DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery.

More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR-metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs.

Effects of a novel mobile health intervention compared to a multi-component behaviour changing program on body mass index, physical capacities and stress parameters in adolescents with obesity: a randomized controlled trial.

BACKGROUND: Less than 2% of overweight children and adolescents in Switzerland can participate in multi-component behaviour changing interventions (BCI), due to costs and lack of time. Stress often hinders positive health outcomes in youth with obesity. Digital health interventions, with fewer on-site visits, promise health care access in remote regions; however, evidence for their effectiveness is scarce. METHODS: This randomized controlled not blinded trial (1:1) was conducted in a childhood obesity center in Switzerland. Forty-one youth aged 10-18 years with body mass index (BMI) > P.90 with risk factors or co-morbidities or BMI > P.97 were recruited. During 5.5 months, the PathMate2 group (PM) received daily conversational agent counselling via mobile app, combined with standardized counselling (4 on-site visits). Controls (CON) participated in a BCI (7 on-site visits). We compared the outcomes of both groups after 5.5 (T1) and 12 (T2) months. Primary outcome was reduction in BMI-SDS (BMI standard deviation score: BMI adjusted for age and sex). Secondary outcomes were changes in body fat and muscle mass (bioelectrical impedance analysis), waist-to-height ratio, physical capacities (modified Dordel-Koch-Test), blood pressure and pulse. Additionally, we hypothesized that less stressed children would lose more weight. Thus, children performed biofeedback relaxation exercises while stress parameters (plasma cortisol, stress questionnaires) were evaluated. RESULTS: At intervention start median BMI-SDS of all patients (18 PM, 13 CON) was 2.61 (obesity > + 2SD). BMI-SDS decreased significantly in CON at T1, but not at T2, and did not decrease in PM during the study. Muscle mass, strength and agility improved significantly in both groups at T2; only PM reduced significantly their body fat at T1 and T2. Average daily PM app usage rate was 71.5%. Cortisol serum levels decreased significantly after biofeedback but with no association between stress parameters and BMI-SDS. No side effects were observed. CONCLUSIONS: Equally to BCI, PathMate2 intervention resulted in significant and lasting improvements of physical capacities and body composition, but not in sustained BMI-SDS decrease. This youth-appealing mobile health intervention provides an interesting approach for youth with obesity who have limited access to health care. Biofeedback reduces acute stress and could be an innovative adjunct to usual care.

[Community-acquired Staphylococcus aureus pneumonia]. / Pneumonie communautaire à staphylocoque doré.

We report a clinical case of a patient hospitalized for community-acquired Staphylococcus aureus pneumonia. A 26-year-old patient with no medical history went to the emergency department for fever. He quickly developed acute respiratory failure and community-acquired Staphylococcus aureus pneumonia as well as bacteremia were confirmed. This pulmonary infection is rare but can affect all age groups and occur in a variety of ways. Patients with community-acquired Staphylococcus aureus pneumonia have more severe clinical outcomes than those with community-acquired pneumonia caused by other germs. The article discusses the main characteristics of community-acquired Staphylococcus aureus pneumonia and recalls the recommendations in case of bacteremia with Staphylococcus aureus.

Nous rapportons le cas clinique d'un patient de 26 ans, sans antécédents médicaux, se présentant au service des urgences pour cause de fièvre persistante depuis plusieurs jours. Dans le décours de son admission à l'hôpital, le patient développe une insuffisance respiratoire aiguë d'installation rapide. Une pneumonie communautaire associée à une bactériémie à staphylocoque doré est alors mise en évidence. Les pneumonies communautaires à staphylocoque doré sont rares, mais peuvent toucher toutes les tranches d'âge et se présenter de façon variée. Leur sévérité est plus importante que celle des pneumonies communautaires imputées à d'autres germes. Par le biais de ce cas clinique, les principales caractéristiques des pneumonies communautaires à staphylocoque doré sont discutées et les recommandations médicales associées sont abordées.

[Paraneoplastic neurological syndromes]. / Les syndromes neurologiques paranéoplasiques.

Neurological paraneoplastic syndromes are non-metastatic complications of systemic cancers, often resulting from an immune response triggered by the crossed expression of neuronal antigens by tumour cells. Several neurological syndromes such as cerebellar degeneration, sensory neuronopathy, limbic encephalitis, encephalomyelitis or the Lambert-Eaton myasthenic syndrome are most often paraneoplastic and require prompt cancer screening, particularly if the patient shows risk factors for cancer. Although there are many exceptions to this rule, a given syndrome is often associated with a particular antibody and the corresponding tumour. A prompt diagnosis of neurological paraneoplastic syndrome is of major importance as it often reveals the underlying tumour. The treatment relies on both the elimination of the neoplasia and the control of the immune response.

Les syndromes neurologiques paranéoplasiques sont des complications neurologiques non métastatiques de cancers systémiques résultant, le plus souvent, d'une réaction immunitaire croisée dirigée contre des antigènes neuronaux membranaires ou intracellulaires. Certains de ces syndromes paranéoplasiques sont classiques comme les ataxies cérébelleuses, les neuronopathies sensitives ou ganglionopathies, l'encéphalite limbique, les encéphalomyélites ou le syndrome de Lambert-Eaton. Devant de tels tableaux cliniques, une étiologie paranéoplasique doit, surtout chez les patients présentant des facteurs de risque, être systématiquement recherchée. Bien que cette règle souffre de nombreuses exceptions, il y a souvent concordance entre un syndrome clinique spécifique, un type d'anticorps et une tumeur associée. Le diagnostic d'un syndrome neurologique paranéoplasique est essentiel puisqu'il révèle souvent le cancer sous-jacent. Le traitement comporte deux axes principaux : celui du cancer responsable et le contrôle de la réponse immunitaire.

[Norovirus nosocomial outbreak : an hospital experience]. / Epidémie nosocomiale à norovirus : à propos d'une expérience hospitalière.

Human noroviruses (NoV) are the main pathogenic agents worldwide responsible for viral sporadic and epidemic gastroenteritis worldwide. A gastroenteritis outbreak broke out in patients hospitalized in several wards located in two different floors of a hospital in Liege, Belgium. The objective was to determine whether a same NoV strain would be involved in the two different floors, and to explore how this outbreak would have spread from a floor to the other. Stool samples from patients and healthcare workers were collected, as well as data from medical files. NoV detection, quantification and characterization were performed using molecular biology methods. A same NoV strain, from genotype GII.4, was detected in two patients hospitalized on the two different floors. This finding allowed to conclude that a same outbreak spread in the two floors, probably due to movements of common healthcare workers. A rapid NoV detection during outbreak is important in the aim to rapidly implement hygiene measures to limit the size of the outbreak.

Les norovirus humains (NoV) sont reconnus mondialement comme les principaux agents étiologiques de gastro-entérites virales sporadiques et épidémiques au niveau mondial. Une épidémie de gastro-entérites s'est déclarée chez des patients hospitalisés dans plusieurs salles d'un hôpital de la région liégeoise, situées à deux étages différents. L'objectif était de déterminer si une même souche de NoV était impliquée aux deux étages, et d'investiguer la manière dont l'épidémie se serait propagée d'un étage à l'autre. Des prélèvements ont été collectés chez les patients et le personnel soignant. Les dossiers médicaux ont été examinés. La détection, la quantification et la caractérisation des souches de NoV ont été réalisées par des méthodes de biologie moléculaire. Une même souche de NoV, du génotype GII.4, a été mise en évidence chez deux patients hospitalisés aux deux étages différents. Ce résultat indique qu'il s'agit de la même épidémie qui s'est étendue à deux étages, probablement transmise par l'intermédiaire du personnel soignant commun. L'identification précoce des NoV lors des épidémies est primordiale afin de mettre en place rapidement les mesures d'hygiène permettant de limiter leur propagation.

New insights into the phenotypes of atopic dermatitis linked with allergies and asthma in children: An overview.

Atopic dermatitis (AD) is a complex disease with multiple causes and complex mechanistic pathways according to age of onset, severity of the illness, ethnic modifiers, response to therapy and triggers. A group of difficult-to-manage patients characterized by early-onset AD and severe lifelong disease associated with allergic asthma and/or food allergy (FA) has been identified. In this study, we focus on these severe phenotypes, analysing their links with other atopic comorbidities, and taking into account the results from recent cohort studies and meta-analyses. The main hypothesis that is currently proposed to explain the onset of allergic diseases is an epithelial barrier defect. Thus, the atopic march could correspond to an epithelial dysfunction, self-sustained by a secondary allergenic sensitization, explaining the transition from AD to allergic asthma. Furthermore, AD severity seems to be a risk factor for associated FA. Results from population-based, birth and patient cohorts show that early-onset and severe AD, male gender, parental history of asthma, and early and multiple sensitizations are risk factors leading to the atopic march and the development of asthma. The importance of environmental factors should be recognized in these high-risk children and prevention programs adapted accordingly. Effective targeted therapies to restore both barrier function and to control inflammation are necessary; early emollient therapy is an important approach to prevent AD in high-risk children. Clinicians should also keep in mind the specific risk of atopic comorbidities in case of filaggrin loss-of-function mutations and the rare phenotypes of orphan syndromes due to heritable mutations in skin barrier components.

Patients with ankylosing spondylitis have been breast fed less often than healthy controls: a case-control retrospective study.

OBJECTIVE: Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the spine and pelvis of young adults. On the HLA-B27 genetic background, the occurrence of AS is influenced by the intestinal microbiota. The goal of our study was to test whether breast feeding, which influences microbiota, can prevent the development of AS. METHODS: First, 203 patients with HLA-B27-positive AS fulfilling the modified New York criteria were recruited in the Department of Rheumatology, Ste Marguerite hospital in Marseilles. A total of 293 healthy siblings were also recruited to make up a control group within the same families. Second, 280 healthy controls, and 100 patients with rheumatoid arthritis and their siblings were recruited. The data collected were age, gender, number of brothers and sisters, age at disease onset, type and duration of feeding (breast or bottle). RESULTS: Patients with AS had been breast fed less often than healthy controls. In families where children were breast fed, the patients with AS were less often breast fed than their healthy siblings (57% vs 72%), giving an OR for AS onset of 0.53 (95% CI (0.36 to 0.77), p value=0.0009). Breast feeding reduced familial prevalence of AS. The frequency of breast feeding was similar in the AS siblings and in the 280 unrelated controls. However, patients with AS were less often breast fed compared with the 280 unrelated controls (OR 0.6, 95% CI (0.42 to 0.89), p<0.01). CONCLUSIONS: Our study suggests a breastfeeding-induced protective effect on the occurrence of AS. To our knowledge, this is the first study of breastfeeding history in patients with AS.

[Therapeutic adherence among asthma patients: Variations according to age groups. How can it be improved? The potential contributions of new technologies]. / Observance thérapeutique dans l'asthme : variation selon les classes d'âge. Comment l'améliorer ? Apport des nouvelles technologies.

While asthma patients' treatment adherence (TA) generally leaves to be desired, few data exist on TA evolution from age group to another. During the meeting of a working group of pneumo-pediatricians and adult pulmonologists, we reviewed the literature on adherence according to age group, examined explanations for poor adherence, and explored ways of improving adherence via new technologies. Asthma is a chronic disease for which TA is particularly low, especially during adolescence, but also among adults. Inhaled medications are the least effectively taken. Several explanations have been put forward: cost and complexity of treatments, difficulties using inhalation devices, poor understanding of their benefits, erroneous beliefs and underestimation of the severity of a fluctuating disease, fear of side effects, neglect, and denial (especially among teenagers). Poor TA is associated with risks of needless treatment escalation, aggravated asthma with frequent exacerbations, increased school absenteeism, degraded quality of life, and excessive mortality. Better compliance is based on satisfactory relationships between caregivers and asthmatics, improved caregiver training, and more efficient transmission to patients of relevant information. The recent evolution of innovative digital technologies opens the way for enhanced communication, via networks and dedicated applications, and thanks to connected inhalation devices, forgetfulness can be limited. Clinical research will also help to ameliorate TA. Lastly, it bears mentioning that analysis of the existing literature is hampered by differences in terms of working definitions and means of TA measurement.

Decreased bone turnover in children and adolescents with well controlled type 1 diabetes.

AIM: Osteoporosis is a common long-term complication of type 1 diabetes (T1DM). We aimed to determine whether bone mineral density (BMD) and turnover are already altered during childhood. PATIENTS AND METHODS: We recruited 27 T1DM children and 32 controls (age 10.5 +/- 2.5 yr.) and measured BMD (dual-energy x-ray absorptiometry); bone biomarkers levels (osteocalcin: OC; procollagen type 1 propeptides amino-terminal: PINP; crosslinking telopeptides of type 1 collagen C-terminal: CTX), glycated hemoglobin (HbA1c), dietary intake and physical activity. RESULTS: Patients with T1DM had lower levels of OC (70.3 +/- 3.3 vs 105.3 +/- 6.8), PINP (556.4 +/- 47.6 vs 716.3 +/- 53.8), CTX(0.97 +/- 0.07 vs 1.20 +/- 0.08), physical activity, and calcium intake. Biomarkers were negatively correlated with HbA1c. Though, BMD was similar among groups and not related to HbA1c, disease duration, physical activity or dietary intakes. CONCLUSIONS: Bone turnover is altered in T1DM children, whereas BMD remains normal during growth. Physical activity and optimal calcium intakes may improve bone metabolism and delay osteoporosis.

Towards a low complexity carbon removal model for the optimal design of compact decentralised wastewater treatment systems.

On-site decentralised wastewater treatment systems can provide a financially attractive alternative to a sewer connection in locations far from existing sewer networks. Operational problems and shortcomings in the design of these systems still occur frequently. The aim of this paper is to provide a low complexity (i.e. easy to calibrate) but still accurate mathematical model that can be used to optimise the operational design of compact individual wastewater treatment systems. An integrated hydraulic and biological carbon removal model of a biofilm-based compact decentralised treatment system is developed. The procedure for drafting the model is generic and can be used for similar types of wastewater treatment systems since (i) the hydraulic model is based on an N-tanks-in-series model inferred from tracer test experiments and (ii) (biofilm) respirometry experiments are exploited to determine the biodegradation kinetics of the biomass. Based on the preliminary validation results of the integrated model, the carbon removal in the system can be predicted quite accurately. While some adjustments could further improve the modelling strategy, the here presented results can already assist the manufacturers of compact treatment systems in efficiently (re)designing their systems.

Evaluation of ultrasound technology for the disinfection of process water and the prevention of biofilm formation in a pilot plant.

In this study, we investigated the use of ultrasound for the disinfection of process water as an alternative for more traditional techniques, like chlorination and UV-irradiation. A pilot plant was constructed to mimic circulating process water in industrial environments. The disinfection efficiency of ultrasound was assessed and compared to UV-treatment and chlorination. In addition, the operational costs for the different technologies were evaluated. Based on disinfection efficiency and operational costs, the pilot plant experiments indicate that chlorination is the method of preference to treat bacteria in suspension. In the prevention of biofilm formation, the results of UV irradiation and ultrasound are comparable, with a slightly higher energy consumption for the ultrasonic treatment. Finally, the use of ultrasound to prevent biofilms was also evaluated in an industrial environment (case study). The results obtained from the case study are in agreement with the results obtained from the pilot plant study. To our knowledge, this is the first study that evaluates the use of ultrasound technology for prevention of biofilm formation in realistic circumstances as encountered in an industrial environment.

Random telegraph signal analysis with a recurrent neural network.

We use an artificial neural network to analyze asymmetric noisy random telegraph signals, and extract underlying transition rates. We demonstrate that a long short-term memory neural network can outperform other methods, particularly for noisy signals and measurements with limited bandwidths. Our technique gives reliable results as the signal-to-noise ratio approaches one, and over a wide range of underlying transition rates. We apply our method to random telegraph signals generated by quasiparticle poisoning in a superconducting double dot, allowing us to extend our measurement of quasiparticle dynamics to new temperature regimes.

New autoantigens in rheumatoid arthritis (RA): screening 8268 protein arrays with sera from patients with RA.

OBJECTIVE: To identify new IgG autoantibodies in sera from patients with rheumatoid arthritis (RA). METHODS: We tested serum samples from 19 patients with RA with given human leukocyte antigen (HLA)-DR genotypes, from 7 patients with spondylarthropathy, 2 patients with lupus, 4 patients with systemic sclerosis and 10 healthy individuals on 8268 human protein arrays. RESULTS: We identified four antigens (peptidyl arginine deiminase 4 (PAD4), protein kinase Cbeta1 (PKCbeta1), phosphatylinositol 4 phosphate 5 kinase type II gamma (PIP4K2C) and v raf murine sarcoma viral oncogene homologue B1 catalytic domain (BRAF)) that were recognised almost uniquely by sera from patients with RA on protein arrays. Using purified proteins, we confirmed that PAD4 and BRAF are recognised almost uniquely by patients with RA. CONCLUSION: We identified PAD4 and BRAF as RA specific autoantigens.

A large multicentre analysis of CTGF -945 promoter polymorphism does not confirm association with systemic sclerosis susceptibility or phenotype.

OBJECTIVE: To conduct a replication study to investigate whether the -945 CTGF genetic variant is associated with systemic sclerosis (SSc) susceptibility or specific SSc phenotype. METHODS: The study population comprised 1180 patients with SSc and 1784 healthy controls from seven independent case-control sets of European ancestry (Spanish, French, Dutch, German, British, Swedish and North American). The -945 CTGF genetic variant was genotyped using a Taqman 5' allelic discrimination assay. RESULTS: An independent association study showed in all the case-control cohorts no association of the CTGF -945 polymorphism with SSc susceptibility. These findings were confirmed by a meta-analysis giving a pooled OR = 1.12 (95% CI 0.99 to 1.25), p = 0.06. Investigation of the possible contribution of the -945 CTGF genetic variant to SSc phenotype showed that stratification according to SSc subtypes (limited or diffuse), selective autoantibodies (anti-topoisomerase I or anticentromere) or pulmonary involvement reached no statistically significant skewing. CONCLUSION: The results do not confirm previous findings and suggest that the CTGF -945 promoter polymorphism does not play a major role in SSc susceptibility or clinical phenotype.

Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

A qualitative study of the food-related experiences of rural village shop customers.

BACKGROUND: In the UK, although food choice in urban contexts has been widely studied, far less empirical information has been gathered from rural settings. With the closure of local services, some rural dwellers were believed to experience difficulties in meeting healthy eating recommendations. The present study aimed to explore perceptions of village store users. METHODS: Semi-structured, in-depth interviews were conducted individually with 40 adults who were frequent users of rural village shops in Norfolk, UK. Participants were purposively selected to ensure that a wide demographic cross-section of customers was obtained. Interviews focused upon food choice strategies; attitudes towards rural food retail; and the provision of healthy foods. Interviews were transcribed verbatim and analysed qualitatively, using an established interpretative phenomenological approach. RESULTS: Four main themes emerged as drivers of food choice in rural villages: (1) village store as icon, which described how the perceived centrality of village shops influenced food choices; (2) village store as a service provider, which described top-up shopping behaviours; (3) alternative food sources, which described store users desires for local foods and their attitudinal conflicts towards supermarkets; and (4) lifestyle factors, which described the influence of factors such as time pressures, access to cars and family structures. CONCLUSIONS: Food choices were strongly influenced by the distinctive characteristics of the rural environment. Village shops were seen as important for community identity ('rural idyll'), as well as providing access to food and services. However, desires were made apparent for a greater range of healthy, fresh and locally-sourced foods.

The molecular chaperone sigma 1 receptor mediates rescue of retinal cone photoreceptor cells via modulation of NRF2.

Sigma 1 receptor (Sig1R), a putative molecular chaperone, has emerged as a novel therapeutic target for retinal degenerative disease. Earlier studies showed that activation of Sig1R via the high-affinity ligand (+)-pentazocine ((+)-PTZ) induced profound rescue of cone photoreceptor cells in the rd10 mouse model of retinitis pigmentosa; however the mechanism of rescue is unknown. Improved cone function in (+)-PTZ-treated mice was accompanied by reduced oxidative stress and normalization of levels of NRF2, a transcription factor that activates antioxidant response elements (AREs) of hundreds of cytoprotective genes. Here, we tested the hypothesis that modulation of NRF2 is central to Sig1R-mediated cone rescue. Activation of Sig1R in 661W cone cells using (+)-PTZ induced dose-dependent increases in NRF2-ARE binding activity and NRF2 gene/protein expression, whereas silencing Sig1R significantly decreased NRF2 protein levels and increased oxidative stress, although (+)-PTZ did not disrupt NRF2-KEAP1 binding. In vivo studies were conducted to investigate whether, in the absence of NRF2, activation of Sig1R rescues cones. (+)-PTZ was administered systemically for several weeks to rd10/nrf2+/+ and rd10/nrf2-/- mice. Through post-natal day 42, cone function was significant in rd10/nrf2+/+, but minimal in rd10/nrf2-/- mice as indicated by electroretinographic recordings using natural noise stimuli, optical coherence tomography and retinal histological analyses. Immunodetection of cones was limited in (+)-PTZ-treated rd10/nrf2-/-, though considerable in (+)-PTZ-treated rd10/nrf2+/+mice. The data suggest that Sig1R-mediated cone rescue requires NRF2 and provide evidence for a previously-unrecognized relationship between these proteins.