Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein

Place de la néphrectomie partielle assistée par robot : revue de la littérature à l’heure d’une demande de nomenclature spécifique

International audienceRobot-assisted surgery is practiced more and more frequently in urology. Besides its place in prostatectomy for cancer, it also concerns partial nephrectomy (NP), in the treatment of renal tumors. The objective of this review is to compare the robot-assisted approach with laparoscopic or open approaches in partial nephrectomy in terms of functional or oncological outcomes and per- and postoperative complications.MATERIAL AND METHODS:A systematic review of the literature published from 2009 was carried out on PubMed. Clinical studies or meta-analyzes comparing robot-assisted surgery versus laparoscopic or open surgery in the NP domain were used.RESULTS:The clinical data presented in this review of the literature are based mainly on meta-analyzes of comparative studies. Patients operated with robotic assistance (NPAR) had significantly fewer postoperative complications than patients operated by open (RR 0.61; P=0.0002) or laparoscopic surgery (RR 0.84; P=0.007). Positive margins, at equivalent pathological stages, are comparable to the open and appear to be lower than the laparoscopic surgery (RR 0.53; P<0.001). After NP, the change in postoperative glomerular filtration rate (GFR) appears to be identical between the 3 pathways. Hot ischemia time is significantly shorter for NPAR compared to NPL. Finally, the estimated blood loss and length of stay are less severe in patients operated by NPAR compared to those operated by open surgery.CONCLUSION:Robot-assisted surgery offers the same oncological results (in the short and medium term) and appears to improve functional outcomes and morbidity. However, these findings need to be carefully analyzed, due to the low level of evidence from the studies presented and included in the meta-analyzes, and the lack of randomized clinical studies

New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis

International audienc

Interaction of the catalytic domain of inositol 1,4,5-trisphosphate 3-kinase A with inositol phosphate analogues.

The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D-2-deoxy-Ins(1,4,5)P3 was devised. D-2-Deoxy-Ins(1,4,5)P3 and D-3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D-2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.Journal ArticleResearch Support, Non-U.S. Gov'tFLWNAinfo:eu-repo/semantics/publishe

Prostatectomie radicale laparoscopique robot-assistée : quelles sont les preuves à l’heure d’une demande de nomenclature spécifique ?

International audienceINTRODUCTION:Despite a decreasing number of radical prostatectomies in France, the number of robot-assisted surgeries increases. The objective of this work is to assess the interest of robotic prostatectomy before asking a specific funding from health authorities.MATERIAL AND METHODS:A systematic review of the literature on PubMed was performed. Prospective studies and meta-analyses comparing robot-assisted radical prostatectomy (RARP), laparoscopic (LRP) and open surgery (OP) were selected.RESULTS:There are only two randomized clinical trials comparing RARP and LRP. Erectile function was significantly better after RARP than after LRP. Compared to OP, sexuality evaluation, based on meta-analyses, was significantly better at 12 months and the absolute risk of erectile dysfunction significantly decreased. Continence after RARP was significantly better than LRP 3 months after surgery. Compared to OP, continence results were discordant, sometimes significantly in favor of RARP, sometimes similar. The rate of positive margins was similar whatever the technique. The long-term oncological outcomes were similar. In terms of perioperative complications, no significant difference was observed between RARP and LRP or OP.CONCLUSION:RARP provides same oncological outcomes as the open and laparoscopic approach. Continence and sexuality are better after RARP than after laparoscopic or open surgery. However, no randomized study comparing RARP and OP is available

Protocol of a multicentric prospective cohort study for the VALIDation of the IBD-disk instrument for assessing disability in inflammatory bowel diseases: the VALIDate study

International audienceAbstract Background Inflammatory Bowel Diseases (IBD) affect psychological, family, social and professional dimensions of patients’ life, leading to disability which is essential to quantify as part of Patient-Reported Outcomes (PROs) newly included in the targets to reach in IBD patients. Up to now, the IBD-Disability Index (IBD-DI) was the only validated tool to assess disability, but it is not appropriate for use in clinical practice. The IBD Disk was developed, a shortened and self-administered tool, adapted from the IBD-DI, in order to give immediate representation of patient-reported disability. However, the IBD Disk has not been validated yet in clinical practice. The aims of the VALIDate study are to validate this tool in a large population of IBD patients and to compare it to the already validated IBD-DI. Methods The VALIDate study is an ongoing multicentric prospective cohort study launched in April 2018 in 3 French University Hospitals (Nantes, Rennes, Angers), with an objective to reach a sample of 400 patients over a period inclusion of 6 months. Each patient will fill in the two questionnaires IBD Disk and IBD-DI at baseline, then between 3 and 12 months later, during a follow-up visit. Clinical and socio-demographic data will also be collected. During these two consultations, gastroenterologists and patients will evaluate disease activity thanks to a semi-quantitative 4-grade scale, named respectively PGA (Physician Global Assessment) and PtGA (Patient Global Assessment). This cohort will allow to evaluate the validity of the IBD Disk with respect to the IBD-DI in order to generalize its use for clinical practice. Other psychometric criteria of the IBD Disk will also be analysed as its reliability or its discriminant capacity. Close attention will nonetheless be needed to minimize the number of lost to follow-up patients between baseline and follow-up. Discussion The VALIDate study is the study designed to validate the IBD Disk, a visual tool easily useable in daily practice to assess disability in IBD patients. The results of this trial should enable the diffusion of this tool. Trial registration The trial is registered in ClinicalTrials.Gov with registration number NCT03590639 . First posted: July 18, 2018

Malignancy after Augmentation Enterocystoplasty: A Nationwide Study of Natural History, Prognosis and Oncogene Panel Analysis

International audienceWe report the natural history and prognosis of tumors after augmentation enterocystoplasty, with a molecular analysis using an oncogene panel to search for potential targeted therapies.Materials and methods: this multicenter, nationwide, retrospective study included 16 patients. A panel of 21 clinically relevant oncogenes was tested on archival tumor specimens using next-generation sequencing. Survival rate was the main clinical outcome and sequences were compared to the reference genome for the genetic outcome.Results: augmentation enterocystoplasties were performed mainly for congenital neurogenic bladder and bladder exstrophy at a median patient age of 17 years (range 4 months to 45 years). Most of the malignancies were diagnosed because of clinical manifestations, with a median latency period of 20 years. Adenocarcinomas were mainly found after gastrocystoplasty, whereas urothelial cell carcinomas were typically found after colocystoplasty. Of the 16 patients 13 were diagnosed at an advanced stage of the disease (positive lymph nodes in 7, distant metastases in 6). The overall 1-year survival rate was 56%. Only 3 patients remained disease-free at a median followup of 70 months. Of the 9 tumors with analyzable DNA 4 were wild-type and 5 harbored missense mutations (KIT-p.Pro573Ser, PDGFRA-p.Glu587Lys, KRAS-p.Gly12Asp, ERBB4p.Arg484Lys, CTNNB1-p.Ser37Phe and p.Ser47Asn).Conclusions: malignancy after augmentation enterocystoplasty is diagnosed late with frequent metastases and a very low 1-year survival rate. More than half the tested samples harbored missense mutations in oncogenes accessible to targeted therapies. An international collaboration to enlarge the genetic panel analysis of these tumors may offer new therapeutic hope to patients