lettuce from the coal-mining area, whereas the levels of Si, P, K, Fe, and Zn were higher in the group consuming kale from the coal-mining area. Our data suggests that people consuming vegetables grown over coal wastes may ingest significant amounts of chemical elements that pose a risk to health, since these plants contain both essential and toxic metals in a wide range of concentrations, which can do more harm than good

Metastatic colorectal cancer outcomes by age among ARCAD first- and second-line Clinical trials

Background We evaluated the time to progression (TTP) and survival outcomes of second-line therapy for metastatic colorectal cancer among adults aged 70 years and older compared with younger adults following progression on first-line clinical trials. Methods Associations between clinical and disease characteristics, time to initial progression, and rate of receipt of second-line therapy were evaluated. TTP and overall survival (OS) were compared between older and younger adults in first- and second-line trials by Cox regression, adjusting for age, sex, Eastern Cooperative Oncology Group Performance Status, number of metastatic sites and presence of metastasis in the lung, liver, or peritoneum. All statistical tests were 2-sided. Results Older adults comprised 16.4% of patients on first-line trials (870 total older adults aged >70 years; 4419 total younger adults aged ≤70 years, on first-line trials). Older adults and those with Eastern Cooperative Oncology Group Performance Status >0 were less likely to receive second-line therapy than younger adults. Odds of receiving second-line therapy decreased by 11% for each additional decade of life in multivariable analysis (odds ratio = 1.11, 95% confidence interval = 1.02 to 1.21, P = .01). Older and younger adults enrolled in second-line trials experienced similar median TTP and median OS (median TTP = 5.1 vs 5.2 months, respectively; median OS = 11.6 vs 12.4 months, respectively). Conclusions Older adults were less likely to receive second-line therapy for metastatic colorectal cancer, though we did not observe a statistical difference in survival outcomes vs younger adults following second-line therapy. Further study should examine factors affecting decisions to treat older adults with second-line therapy. Inclusion of geriatric assessment may provide better criteria regarding the risks and benefits of second-line therapy

Particulate matter exposure during pregnancy is associated with birth weight, but not gestational age, 1962-1992: a cohort study

<p>Abstract</p> <p>Background</p> <p>Exposure to air pollutants is suggested to adversely affect fetal growth, but the evidence remains inconsistent in relation to specific outcomes and exposure windows.</p> <p>Methods</p> <p>Using birth records from the two major maternity hospitals in Newcastle upon Tyne in northern England between 1961 and 1992, we constructed a database of all births to mothers resident within the city. Weekly black smoke exposure levels from routine data recorded at 20 air pollution monitoring stations were obtained and individual exposures were estimated via a two-stage modeling strategy, incorporating temporally and spatially varying covariates. Regression analyses, including 88,679 births, assessed potential associations between exposure to black smoke and birth weight, gestational age and birth weight standardized for gestational age and sex.</p> <p>Results</p> <p>Significant associations were seen between black smoke and both standardized and unstandardized birth weight, but not for gestational age when adjusted for potential confounders. Not all associations were linear. For an increase in whole pregnancy black smoke exposure, from the 1^st(7.4 μg/m³) to the 25^th(17.2 μg/m³), 50^th(33.8 μg/m³), 75^th(108.3 μg/m³), and 90^th(180.8 μg/m³) percentiles, the adjusted estimated decreases in birth weight were 33 g (SE 1.05), 62 g (1.63), 98 g (2.26) and 109 g (2.44) respectively. A significant interaction was observed between socio-economic deprivation and black smoke on both standardized and unstandardized birth weight with increasing effects of black smoke in reducing birth weight seen with increasing socio-economic disadvantage.</p> <p>Conclusions</p> <p>The findings of this study progress the hypothesis that the association between black smoke and birth weight may be mediated through intrauterine growth restriction. The associations between black smoke and birth weight were of the same order of magnitude as those reported for passive smoking. These findings add to the growing evidence of the harmful effects of air pollution on birth outcomes.</p

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Determinants of Physical Activity among Patients with Colorectal Cancer: From Diagnosis to 5 Years after Diagnosis

Introduction  Physical activity (PA) is associated with higher quality of life and probably better prognosis among colorectal cancer (CRC) patients. This study focuses on determinants of PA among CRC patients from diagnosis until 5 yr postdiagnosis. Methods  Sociodemographic and disease-related factors of participants of two large CRC cohort studies were combined. Moderate-to-vigorous PA during sport and leisure time (MVPA-SL) was measured at diagnosis (T0) and 6, 12, 24, and 60 months (T6 to T60) postdiagnosis, using the SQUASH questionnaire. Mixed-effects models were performed to identify sociodemographic and disease-related determinants of MVPA-SL, separately for stage I–III colon (CC), stage I–III rectal cancer (RC), and stage IV CRC (T0 and T6 only). Associations were defined as consistently present when significant at ≥4 timepoints for the stage I–III subsets. MVPA-SL levels were compared with an age- and sex-matched sample of the general Dutch population. Results  In total, 2905 CC, 1459 RC and 436 stage IV CRC patients were included. Patients with higher fatigue scores, and women compared with men had consistently lower MVPA-SL levels over time, regardless of tumor type and stage. At T6, having a stoma was significantly associated with lower MVPA-SL among stage I-III RC patients. Systemic therapy and radiotherapy were not significantly associated with MVPA-SL changes at T6. Compared with the general population, MVPA-SL levels of CRC patients were lower at all timepoints, most notably at T6. Conclusions  Female sex and higher fatigue scores were consistent determinants of lower MVPA-SL levels among all CRC patients, and MVPA-SL levels were lowest at 6 months postdiagnosis. Our results can inform the design of intervention studies aimed at improving PA, and guide healthcare professionals in optimizing individualized support

Severe Pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children from Wild-type to Population Immunity: A Prospective Multicenter Cohort Study with Real-time Reporting

Background: SARS-CoV-2 variant evolution and increasing immunity altered the impact of pediatric SARS-CoV-2 infection. Public health decision-making relies on accurate and timely reporting of clinical data. Methods: This international hospital-based multicenter, prospective cohort study with real-time reporting was active from March 2020 to December 2022. We evaluated longitudinal incident rates and risk factors for disease severity. Results: We included 564 hospitalized children with acute COVID-19 (n = 375) or multisystem inflammatory syndrome in children (n = 189) from the Netherlands, Curaçao and Surinam. In COVID-19, 134/375 patients (36%) needed supplemental oxygen therapy and 35 (9.3%) required intensive care treatment. Age above 12 years and preexisting pulmonary conditions were predictors for severe COVID-19. During omicron, hospitalized children had milder disease. During population immunity, the incidence rate of pediatric COVID-19 infection declined for older children but was stable for children below 1 year. The incidence rate of multisystem inflammatory syndrome in children was highest during the delta wave and has decreased rapidly since omicron emerged. Real-time reporting of our data impacted national pediatric SARS-CoV-2 vaccination- and booster-policies. Conclusions: Our data supports the notion that similar to adults, prior immunity protects against severe sequelae of SARS-CoV-2 infections in children. Real-time reporting of accurate and high-quality data is feasible and impacts clinical and public health decision-making. The reporting framework of our consortium is readily accessible for future SARS-CoV-2 waves and other emerging infections