Functional metagenomics of the thioredoxin superfamily.

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

Collapsin response mediator protein 2: high-resolution crystal structure sheds light on small-molecule binding, post-translational modifications, and conformational flexibility.

Collapsin response mediator protein 2 (CRMP-2) is a neuronal protein involved in axonal pathfinding. Intense research is focusing on its role in various neurological diseases. Despite a wealth of studies, not much is known about the molecular mechanisms of CRMP-2 function in vivo. The detailed structure-function relationships of CRMP-2 have also largely remained unknown, in part due to the fact that the available crystal structures lack the C-terminal tail, which is known to be a target for many post-translational modifications and protein interactions. Although CRMP-2, and other CRMPs, belong to the dihydropyrimidinase family, they have lost the enzymatic active site. Drug candidates for CRMP-2-related processes have come up during the recent years, but no reports of CRMP-2 complexes with small molecules have emerged. Here, CRMP-2 was studied at 1.25-Å resolution using X-ray crystallography. In addition, ligands were docked into the homotetrameric structure, and the C-terminal tail of CRMP-2 was produced recombinantly and analyzed. We have obtained the human CRMP-2 crystal structure at atomic resolution and could identify small-molecule binding pockets in the protein. Structures obtained in different crystal forms highlight flexible regions near possible ligand-binding pockets. We also used the CRMP-2 structure to analyze known or suggested post-translational modifications at the 3D structural level. The high-resolution CRMP-2 structure was also used for docking experiments with the sulfur amino acid metabolite lanthionine ketimine and its ester. We show that the C-terminal tail is intrinsically disordered, but it has conserved segments that may act as interaction sites. Our data provide the most accurate structural data on CRMPs to date and will be useful in further computational and experimental studies on CRMP-2, its function, and its binding to small-molecule ligands.

Arc is a flexible modular protein capable of reversible self-oligomerization.

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.

Vitellogenin recognizes cell damage through membrane binding and shields living cells from reactive oxygen species.

Large lipid transfer proteins are involved in lipid transportation and diverse other molecular processes. These serum proteins include vitellogenins, which are egg yolk precursors and pathogen pattern recognition receptors, and apolipoprotein B, which is an anti-inflammatory cholesterol carrier. In the honey bee, vitellogenin acts as an antioxidant, and elevated vitellogenin titer is linked to prolonged life span in this animal. Here, we show that vitellogenin has cell and membrane binding activity and that it binds preferentially to dead and damaged cells. Vitellogenin binds directly to phosphatidylcholine liposomes and with higher affinity to liposomes containing phosphatidylserine, a lipid of the inner leaflet of cell membranes that is exposed in damaged cells. Vitellogenin binding to live cells, furthermore, improves cell oxidative stress tolerance. This study can shed more light on why large lipid transfer proteins have a well conserved α-helical domain, because we locate the lipid bilayer-binding ability of vitellogenin largely to this region. We suggest that recognition of cell damage and oxidation shield properties are two mechanisms that allow vitellogenin to extend honey bee life span.

Investigation of the biotransformation of melarsoprol by electrochemistry coupled to complementary LC/ESI-MS and LC/ICP-MS analysis.

Melarsoprol is the only currently available drug for treatment of the late stage of African trypanosomiasis (sleeping sickness). Unfortunately, the arsenic-containing drug causes serious side effects, for which the mechanisms have not been elucidated so far. This investigation describes the study of the melarsoprol biotransformation processes by electrochemical (EC) techniques. Based on EC, potential oxidation reactions of melarsoprol are examined. Moreover, the reactivity of melarsoprol, its metabolite melarsen oxide, and their oxidation products toward the tripeptide glutathione and the proteins hemoglobin and human serum albumin is evaluated. The combination of different analytical techniques allows the identification as well as the quantification of the biotransformation products. The hyphenation of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS) is applied for identification and structure elucidation, which implies the determination of exact masses and fragmentation patterns. For the selective detection of arsenic containing metabolites, LC coupled to inductively coupled plasma mass spectrometry is utilized. Based on the obtained data, the oxidative biotransformation of melarsoprol can be predicted, revealing novel species which have been suspected, but not been identified up to now. The results of the protein studies prove that melarsen oxide, the active derivative of melarsoprol, strongly binds to human hemoglobin and forms different adducts via the free cysteinyl groups of the hemoglobin α- and ß-chain.

Acetylcholine content and viability of cholinergic neurons are influenced by the activity of protein histidine phosphatase.

BACKGROUND: The first mammalian protein histidine phosphatase (PHP) was discovered in the late 90s of the last century. One of the known substrates of PHP is ATP-citrate lyase (ACL), which is responsible--amongst other functions--for providing acetyl-CoA for acetylcholine synthesis in neuronal tissues. It has been shown in previous studies that PHP downregulates the activity of ACL by dephosphorylation. According to this our present work focused on the influence of PHP activity on the acetylcholine level in cholinergic neurons. RESULTS: The amount of PHP in SN56 cholinergic neuroblastoma cells was increased after overexpression of PHP by using pIRES2-AcGFP1-PHP as a vector. We demonstrated that PHP overexpression reduced the acetylcholine level and induced cell death. The acetylcholine content of SN56 cells was measured by fast liquid chromatography-tandem mass spectrometry method. Overexpression of the inactive H53A-PHP mutant also induced cell damage, but in a significantly reduced manner. However, this overexpression of the inactive PHP mutant did not change the acetylcholine content of SN56 cells significantly. In contrast, PHP downregulation, performed by RNAi-technique, did not induce cell death, but significantly increased the acetylcholine content in SN56 cells. CONCLUSIONS: We could show for the first time that PHP downregulation increased the acetylcholine level in SN56 cells. This might be a potential therapeutic strategy for diseases involving cholinergic deficits like Alzheimer's disease.

Electrochemical oxidation and protein adduct formation of aniline: a liquid chromatography/mass spectrometry study.

Historically, skin sensitization tests are typically based on in vivo animal tests. However, for substances used in cosmetic products, these tests have to be replaced according to the European Commission regulation no. 1223/2009. Modification of skin proteins by electrophilic chemicals is a key process associated with the induction of skin sensitization. The present study investigates the capabilities of a purely instrumental setup to determine the potential of commonly used non-electrophilic chemicals to cause skin sensitization by the generation of electrophilic species from the parent compound. In this work, the electrophiles were generated by the electrochemical oxidation of aniline, a basic industrial chemical which may also be released from azo dyes in cosmetics. The compound is a known sensitizer and was oxidized in an electrochemical thin-layer cell which was coupled online to electrospray ionization-mass spectrometry. The electrochemical oxidation was performed on a boron-doped diamond working electrode, which is able to generate hydroxyl radicals in aqueous solutions at high potentials. Without any pretreatment, the oxidation products were identified by electrospray ionization/time-of-flight mass spectrometry (ESI-ToF-MS) using their exact masses. A mass voltammogram was generated by plotting the obtained mass spectra against the applied potential. Oligomerization states with up to six monomeric units in different redox states of aniline were observed using this setup. This approach was extended to generate adducts between the oxidation products of aniline and the tripeptide glutathione. Two adducts were identified with this trapping experiment. Protein modification was carried out subsequently: Aniline was oxidized at a constant potential and was allowed to react with ß-lactoglobulin A (ß-LGA) or human serum albumin (HSA), respectively. The generated adducts were analyzed by liquid chromatography coupled to ESI-ToF-MS. For both ß-LGA and HSA, aniline adducts were successfully generated and identified.

A ferrocene-based reagent for the conjugation and quantification of reactive metabolites.

In the present study, a method for the analysis of reactive metabolites via liquid chromatography (LC) with inductively coupled plasma-mass spectrometry (MS) was developed. A ferrocenyl-modified glutathione (GSH) reagent, consisting of GSH and succinimidyl-3-ferrocenylpropionate, was synthesized. Derivatization of the tripeptide was performed at the N-terminus, leaving the nucleophilic thiol group vacant for the attack of electrophilic compounds. The potential of ferrocenylpropionate (FP)-GSH as a trapping agent for reactive metabolites was investigated using an electrochemical flow-through cell for metabolism simulation coupled online to a LC system with electrospray ionization mass spectrometric detection. The pharmaceuticals amodiaquine, an antimalarial agent, and clozapine, an antipsychotic compound, served as model substances. By proving the successful adduct formation between the reactive metabolite and ferrocene-labeled GSH, it could be shown that FP-GSH is an effective trapping agent which eases routine reversed-phase LC analyses. In contrast to GSH, which is usually used for the conjugation of reactive metabolites and where the resulting adducts often show no or only very little retention, FP-GSH facilitates the detection of the corresponding metabolite adducts due to higher retention times.

Metabolically-incorporated deuterium in myelin localized by neutron diffraction and identified by mass spectrometry.

Myelin is a natural and dynamic multilamellar membrane structure that continues to be of significant biological and neurological interest, especially with respect to its biosynthesis and assembly during its normal formation, maintenance, and pathological breakdown. To explore the usefulness of neutron diffraction in the structural analysis of myelin, we investigated the use of in vivo labeling by metabolically incorporating non-toxic levels of deuterium (2H; D) via drinking water into a pregnant dam (D-dam) and her developing embryos. All of the mice were sacrificed when the pups (D-pups) were 55 days old. Myelinated sciatic nerves were dissected, fixed in glutaraldehyde and examined by neutron diffraction. Parallel samples that were unfixed (trigeminal nerves) were frozen for mass spectrometry (MS). The diffraction patterns of the nerves from deuterium-fed mice (D-mice) vs. the controls (H-mice) had major differences in the intensities of the Bragg peaks but no appreciable differences in myelin periodicity. Neutron scattering density profiles showed an appreciable increase in density at the center of the lipid-rich membrane bilayer. This increase was greater in D-pups than in D-dam, and its localization was consistent with deuteration of lipid hydrocarbon, which predominates over transmembrane protein in myelin. MS analysis of the lipids isolated from the trigeminal nerves demonstrated that in the pups the percentage of lipids that had one or more deuterium atoms was uniformly high across lipid species (97.6% â€‹± â€‹2.0%), whereas in the mother the lipids were substantially less deuterated (60.6% â€‹± â€‹26.4%) with levels varying among lipid species and subspecies. The mass distribution pattern of deuterium-containing isotopologues indicated the fraction (in %) of each lipid (sub-)species having one or more deuteriums incorporated: in the D-pups, the pattern was always bell-shaped, and the average number of D atoms ranged from a low of ∼4 in fatty acid to a high of ∼9 in cerebroside. By contrast, in D-dam most lipids had more complex, overlapping distributions that were weighted toward a lower average number of deuteriums, which ranged from a low of ∼3-4 in fatty acid and in one species of sulfatide to a high of 6-7 in cerebroside and sphingomyelin. The consistently high level of deuteration in D-pups can be attributed to their de novo lipogenesis during gestation and rapid, postnatal myelination. The widely varying levels of deuteration in D-dam, by contrast, likely depends on the relative metabolic stability of the particular lipid species during myelin maintenance. Our current findings demonstrate that stably-incorporated D label can be detected and localized using neutron diffraction in a complex tissue such as myelin; and moreover, that MS can be used to screen a broad range of deuterated lipid species to monitor differential rates of lipid turnover. In addition to helping to develop a comprehensive understanding of the de novo synthesis and turnover of specific lipids in normal and abnormal myelin, our results also suggest application to studies on myelin proteins (which constitute only 20-30% by dry mass of the myelin, vs. 70-80% for lipid), as well as more broadly to the molecular constituents of other biological tissues.

Metabolite identification of a radiotracer by electrochemistry coupled to liquid chromatography with mass spectrometric and radioactivity detection.

Radioligands, which specifically bind to a receptor or enzyme (target), enable molecular imaging of the target expression by positron emission tomography (PET). One very promising PET tracer is (S)-1-(4-(2-[(18)F]-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (isatin), a caspase-3 inhibitor, which has been developed at the University Hospital of Münster to image cell death (apoptosis). The translation of this novel tracer from preclinical evaluation to clinical examinations requires biodistribution studies, which characterize the pharmakodynamics and metabolic fate of the compound. This information is used to further optimize the radioligands and to interpret radioactive signals from tissues upon injection of the radioligand in vivo with respect to their specificity. The analysis of the metabolism of radioligands is hampered by the low amount of the compound being typically injected (nano/picomolar amount per injection). In the present study, electrochemistry (EC) is applied to elucidate the oxidative metabolism pathway of the radiotracer. Previous studies have demonstrated that EC can be utilized as a complementary tool to conventional in vitro approaches in drug metabolism studies. Thereby, potential oxidative metabolites of the isatin are determined by EC coupled to electrospray ionization mass spectrometry (EC/ESI-MS). Moreover, using EC/liquid chromatography (LC) and ESI-ion trap MS(n), structural elucidation of the oxidation products is performed. Comparatively to EC, in vitro metabolism studies with rat liver microsomes are conducted. Finally, the developed LC/ESI-MS method is applied to determine metabolites in body fluids and cell extracts from in vivo studies with the nonradioactive ((19)F) and radioactive isatin ((18)F). On the basis of the electrochemically generated oxidation products of the radioligand, the major radioactive metabolite occurring in vivo was successfully identified.

Limitation of treatment at the end of life: an empirical-ethical analysis regarding the practices of physician members of the German Society for Palliative Medicine.

OBJECTIVES: To determine the frequencies and types of limitation of medical treatment performed by physician members of the German Society for Palliative Medicine and to analyse the findings with respect to clinical and ethical aspects of end-of-life practices. DESIGN: Cross-sectional postal survey. SETTING: Data collection via the secretary of the German Society for Palliative Medicine using the German language version of the EURELD survey instrument. SUBJECTS: All 1645 physician members of the German Society for Palliative Medicine. Main outcome measures Types and frequencies of limitation of treatment and possible determinants. RESULTS: 901 physicians participated in the study (response rate 55.8%). Participants reported limitation of treatment in 69.1% of cases. These decisions most often affected artificial nutrition (19%), chemotherapy (14%), antibiotics (11%) and medication other than antibiotics (11%). In the majority of eligible cases, physicians estimated the life-shortening effect of limitation of treatment to be <7 days. However, estimations differ depending on the medical measures in question. Bivariate statistical analysis indicated that withholding of treatment was performed significantly more frequently for patients aged ≥65 years (p=0.019). In addition, there were significant associations between the incidence of limitation of treatment and the different diseases reported by respondents as the underlying cause of death. CONCLUSION: The findings of this study provide information on the current state of an ethically and clinically challenging aspect of clinical practice and can serve as a starting point for further interdisciplinary research on normative and empirical aspects of treatment decision-making at the end of life.

Hepatocellular carcinoma: French Intergroup Clinical Practice Guidelines for diagnosis, treatment and follow-up (SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO, AFEF, SIAD, SFR/FRI).

INTRODUCTION: This document is a summary of the French Intergroup guidelines regarding the management of hepatocellular carcinoma (HCC) published in March 2019. METHOD: It is a collaborative work under the auspices of most of the French medical societies involved in the management of HCC. It is based on the previous guidelines published in 2017. Recommendations are graded in 3 categories according to the level of evidence of data found in the literature. RESULTS: The diagnosis and staging of HCC is essentially based on clinical, biological and imaging features. A pathological analysis obtained by a biopsy of tumoral and non-tumoral liver is recommended. HCCs can be divided into 2 groups, taking into account not only the tumor stage, but also liver function. HCCs accessible to curative treatments are tumors that are in Milan criteria or with an AFP score ≤ 2, mainly treated by surgical resection, local ablation or liver transplantation. Intermediate and advanced HCCs with no liver insufficiency, accessible only to palliative treatments, benefit from TACE, SIRT or systemic therapy according to the presence or absence of macrovascular invasion or extrahepatic spread. CONCLUSION: Such recommendations are in permanent optimization and each individual case must be discussed in a multidisciplinary expert board.

Three-way interaction between 14-3-3 proteins, the N-terminal region of tyrosine hydroxylase, and negatively charged membranes.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1-43)) and Ser(19)-phosphorylated (THp-(1-43)) states by surface plasmon resonance. THp-(1-43) showed high affinity for 14-3-3 proteins (K(d) approximately 0.5 microM for 14-3-3gamma and -zeta and 7 microM for 14-3-3eta). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S(0.5) = 25-58 microM (TH-(1-43)) and S(0.5) = 135-475 microM (THp-(1-43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3gamma showed a preferential binding to membranes, compared with 14-3-3zeta, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3gamma for negatively charged membranes (S(0.5) = 1-9 microM) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser(19)-phosphorylated TH, 14-3-3gamma, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of L-DOPA and dopamine synthesis.

Curative Irradiation Treatment of Hepatocellular Carcinoma: A Multicenter Phase 2 Trial.

PURPOSE: Liver transplantation is the standard definitive treatment for nonmetastatic hepatocellular carcinoma (HCC). However, less than 5% of patients are ultimately candidates as a result of frequent comorbidities and graft shortage. The aim of this study was to evaluate stereotactic body radiation therapy (SBRT) as an ablative treatment for inoperable HCC. METHODS AND MATERIALS: A prospective phase 2 trial included newly diagnosed single HCC lesions that were without extrahepatic extension and that were deemed unsuitable for standard locoregional therapies, with a tumor size ranging from 1 to 6 cm. The SBRT dose was 45 Gy in 3 fractions. Primary endpoint was the local control of irradiated HCC at 18 months, defined by Response Evaluation Criteria in Solid Tumors. RESULTS: Forty-three patients were treated and evaluable. Median follow-up was 4.0 years (range, 1.2-4.6 years). All 43 patients had cirrhosis; 37 (88%) were Child-Pugh grade A and 5 (12%) grade B (1 missing data). No patients had received prior local treatment. Thirteen patients (31%) presented grade ≥3 acute adverse events, including 8 patients with an abnormality of the liver function tests (19%). Three patients (10%) experienced a decline in Child-Pugh at 3 months post-SBRT. The 18-month local control rate was 98% (95% confidence interval, 85%-99%). The 18-month overall survival rate was 72% (range, 56%-83%). Median overall survival was 3.5 years. CONCLUSIONS: Local control and overall survival after SBRT for untreated solitary HCC were excellent despite candidates being unfit for transplantation, resection, ablation, or embolization treatments. SBRT should be considered as a bridge to transplant or as definitive therapy for those ineligible for transplant.

GADL1 is a multifunctional decarboxylase with tissue-specific roles in ß-alanine and carnosine production.

Carnosine and related ß-alanine-containing peptides are believed to be important antioxidants, pH buffers, and neuromodulators. However, their biosynthetic routes and therapeutic potential are still being debated. This study describes the first animal model lacking the enzyme glutamic acid decarboxylase-like 1 (GADL1). We show that Gadl1-/- mice are deficient in ß-alanine, carnosine, and anserine, particularly in the olfactory bulb, cerebral cortex, and skeletal muscle. Gadl1-/- mice also exhibited decreased anxiety, increased levels of oxidative stress markers, alterations in energy and lipid metabolism, and age-related changes. Examination of the GADL1 active site indicated that the enzyme may have multiple physiological substrates, including aspartate and cysteine sulfinic acid. Human genetic studies show strong associations of the GADL1 locus with plasma levels of carnosine, subjective well-being, and muscle strength. Together, this shows the multifaceted and organ-specific roles of carnosine peptides and establishes Gadl1 knockout mice as a versatile model to explore carnosine biology and its therapeutic potential.

Molecular structure and function of myelin protein P0 in membrane stacking.

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

Publisher Correction: Molecular mechanisms of Charcot-Marie-Tooth neuropathy linked to mutations in human myelin protein P2.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Gastric cancer: French intergroup clinical practice guidelines for diagnosis, treatments and follow-up (SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO).

INTRODUCTION: This document is a summary of the French Intergroup guidelines regarding the management of gastric cancer published in October 2016, available on the website of the French Society of Gastroenterology (SNFGE) (www.tncd.org), updated in October 2017. METHODS: This collaborative work was realized under the auspices of several French medical societies involved in management of gastric cancer. Recommendations are graded in three categories (A-C), according to the amount of evidence found in the literature until July 2017. RESULTS: There are several known risk factors for gastric cancer, including Helicobacter pylori and genetic predispositions, both requiring a specific screening for patients and their relatives. The diagnosis and staging evaluation are essentially based on gastroscopy plus biopsies and computed tomography scan. The endoscopic ultrasonography can be used for superficial tumors in case of discussion for endoscopic resection (T1N0). For local disease (N+ and/or T > T1), the strategic therapy is based on surgery associated with perioperative chemotherapy. In the absence of preoperative treatment (for any raison), the postoperative chemoradiotherapy (or chemotherapy) should be discussed for patients with stage II or III tumor. For metastatic disease, the treatment is based on "palliative" chemotherapy consisting in a doublet or triplet regimens depending of age, performance status and HER2 tumor status. For patients with limited metastatic disease, surgical resection could be discussed in multidisciplinary meeting in case of stable disease after chemotherapy. CONCLUSION: These guidelines in gastric cancer are done to help decision for daily clinical practice. These recommendations are permanently being reviewed. Each individual case must be discussed within a multidisciplinary team.

Intensity-modulated radiation therapy from 70Gy to 80Gy in prostate cancer: six- year outcomes and predictors of late toxicity.

OBJECTIVE: To report grade ≥2 overall late rectal and urinary toxicities in patients (pts) with prostate cancer treated by intensity-modulated radiotherapy (IMRT) at 3 dose-levels. Identify predictors of radiation toxicity and report biochemical progression free survival (bPFS). METHODS: A total of 277 pts were treated with 70Gy (10.8%), 74Gy (63.9%) and 80 Gy (25.3%) using IMRT without pelvic irradiation were analyzed. Short or long-course androgen deprivation therapy (ADT) was allowed in 46.1% of pts. The toxicity was described using the Common Terminology Criteria for Adverse Events (CTCAE) v4.0 scale. Cox regression models addressed demographics, disease and dosimetry characteristics as potential predictors of late grade ≥2 toxicity after adjusting for other modifying factors. RESULTS: The median follow-up was 77 months (range 15; 150). There was no grade ≥4 toxicity. The 5-year cumulative rate of grade ≥2 late rectal and urinary toxicities was 6.3% (95% CI = 3.8%; 10.3%) and 25.3% (95% CI = 19.8%; 31.8%) respectively. In multivariate analysis, only the dose (80Gy vs 74 and 70Gy) was found to increase the risk of rectal toxicity (HR = 2.96 [1.07; 8.20]). For pts receiving 74 Gy, International Prostate Symptom Score (IPSS) at baseline ≥8 (HR = 2.40 [1.08; 5.35]) and dose ≥73Gy delivered in more than 2% of bladder (D2%) were found to be predictors of bladder toxicity (HR = 3.29 [1.36; 7.98]). The 5-year biochemical relapse free survival was 81.0% [74.5%; 86.0%] in the entire population, 97.5% [83.5%; 99.6%] in the low risk group, 84.9% [76.7%; 90.3%] in the intermediate risk group and 66.4% [51.8%; 77.4%] in the high-risk group. D'Amico low (HR = 0.09 [0.01; 0.69]) and intermediate risk groups (HR = 0.50 [0.28; 0.88]) as well as PSA nadir ≥0.2 ng/ml (HR = 1.79 [1.01; 3.21]) were predictive of biochemical relapse. CONCLUSIONS: The rate of late rectal toxicity increased with higher doses, while Dmax ≥74Gy, D2% ≥ 73Gy for bladder wall and baseline IPSS ≥8 increased late urinary toxicity.

Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line.

Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBP-driven formation of the MDL remains elusive at the biomolecular level. We employed complementary biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions.