Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1–4Rs

Magnetofection enhances adenoviral vector-based gene delivery in skeletal muscle cells

The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models.Instituto de Investigaciones Bioquímicas de La Plat

Intrauterine Growth Restriction following Ligation of the Uterine Arteries Leads to More Severe Glomerulosclerosis after Mesangioproliferative Glomerulonephritis in the Offspring

Background: Low birth weight is a risk factor for the development of a more severe course of secondary renal diseases. We tested the hypothesis that experimental mesangioproliferative glomerulonephritis (GN) shows an aggravated course in rats inflicted with experimental uteroplacental insufficiency during gestation. Methods: Intrauterine growth restriction (IUGR) was induced by ligation of both uterine arteries on day 19 in pregnant Wistar rat dams. GN was induced in male offspring at the age of 9 weeks by intravenous injection of an anti-Thy-1.1 antibody. At day 14 of GN, kidneys were taken and analyzed for glomerular morphometry, markers of inflammation, glomerulosclerosis and tubulointerstitial fibrosis. Results: Despite a similar extent of mesangiolysis, former IUGR animals presented with a higher level of glomerulosclerosis and increased deposition of glomerular collagens I and IV compared to nephritic control animals. Arterial blood pressure, renal function, and proteinuria after 14 days of GN were not influenced by former IUGR. Conclusion: Ligation of the uterine arteries in the rat leads to more pronounced sclerotic changes in the glomerulus in the offspring suffering from acute GN. This finding supports the hypothesis that former IUGR increases the susceptibility for a more severe course of secondary renal diseases

Isogeometric approximation of cardiac electrophysiology models on surfaces: An accuracy study with application to the human left atrium

We consider Isogeometric Analysis in the framework of the Galerkin method for the spatial approximation of cardiac electrophysiology models defined on NURBS surfaces; specifically, we perform a numerical comparison between basis functions of degree p ≥ 1 and globally C k -continuous, with k = 0 or p − 1, to find the most accurate approximation of a propagating front with the minimal number of degrees of freedom. We show that B-spline basis functions of degree p ≥ 1, which are C p−1 -continuous capture accurately the front velocity of the transmembrane potential even with moderately refined meshes; similarly, we show that, for accurate tracking of curved fronts, high-order continuous B-spline basis functions should be used. Finally, we apply Isogeometric Analysis to an idealized human left atrial geometry described by NURBS with physiologically sound fiber directions and anisotropic conductivity tensor to demonstrate that the numerical scheme retains its favorable approximation properties also in a more realistic setting

The application of online transcranial random noise stimulation and perceptual learning in the improvement of visual functions in mild myopia

It has recently been demonstrated how perceptual learning, that is an improvement in a sensory/perceptual task upon practice, can be boosted by concurrent high-frequency transcranial random noise stimulation (tRNS). It has also been shown that perceptual learning can generalize and produce an improvement of visual functions in participants with mild refractive defects. By using three different groups of participants (single-blind study), we tested the efficacy of a short training (8 sessions) using a single Gabor contrast-detection task with concurrent hf-tRNS in comparison with the same training with sham stimulation or hf-tRNS with no concurrent training, in improving visual acuity (VA) and contrast sensitivity (CS) of individuals with uncorrected mild myopia. A short training with a contrast detection task is able to improve VA and CS only if coupled with hf-tRNS, whereas no effect on VA and marginal effects on CS are seen with the sole administration of hf-tRNS. Our results support the idea that, by boosting the rate of perceptual learning via the modulation of neuronal plasticity, hf-tRNS can be successfully used to reduce the duration of the perceptual training and/or to increase its efficacy in producing perceptual learning and generalization to improved VA and CS in individuals with uncorrected mild myopia

Early and Late Postnatal Myocardial and Vascular Changes in a Protein Restriction Rat Model of Intrauterine Growth Restriction

Intrauterine growth restriction (IUGR) is a risk factor for cardiovascular disease in later life. Early structural and functional changes in the cardiovascular system after IUGR may contribute to its pathogenesis. We tested the hypothesis that IUGR leads to primary myocardial and vascular alterations before the onset of hypertension. A rat IUGR model of maternal protein restriction during gestation was used. Dams were fed low protein (LP; casein 8.4%) or isocaloric normal protein diet (NP; casein 17.2%). The offspring was reduced to six males per litter. Immunohistochemical and real-time PCR analyses were performed in myocardial and vascular tissue of neonates and animals at day 70 of life. In the aortas of newborn IUGR rats expression of connective tissue growth factor (CTGF) was induced 3.2-fold. At day 70 of life, the expression of collagen I was increased 5.6-fold in aortas of IUGR rats. In the hearts of neonate IUGR rats, cell proliferation was more prominent compared to controls. At day 70 the expression of osteopontin was induced 7.2-fold. A 3- to 7-fold increase in the expression of the profibrotic cytokines TGF-β and CTGF as well as of microfibrillar matrix molecules was observed. The myocardial expression and deposition of collagens was more prominent in IUGR animals compared to controls at day 70. In the low-protein diet model, IUGR leads to changes in the expression patterns of profibrotic genes and discrete structural abnormalities of vessels and hearts in adolescence, but, with the exception of CTGF, not as early as at the time of birth. Invasive and non-invasive blood pressure measurements confirmed that IUGR rats were normotensive at the time point investigated and that the changes observed occurred independently of an increased blood pressure. Hence, altered matrix composition of the vascular wall and the myocardium may predispose IUGR animals to cardiovascular disease later in life

Prediction model to estimate presence of coronary artery disease: retrospective pooled analysis of existing cohorts

Objectives To develop prediction models that better estimate the pretest probability of coronary artery disease in low prevalence populations

A partially supervised physical activity program for adult and adolescent survivors of childhood cancer (SURfit): study design of a randomized controlled trial [NCT02730767].

BACKGROUND Beyond survival of nowadays >80%, modern childhood cancer treatment strives to preserve long-term health and quality of life. However, the majority of today's survivors suffer from short- and long-term adverse effects such as cardiovascular and pulmonary diseases, obesity, osteoporosis, fatigue, depression, and reduced physical fitness and quality of life. Regular exercise can play a major role to mitigate or prevent such late-effects. Despite this, there are no data on the effects of regular exercise in childhood cancer survivors from randomized controlled trials (RCTs). Primary outcome of the current RCT is therefore the effect of a 12-months exercise program on a composite cardiovascular disease risk score in childhood cancer survivors. Secondary outcomes are single cardiovascular disease risk factors, glycaemic control, bone health, body composition, physical fitness, physical activity, quality of life, mental health, fatigue and adverse events (safety). METHODS A total of 150 childhood cancer survivors aged ≥16 years and diagnosed ≥5 years prior to the study are recruited from Swiss paediatric oncology clinics. Following the baseline assessments patients are randomized 1:1 into an intervention and control group. Thereafter, they are seen at month 3, 6 and 12 for follow-up assessments. The intervention group is asked to add ≥2.5 h of intense physical activity/week, including 30 min of strength building and 2 h of aerobic exercises. In addition, they are told to reduce screen time by 25%. Regular consulting by physiotherapists, individual web-based activity diaries, and pedometer devices are used as motivational tools for the intervention group. The control group is asked to keep their physical activity levels constant. DISCUSSION The results of this study will show whether a partially supervised exercise intervention can improve cardiovascular disease risk factors, bone health, body composition, physical activity and fitness, fatigue, mental health and quality of life in childhood cancer survivors. If the program will be effective, all relevant information of the SURfit physical activity intervention will be made available to interested clinics that treat and follow-up childhood cancer patients to promote exercise in their patients. TRIAL REGISTRATION Prospectively registered in clinicaltrials.gov [ NCT02730767 ], registration date: 10.12.2015

Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial

BackgroundAnterior cruciate ligament (ACL) rupture is a common debilitating injury that can cause instability of the knee. We aimed to investigate the best management strategy between reconstructive surgery and non-surgical treatment for patients with a non-acute ACL injury and persistent symptoms of instability.MethodsWe did a pragmatic, multicentre, superiority, randomised controlled trial in 29 secondary care National Health Service orthopaedic units in the UK. Patients with symptomatic knee problems (instability) consistent with an ACL injury were eligible. We excluded patients with meniscal pathology with characteristics that indicate immediate surgery. Patients were randomly assigned (1:1) by computer to either surgery (reconstruction) or rehabilitation (physiotherapy but with subsequent reconstruction permitted if instability persisted after treatment), stratified by site and baseline Knee Injury and Osteoarthritis Outcome Score—4 domain version (KOOS4). This management design represented normal practice. The primary outcome was KOOS4 at 18 months after randomisation. The principal analyses were intention-to-treat based, with KOOS4 results analysed using linear regression. This trial is registered with ISRCTN, ISRCTN10110685, and ClinicalTrials.gov, NCT02980367.FindingsBetween Feb 1, 2017, and April 12, 2020, we recruited 316 patients. 156 (49%) participants were randomly assigned to the surgical reconstruction group and 160 (51%) to the rehabilitation group. Mean KOOS4 at 18 months was 73·0 (SD 18·3) in the surgical group and 64·6 (21·6) in the rehabilitation group. The adjusted mean difference was 7·9 (95% CI 2·5–13·2; p=0·0053) in favour of surgical management. 65 (41%) of 160 patients allocated to rehabilitation underwent subsequent surgery according to protocol within 18 months. 43 (28%) of 156 patients allocated to surgery did not receive their allocated treatment. We found no differences between groups in the proportion of intervention-related complications.InterpretationSurgical reconstruction as a management strategy for patients with non-acute ACL injury with persistent symptoms of instability was clinically superior and more cost-effective in comparison with rehabilitation management