Identification de nouveaux partenaires d'intéraction de TRPC3 et TRPC6 par spectrométrie de masse

Les protéines de type TRPC ( transient receptor potential canonical ) forment les canaux ioniques responsables de l'entrée de calcium chez les cellules non excitables suite à la stimulation d'un récepteur couplé à une protéine Gq ou d'un récepteur de type tyrosine kinase. De récentes études ont mis en évidence l'association de certaines protéines avec ces canaux permettant leur modulation, tels la calmoduline, l'IP[indice inférieur 3]R, la cavéoline, etc. La partie C-terminale des TRPCs possède plusieurs motifs dont un domaine TRP, un domaine riche en proline, un domaine d'interaction pour la calmoduline et l'IP[indice inférieur 3]R ainsi qu'un domaine super hélice qui sont des sites d'interaction potentiels. Dans le but d'identifier des protéines qui interagissent avec ces motifs de la sous-famille de TRPC3, 6 et 7, nous avons produit des colonnes d'affinité où des protéines chimériques contenant les acides aminés Glu[indice supérieur 676] à Lys[indice supérieur 795] de TRPC3 ou Met[indice supérieur 725] à Arg[indice supérieur 931]" de TRPC6, fusionnées à la glutathione S-transférase (GST-CT3a et GST-CT6), ont été attachées sur des billes de glutathione-sépharose. Des protéines extraites de coeurs/poumons, de reins ou de cerveaux de souris, ou encore de lysats de cellules surexprimant de façon stable TRPC6, ont été incubées avec ces colonnes d'affinité.Les protéines retenues sur la colonne ont été éluées, séparées par SDS-PAGE, extraites du gel et digérées à la trypsine.Les extraits peptidiques obtenus ont été analysés par spectrométrie de masse grâce à un système nano-LC-ESI-TOF-MSMS. Nous avons identifié de cette façon 14 partenaires d'interaction potentiels des TRPCs, dont des protéines du cytosquelette, des chaperonnes, des protéines membranaires, des enzymes mithocondriales ainsi que d'autres protéines cytosoliques dont le rôle est plus ou moins bien connu. Deux de ces protéines ont capté notre intérêt soit la pompe à protons mitochondriale de type F[indice inférieur 0]F[indice inférieur 1] ainsi que le N -Ethylmaleimide-sensitive factor (NSF). Par des essais de liaison à la GST de type pull down, nous démontrons dans cette étude que NSF interagit directement avec TRPC3 et TRPC6, soit sans intermédiaire, et possiblement au niveau du domaine CIRB de ces canaux. Nous démontrons également, grâce à l'utilisation d'un dominant négatif de NSF, que cette protéine module l'entrée de Ca[indice supérieur 2+] par TRPC6, suite à la stimulation de la cellule au CCh, en plus de jouer un rôle dans la maturation du canal

Analysis of Multiple Sarcoma Expression Datasets: Implications for Classification, Oncogenic Pathway Activation and Chemotherapy Resistance

Background: Diagnosis of soft tissue sarcomas (STS) is challenging. Many remain unclassified (not-otherwise-specified, NOS) or grouped in controversial categories such as malignant fibrous histiocytoma (MFH), with unclear therapeutic value. We analyzed several independent microarray datasets, to identify a predictor, use it to classify unclassifiable sarcomas, and assess oncogenic pathway activation and chemotherapy response. Methodology/Principal Findings: We analyzed 5 independent datasets (325 tumor arrays). We developed and validated a predictor, which was used to reclassify MFH and NOS sarcomas. The molecular “match” between MFH and their predicted subtypes was assessed using genome-wide hierarchical clustering and Subclass-Mapping. Findings were validated in 15 paraffin samples profiled on the DASL platform. Bayesian models of oncogenic pathway activation and chemotherapy response were applied to individual STS samples. A 170-gene predictor was developed and independently validated (80-85% accuracy in all datasets). Most MFH and NOS tumors were reclassified as leiomyosarcomas, liposarcomas and fibrosarcomas. “Molecular match” between MFH and their predicted STS subtypes was confirmed both within and across datasets. This classification revealed previously unrecognized tissue differentiation lines (adipocyte, fibroblastic, smooth-muscle) and was reproduced in paraffin specimens. Different sarcoma subtypes demonstrated distinct oncogenic pathway activation patterns, and reclassified MFH tumors shared oncogenic pathway activation patterns with their predicted subtypes. These patterns were associated with predicted resistance to chemotherapeutic agents commonly used in sarcomas. Conclusions/Significance: STS profiling can aid in diagnosis through a predictor tracking distinct tissue differentiation in unclassified tumors, and in therapeutic management via oncogenic pathway activation and chemotherapy response assessment

MicroRNA paraffin-based studies in osteosarcoma reveal reproducible independent prognostic profiles at 14q32

Background: Although microRNAs (miRNAs) are implicated in osteosarcoma biology and chemoresponse, miRNA prognostic models are still needed, particularly because prognosis is imperfectly correlated with chemoresponse. Formalin-fixed, paraffin-embedded tissue is a necessary resource for biomarker studies in this malignancy with limited frozen tissue availability. Methods: We performed miRNA and mRNA microarray formalin-fixed, paraffin-embedded assays in 65 osteosarcoma biopsy and 26 paired post-chemotherapy resection specimens and used the only publicly available miRNA dataset, generated independently by another group, to externally validate our strongest findings (n = 29). We used supervised principal components analysis and logistic regression for survival and chemoresponse, and miRNA activity and target gene set analysis to study miRNA regulatory activity. Results: Several miRNA-based models with as few as five miRNAs were prognostic independently of pathologically assessed chemoresponse (median recurrence-free survival: 59 months versus not-yet-reached; adjusted hazards ratio = 2.90; P = 0.036). The independent dataset supported the reproducibility of recurrence and survival findings. The prognostic value of the profile was independent of confounding by known prognostic variables, including chemoresponse, tumor location and metastasis at diagnosis. Model performance improved when chemoresponse was added as a covariate (median recurrence-free survival: 59 months versus not-yet-reached; hazard ratio = 3.91; P = 0.002). Most prognostic miRNAs were located at 14q32 - a locus already linked to osteosarcoma - and their gene targets display deregulation patterns associated with outcome. We also identified miRNA profiles predictive of chemoresponse (75% to 80% accuracy), which did not overlap with prognostic profiles. Conclusions: Formalin-fixed, paraffin-embedded tissue-derived miRNA patterns are a powerful prognostic tool for risk-stratified osteosarcoma management strategies. Combined miRNA and mRNA analysis supports a possible role of the 14q32 locus in osteosarcoma progression and outcome. Our study creates a paradigm for formalin-fixed, paraffin-embedded-based miRNA biomarker studies in cancer

Bat pluripotent stem cells reveal unique entanglement between host and viruses

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats’ unique traits.N

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance. Keywords: immunometabolism, Klebsiella pneumoniae, immunosuppressive, anti-inflammatory, itaconate, Type Six Secretion Syste

The PsychENCODE project

Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

PREFABL: predictors of failure of antibiotic locks for the treatment of catheter-related bacteraemia

Antibiotic lock (ABL) solutions can effectively treat catheter-related bacteraemia (CRB) without the need for catheter exchange. This approach does not increase secondary infectious complications. We evaluated the risk factors that contribute to failure when CRB is treated with ABLs and systemic antibiotics in paediatric haemodialysis patients. A retrospective chart review of 72 children on haemodialysis between January 2004 and June 2006 was performed. We evaluated risk factors for ABL treatment using patients' characteristics, CRB/catheter characteristics and patients' biochemical profiles. The first CRB of each catheter was included in the statistical analysis. Our end points were outcome at 2 weeks of treatment and at 6 weeks following treatment. Compound symmetry covariance structure was employed for statistical analysis. We treated 149 CRB in 50 patients. The incidence was 3.4 CRB/1000 catheter days. Thirty CRB failed to be cleared with the use of ABL and systemic antibiotics at 2 weeks of treatment (30/149, 20 vs 80%, P < 0.001). Twenty-four of these catheters required exchange. Thirty-nine of the treated catheters got re-infected within the next 6 weeks (39/125, 31 vs 69%, P < 0.001). CRB aetiology was the only statistically significant independent variable for 2-week outcome (P = 0.033). Coagulase-negative Staphylococcus CRB had higher odds of being cleared at 2 weeks compared with other CRB aetiologies. For the 6-week outcome, the statistically significant independent variables in the final model included age (P = 0.048) and serum phosphorous level (P < 0.001). Younger age and higher serum phosphorous levels were independent risk factors for failure at 6 weeks with re-infection. Area under the receiver operating characteristic (ROC) curve for the model of the 2-week outcome was 0.736 with the percentage of correct predictions at 81.2%. Area under the ROC curve for the model of the 6-week outcome was 0.689 with the percentage of correct predictions at 75.5%. CRB can effectively be treated with ABLs and systemic antibiotics. CRB aetiology is the only independent variable of early treatment failure. Younger age and higher serum phosphorous levels are independent risk factors for re-infection at 6 weeks

Prophylaxis of catheter-related bacteremia using tissue plasminogen activator-tobramycin locks

This retrospective study was designed to investigate the effectiveness of tissue plasminogen activator-tobramycin antibiotic lock solutions (TPA/tobra ABLs) for prophylaxis of catheter-related bacteremia (CRB) in high-risk children on long-term hemodialysis. During the first 6 months (Era 1), the high-risk group was defined. These patients received TPA/tobra ABL prophylaxis after every hemodialysis treatment for the next 6 months (Era 2). The prophylaxis regimen was applied once a week for the third 6-months period (Era 3). Primary endpoints were CRB and infection-free catheter survival. There were 16,412 catheter days, and 95 cases of CRB in 43 children. The incidence of CRB was 5.8/1,000 catheter days. Significant decrease in the incidence of CRB was observed when prophylactic TPA/tobra ABL was used in the high-risk group (P = 0.0201). There was a tendency for less CRB when prophylactic ABL was applied after every hemodialysis session compared with once a week (P = 0.0947). The catheters in the high-risk group had shorter survival times than those in the average-risk group in Era 1 (P < 0.0001). However, both the overall and infection-free survival of the catheters in the high-risk group significantly improved while the patients were receiving TPA/tobra ABL prophylaxis, becoming similar to the outcomes of the catheters in the average-risk group and exhibiting statistically non-significant differences (P = 0.5571 and P = 0.9711, respectively). In conclusion, the TPA/tobra ABLs may effectively reduce the rate of CRB, and this may prolong both the overall and infection-free survival times of the catheters in the high-risk group