Palmitoylation at Cys 1145 in the Carboxyl Terminus of Human Type 6 Adenylyl Cyclase is Not Required for Targeting to Lipid Rafts and Caveolae

Palmitoylation is important for targeting certain membrane-associated and integral membrane proteins to lipid rafts and caveolae. Previous data have shown that adenylyl cyclase type 6 (AC6) is enriched in lipid rafts or caveolae while other isoforms of AC, such as AC2 and AC4, are excluded from these domains. We hypothesized that palmitoylation on a cysteine residue in the carboxyl terminus (C-terminus) of AC6 or other elements encoded in this region are required for AC6 expression in lipid rafts and caveolae. Thus, we expressed in Cos-7 cells epitope-tagged full length human AC6 and three different C-terminally truncated AC6 proteins, one (AC6 1-1148) retaining Cys 1145, a putative palmitoylation site, and two others (AC6 1-1144 and AC6 1-1127) lacking this residue. We used several approaches for assessing the subcellular localization of these expressed proteins: non-detergent biochemical isolation of lipid rafts and immunoblotting, immunoprecipitation of caveolin-1, Triton-X-100 insolubility, and immunoisolation of caveolae followed by adenylyl cyclase activity assays. We found that AC6 1-1144, AC6 1-1127 and AC6 1–1148 truncation proteins were each localized similarly to full-length AC6. We conclude that neither the putative palmitoylation site Cys 1145, nor other elements in the distal portion of the carboxyl terminus of AC6, are important for targeting of this effector enzyme to lipid rafts and caveolae

A Novel Graphene Quantum Dot-Based mRNA Delivery Platform

During the last decades, there has been growing interest in using therapeutic messager RNA (mRNA) together with drug delivery systems. Naked, unformulated mRNA is, however, unable to cross the cell membrane and is susceptible to degradation. Here we use graphene quantum dots (GQDs) functionalized with polyethyleneimine (PEI) as a novel mRNA delivery system. Our results show that these modified GQDs can be used to deliver intact and functional mRNA to Huh-7 hepatocarcinoma cells at low doses and, that the GQDs are not toxic, although cellular toxicity is a problem for these first-generation modified particles. Functionalized GQDs represent a potentially interesting delivery system that is easy to manufacture, stable and effective

High-Resolution 3D Heart Models of Cardiomyocyte Subpopulations in Cleared Murine Heart

Biological tissues are naturally three-dimensional (3D) opaque structures, which poses a major challenge for the deep imaging of spatial distribution and localization of specific cell types in organs in biomedical research. Here we present a 3D heart imaging reconstruction approach by combining an improved heart tissue-clearing technique with high-resolution light-sheet fluorescence microscopy (LSFM). We have conducted a three-dimensional and multi-scale volumetric imaging of the ultra-thin planes of murine hearts for up to 2,000 images per heart in x-, y-, and z three directions. High-resolution 3D volume heart models were constructed in real-time by the Zeiss Zen program. By using such an approach, we investigated detailed three-dimensional spatial distributions of two specific cardiomyocyte populations including HCN4 expressing pacemaker cells and Pnmt(+) cell-derived cardiomyocytes by using reporter mouse lines Hcn4(DreER/tdTomato) and Pnmt(Cre/ChR2−tdTomato). HCN4 is distributed throughout right atrial nodal regions (i.e., sinoatrial and atrioventricular nodes) and the superior-inferior vena cava axis, while Pnmt(+) cell-derived cardiomyocytes show distinct ventral, left heart, and dorsal side distribution pattern. Our further electrophysiological analysis indicates that Pnmt + cell-derived cardiomyocytes rich left ventricular (LV) base is more susceptible to ventricular arrhythmia under adrenergic stress than left ventricular apex or right ventricle regions. Thus, our 3D heart imaging reconstruction approach provides a new solution for studying the geometrical, topological, and physiological characteristics of specific cell types in organs

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis

Peptidylarginine deiminase 1-catalyzed histone citrullination is essential for early embryo development

Peptidylarginine deiminase (PADI) enzymes are increasingly being associated with the regulation of chromatin structure and gene activity via histone citrullination. As one of the PADI family members, PADI1 has been mainly reported to be expressed in the epidermis and uterus, where the protein in keratinocytes is thought to promote differentiation by citrullinating filament proteins. However, the roles of PADI1 in preimplantation development have not been addressed. Using a PADI1-specific inhibitor and Padi1-morpholino knockdown, we found that citrullination of histone tails at H4R3 and H3R2/8/17 were markedly reduced in the 2- and 4-cell embryos. Consistent with this observation, early embryo development was also arrested at the 4-cell stage upon depletion of PADI1 or inhibition of PADI1 enzyme activity. Additionally, by employing 5-ethynyl uridine (EU) incorporation analysis, ablation of PADI1 function led to a dramatic decrease in overall transcriptional activity, correlating well with the reduced levels of phosphorylation of RNA Pol II at Ser2 observed at 2- or 4-cell stage of embryos under Padi1 knockdown or inhibiting PADI1. Thus, our data reveal a novel function of PADI1 during early embryo development transitions by catalyzing histone tail citrullination, which facilitates early embryo genome transactivation

Temporal Factors and Missed Doses of Tuberculosis Treatment: A Causal Associations Approach to Analyses of Digital Adherence Data

Rationale: Tuberculosis treatment lasts for 6 months or more. Treatment adherence is critical; regimen length, among other factors, makes this challenging. Globally, analyses mapping common types of nonadherence are lacking. For example, is there a greater challenge resulting from early treatment cessation (discontinuation) or intermittent missed doses (suboptimal dosing implementation)? This is essential knowledge for the development of effective interventions and more "forgiving" regimens, as well as to direct national tuberculosis programs.Objectives: To granularly describe how patients take their tuberculosis medication and the temporal factors associated with missed doses.Methods: The present study included patients with pulmonary tuberculosis enrolled in the control arm of a pragmatic, cluster-randomized trial in China of electronic reminders to improve treatment adherence. Treatment was the standard 6-month course (180 d), dosed every other day (90 doses). Medication monitor boxes recorded adherence (box opening) without prompting reminders. Patterns of adherence were visualized and described. Mixed-effects logistic regression models examined the temporal factors associated with per-dose suboptimal dosing implementation, adjusting for clustering within a participant. Cox regression models were used to examine the association between early suboptimal dosing implementation and permanent discontinuation.Results: Across 780 patients, 16,794 (23.9%) of 70,200 doses were missed, 9,487 of which were from suboptimal dosing implementation (56.5%). By 60 days, 5.1% of participants had discontinued, and 14.4% had discontinued by 120 days. Most participants (95.9%) missed at least one dose. The majority of gaps were of a single dose (71.4%), although 22.6% of participants had at least one gap of 2 weeks or more. In adjusted models, the initiation-continuation phase transition (odds ratio, 3.07 [95% confidence interval, 2.68-3.51]) and national holidays (1.52 [1.39-1.65]) were associated with increased odds of suboptimal dosing implementation. Early-stage suboptimal dosing implementation was associated with increased discontinuation rates.Conclusions: Digital tools provide an unprecedented step change in describing and addressing nonadherence. In our setting, nonadherence was common; patients displayed a complex range of patterns. Dividing nonadherence into suboptimal dosing implementation and discontinuation, we found that both increased over time. Discontinuation was associated with early suboptimal dosing implementation. These apparent causal associations between temporal factors and nonadherence present opportunities for targeted interventions.Clinical trial registered with the ISRCTN Registry (ISRCTN46846388)

Causal association of gastroesophageal reflux disease with obstructive sleep apnea and sleep-related phenotypes: a bidirectional two-sample Mendelian randomization study

BackgroundThe interactions and associations between obstructive sleep apnea (OSA), sleep-related phenotypes (SRPs), and gastroesophageal reflux disease (GERD) are complex, thus it is hard to explore the effect and direction of causalities.Study objectivesA bidirectional Mendelian randomization (MR) study was performed to explore causal associations of GERD with OSA and SRPs (including insomnia, morningness, sleep duration, ease of getting up, daytime napping, daytime dozing, and snoring).MethodsFirst, we gathered summary statistics from publicly available databases. Subsequently, we identified single-nucleotide polymorphisms without strong linkage (r2 ≤ 0.001) by referencing relevant genome-wide association studies that met genome-wide significance criteria. Our primary analysis relied on inverse variance weighted to estimate the causal relationship. To ensure the validity of our findings, we also conducted several sensitivity analyses. These included MR Pleiotropy RESidual Sum and Outlier to detect and correct for potential pleiotropic effects, MR-Egger to assess directional pleiotropy, and weighted median analysis to further evaluate heterogeneity and pleiotropy. For the initial MR analysis, when causality was indicated by the results, instrumental variables that were significantly linked to the aforementioned confounding factors were removed. We will re-analyze the data after excluding outcome-related single nucleotide polymorphisms to confirm that the results are still consistent with the previous results.ResultsGERD was found to increase the risk of OSA (OR = 1.53, 95% CI = 1.37–1.70, p = 5.3 × 10−15), insomnia (OR = 1.14, 95% CI = 1.10–1.19, p = 1.3 × 10−10), snoring (OR = 1.09, 95% CI = 1.04–1.13, p = 6.3 × 10−5) and less sleep duration (OR = 0.94, 95% CI = 0.91–0.97, p = 3.7 × 10−4). According to the reverse-direction analysis, there is an elevated risk of GERD associated with OSA (OR = 1.07, 95% CI = 1.02–1.12, p = 0.005), insomnia (OR = 1.95, 95% CI = 1.60–2.37, p = 1.92 × 10−11) and snoring (OR = 1.74, 95% CI = 1.37–2.21, p = 4.4 × 10−6).ConclusionGenetic susceptibility to GERD can elevate the likelihood of experiencing insomnia, snoring, and OSA, in addition to diminishing sleep duration. Conversely, a reverse MR analysis indicates that ameliorating any one of insomnia, snoring, or OSA can mitigate the risk of developing GERD

Secondhand smoke presence in outdoor areas in 12 European countries

Introduction: Secondhand smoke (SHS) causes morbidity and mortality among non-smokers. Objectives: To investigate SHS presence in outdoor areas from 12 European countries and its association with country-level characteristics. Methods: Cross-sectional study performed in 2017-2018 within the TackSHS project. We conducted a face-to-face survey on a representative sample of the population aged 15 years and older from 12 European countries: Bulgaria, England, France, Germany, Greece, Ireland, Italy, Latvia, Poland, Portugal, Romania, and Spain. Out of 11,902 participants, 8,562 were non-smokers. SHS presence was assessed in selected outdoor areas and defined as respondents viewing someone smoking the last time they visited each setting within the last 6 months. A ranking score for outdoor SHS presence was assigned to each country based on the SHS presence in each setting. We used Spearman's correlation (r) and the Chi-squared tests to assess the relationship between SHS presence and country-level characteristics. Results: Except for children's playgrounds (39.5%; 95% confidence interval, CI: 37.6%-41.3%), more than half of non-smokers reported SHS presence in outdoor areas: schools (52.0%; 95%CI: 50.2%-53.7%), stadia (57.4%; 95%CI: 55.4%-59.4%), parks (67.3%; 95%CI: 66.0%-68.5%), hospitals (67.3%; 95%CI: 65.8%-68.7%), public transport stops (69.9%; 95%CI: 68.6%-71.2%), bar/restaurant terraces (71.4%; 95%CI: 70.2%-72.6%), and beaches (72.8%; 95%CI: 71.4%-74.1%). Residents in Latvia showed the highest overall outdoor SHS presence rank, followed by Greece, and Portugal. Outdoor SHS presence was directly correlated to the country's smoking prevalence (r = 0.64), and inversely correlated to the Tobacco Control Scale 2016 overall score (r = -0.62), the socio-demographic index 2017 (r = -0.56), and Gross Domestic Product per capita 2018 (r = -0.47) (p < 0.001). Conclusions: SHS presence is high in most outdoor areas in Europe, especially in countries with higher smoking prevalence and lower tobacco control performance. To address outdoor SHS exposure, our findings require considering smoking bans along with other strategies to reduce smoking prevalence

Severe Maternal Hyperglycemia Exacerbates the Development of Insulin Resistance and Fatty Liver in the Offspring on High Fat Diet

Background. Adverse maternal environments may predispose the offspring to metabolic syndrome in adulthoods, but the underlying mechanism has not been fully understood. Methods. Maternal hyperglycemia was induced by streptozotocin (STZ) injection while control (CON) rats received citrate buffer. Litters were adjusted to eight pups per dam and then weaned to standard diet. Since 13 weeks old, a subset of offspring from STZ and CON dams were switched to high fat diet (HFD) for another 13 weeks. Glucose and insulin tolerance tests (GTT and ITT) and insulin secretion assay were performed; serum levels of lipids and leptin were measured. Hepatic fat accumulation and islet area were evaluated through haematoxylin and eosin staining. Results. STZ offspring exhibited lower survival rate, lower birth weights, and growth inhibition which persisted throughout the study. STZ offspring on HFD showed more severe impairment in GTT and ITT, and more profound hepatic steatosis and more severe hyperlipidemia compared with CON-HFD rats. Conclusions. Offspring from diabetic dams would be prone to exhibit low birth weight and postnatal growth inhibition, but could maintain normal glucose tolerance and insulin sensitivity. HFD accelerates development of insulin resistance in the offspring of diabetic dams mainly via a compensatory response of islets