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BACKGROUNDDelayed cerebral ischaemia (DCI) following aneurysmal subarachnoid haemorrhage (aSAH) is a major cause of complications and death. Here we set out to identify high-performance predictive biomarkers of DCI and its underlying metabolic disruptions using metabolomics and lipidomics approaches.METHODSThis single-centre retrospective observational study enrolled 61 consecutive patients with severe aSAH requiring external ventricular drainage between 2013 and 2016. Of these 61 patients, 22 experienced a DCI and were classified as DCI+ and the other 39 patients were classified as DCI-. A further 9 patients with other neurological features were included as non aSAH controls. Blood and cerebrospinal fluid (CSF) were sampled within the first 24 h after admission. We carried out LC-MS/MS-based plasma and CSF metabolomic profiling together with total lipid fatty acids analysis.RESULTSWe identified a panel of 20 metabolites that together showed high predictive performance for DCI (area under the receiver operating characteristic curve: 0.968, specificity: 0.88, sensitivity: 0.94). This panel of metabolites included lactate, cotinine, salicylate, 6 phosphatidylcholines, and 4 sphingomyelins. Analysis of the whole set of metabolites to highlight early biological disruptions that might explain the subsequent DCI found peripheral hypoxia driven mainly by higher blood lactate, arginine and proline metabolism likely associated to vascular NO, dysregulation of the citric acid cycle in the brain, defective peripheral energy metabolism and disrupted ceramide/sphingolipid metabolism. We also unexpectedly found a potential influence of gut microbiota on the onset of DCI.CONCLUSIONWe identified a high-performance predictive metabolomic/lipidomic signature of further DCI in aSAH patients at admission to a NeuroCritical Care Unit. This signature is associated with significant peripheral and cerebral biological dysregulations. We also found evidence, for the first time, pointing to a possible gut microbiota/brain DCI axis, and proposed the putative microorganisms involved.Clinical trial registrationClinicaltrials.govidentifier:NCT02397759

Many studies have been conducted to identify potentially useful behavioural markers of harmful behaviour using online gambling data sourced from operators. However, less is known about how such markers could be used to identify higher risk products. The study examined whether certain categories and subcategories of product are more strongly associated with behavioural markers of harm than others. Analyses were based on 6 months of data (N = 100,000 individual gamblers) drawn from the population of UK users of an online gambling website in 2022. Measures included individual-level expenditure data across multiple forms of gambling and a series of literature-informed behavioural markers of harm including declined deposits, easing/removing responsible gambling settings, within-session repeat deposits (or ‘top-ups’), bonus-seeking behaviour and gambling at unusual hours. Negative binomial models examined how well the number of active days playing different products predicted behavioural markers of harm. All markers apart from easing/removing responsible gambling settings appeared to covary with the number of active days engaging in specific product types, most notably slots, in-playing betting and some most forms of combination bets on sports. These findings highlight the potential value of using measurable markers to differentiate the risk and potential harm associated with different online products.

Heterogeneous catalysts were used in several production processes for the synthesis of different products such as chemicals, drugs, energy resources, fuels and other specific products. In recent years, mesoporous silica, in particular MCM-41 was widely used in several fields, including catalysis and catalytic support, due to its remarkable textural properties. This material cannot be used directly in catalysis due to its inner surface containing only silanol groups. Therefore, several attempts were proposed in order to obtain an effective catalyst. The immobilization of metal complexes on the surface of mesoporous silica is one of the most popular methods. The metal complexes immobilized on the mesoporous silica MCM-41 exhibit significant catalytic activities due to the increased dispersion of the active sites and the faster diffusion of large organic molecules. The strong interactions between the metal and the ligand generate significant stability for this kind of catalyst, which allows them to be reused in several cycles without losing their efficiency. This review mainly discusses the immobilization strategies of different metal complexes supported on the mesoporous silica MCM-41. A recent study was presented and well detailed based on the different preparation methods, catalytic applications and the stability of metal complexes supported on mesoporous silica MCM-41.

Background Thyroglobulin (Tg) assay in washout fluids of fine needles, after cervical lymph nodes aspiration, is used for detecting metastases from differentiated thyroid carcinomas. Assay methods are the same as for Tg in serum. However, with non-serum samples, methods require extensive validation to notably check for the absence of matrix effect. This study fits this context. Our objectives were to assess analytic performances, in washout fluid, of eight different Tg assay methods and to compare them to validated data in serum. Methods Eleven medical laboratories participated in this study. The matrix tested was phosphate-buffer saline containing 1% bovine serum albumin (PBS-1% BSA). Samples used were dilutions, in this buffer, of Certified Reference Material (CRM 457). We verified, for all methods, the limit of detection, precision, linearity, trueness and accuracy. Results In PBS-1% BSA, the functional sensitivities (FS) were comparable to those expected for serum. All the methods were linear. The relative biases of trueness were between –24.5 and 10.2% around 1 µg/L. Total analytical error was ≤40% near the functional sensitivity values. Conclusion No quantitatively important matrix effect was observed. All the methods showed their ability to measure Tg in PBS-1% BSA, over the concentration range of interest, with acceptable total analytical error. We validated the functional sensitivity value as a decision threshold in thyroidectomized patients after treatment and with low concentrations of serum Tg.

Introduction: Maternal TSH receptor antibodies (TRAbs) can cross the placenta and affect fetal and neonatal thyroid function. Maternal TSH receptor-blocking antibodies (TBAbs) are a rare cause of congenital hypothyroidism. Case Report: Following the discovery of a highly elevated TSH on her neonatal screening test, a 10-day-old girl with no familial history of thyroid disorder was referred to the pediatric endocrinology unit. Hypothyroidism was confirmed with a highly elevated TSH (817 mIU/L, reference range 0.4–3.1) and very low levels of FT4 (1.8 pmol/L, reference range 12–22). Anti-TPO antibodies were at 81 IU/mL (reference range Conclusion: We report herein a case of transient congenital hypothyroidism with a normal neonatal TRAbs level. In case of maternal TBAbs, similar activity of maternal TBAbs must be expected in the neonate, independently of the neonatal level of TRAbs.

To improve the properties of copolymer PPB (Poly[(pyrrole-2,5-diyl)-co-(para-hydroxylbenzylidene)]), a novel nanocomposite bonded with different percentages of CuO was prepared via simple one-pot method. The structural, textural, thermal and morphological characteristics of obtained samples were analyzed by various physico-chemical techniques such as X-ray diffraction (XRD), nitrogen sorption at 77 K, thermogravimetric analysis TGA, Fourier transform infrared spectroscopy (FTIR) and scanning electronic microscopy (SEM). The resulting materials were tested for the adsorption of CO2 at 25 °C using the volumetric method. The results show that higher thermal stability and CO2 adsorption capacity were achieved by the nanocomposite containing low percentage of CuO(1%). Higher percentages of CuO leads to a gradual decrease in product yield, thermal stability and CO2 adsorption capacity. The percentage of CuO, the content and the functional groups containing copolymer were found to be correlated with the CO2 adsorption capacities. The reuse of PPB and nanocomposite CuO/PPB(1%) in five cycles showed a very good stability with a slight decrease in the CO2 adsorption capacity recorded for CuO/PPB(1%).

The present research focuses on the synthesis and the modification of mesoporous silica SBA-15 using in situ polymerization of pyrrole. The structural/textural and morphological features of adsorbents were investigated through X-ray diffraction, nitrogen sorption at 77 K, thermogravimetric analysis, Fourier transform infrared spectroscopy, zeta potential measurements and scanning electronic microscopy. Several nanocomposites containing different percentages of polypyrrole were tested for the adsorption of both anionic and cationic dyes. The effect of adsorbent nature, pH, contact time, adsorbent dose and initial dye concentration were investigated and discussed in terms of adsorption efficiency. The adsorption efficiency of MO dye increased in the following sequence: SBA-15

The polymerization of octamethylcyclotetrasiloxane (D4) catalyzed by Maghnite-H+, a nontoxic and green solid catalyst, is studied. The Maghnite-H+ is a montmorillonite type 2:1 dioctahedral phyllosilicates whose interlayer ions are exchanged by hydronium ions after activation with sulfuric acid which gives it its catalytic appearance. D4 was polymerized cationically by ring opening at 60 °C without solvent using Maghnite-H+ contents less than 5% by weight. The molecular structure of the polymer obtained was identified by IR, 1H NMR and 13C NMR. The DSC was used to study the thermal properties. The operating conditions were optimized so that we can achieve best performance for obtaining a linear polymer with high average molecular mass. The variation of the molecular mass distribution was verified by GPC. Finally, a reaction mechanism was proposed to show the role of the Maghnite-H+ during the different steps of the reaction.

Introduction Les thyroidites induites par les immunotherapies anti-neoplasiques (IT) sont frequentes et se presentent generalement comme une thyroidite biphasique. Le dosage d’anticorps anti-recepteurs de la TSH (TRAb) est systematique dans le bilan de thyrotoxicose. Objectif Devenir des patients presentant une hyperthyroidie avec presence de TRAb dans un contexte d’IT. Methode Etude retrospective de patients ayant eu un dosage de TRAb positif au sein d’une cohorte suivie pour complications endocriniennes des IT aux Hospices Civils de Lyon. Resultats Sept patients ont ete inclus (5H/2F ; âge median 67 ans [44-76]), tous ont recu une immunotherapie par anti-PD1. Quatre ont developpe une thyrotoxicose avec presence de TRAb apres une duree mediane de 27 jours [20-43] apres debut de l’IT : L’un a developpe une authentique maladie de Basedow (MB) (scintigraphie hyperfixante) necessitant un traitement antithyroidien de synthese (ATS) d’un an. Les trois autres ont evolue comme une thyroidite avec normalisation du bilan thyroidien en 34 jours, puis hypothyroidie definitive pour deux. L’un avait eu une scintigraphie hypofixante. Pour trois patients une MB a ete mise en evidence au bilan pre-therapeutique de l’IT, ils ont ete traites par ATS pour des durees de 8 a 160 jours et ont evolue vers une hypothyroidie permettant un arret precoce des ATS et une substitution definitive. Aucun de ces patients n’a developpe d’orbitopathie. Conclusion Evolution favorable des MB sous IT dans cette courte serie. En presence de TRAb lors d’une thyroidite induite par IT l’evolution et la scintigraphie permettent de discriminer entre authentique MB et thyroidite.

International audience; Background: Thyroid-stimulating hormone (TSH) receptor (TSHR) antibodies (TRAb) can be present in chronic autoimmune thyroiditis. Transplacental TRAb transfer can lead to fetal thyroid dysfunction and serious complications. Patient Findings: We report the case of a woman with autoimmune hypothyroidism and extremely high TRAb levels, with blocking and stimulating activities (biological activities characterized with Chinese hamster ovary cells expressing TSHR). At week 22 of her first pregnancy, sonography detected fetal growth retardation and cardiac abnormalities (extreme tachycardia, right ventricular dilatation, pericardial effusion). The mother's TRAb level, assayed later, was 4030 IU/L (n

Objectives: Measuring protein markers with variable glycosylation, such as thyroid-stimulating hormone (TSH), with high accuracy is not an easy task. Despite highly sensitive third-generation tests, discrepancies among TSH assays still remain unsolved and are the focus of important standardization efforts. Earlier work from our group showed that a lack of similarity in epitope expression between standards and samples may account for discordant hormone measurements. In this study, we aimed at producing a glycoengineered TSH with serum-type glycosylation and compared its immunological behavior to that of the international standards. Study Design: Recombinant glycoengineered TSH (rgTSH) was produced in glycoengineered Chinese hamster ovary cells to express a highly sialylated TSH and tested in newly designed assays. Two groups of assays targeting defined epitopes were constructed and TSH levels were estimated in a panel of 84 clinical samples (2.1-22.4 mIU/l) based on the use of the current 3rd IS 81/565, the 1st IRP 94/674 and rgTSH calibrations. Results: Calibration based on rgTSH was found to significantly reduce the percentage difference means of assays compared to the pituitary standard. We also found that a switch from a mIU/l (3rd IS 81/565) to ng/l (rgTSH) basis can be established within the normal as well as in the mid to upper normal range of TSH levels. Of interest, TSH assays targeting the main immunogenic region displayed variable TSH values, indicating that, in this region, epitopes should be defined for assays to deliver similar values. Conclusions: A glycoengineered TSH with serum-type glycosylation proved to be a new calibrator efficient in harmonizing TSH values.

Objectifs Presenter un cas de deces impliquant un analogue de l’insuline et discuter les apports respectifs et limites des analyses immunochimiques et chromatographiques de l’insuline humaine (IH) et de ses analogues. Methode Description du cas Une infirmiere de 47 ans est retrouvee decedee dans un champ. Dans son vehicule, stationne proche du corps, ont ete retrouves differents contenants de medicaments et une seringue d’insuline asparte. La victime n’etait pas diabetique. Les constatations autopsiques ont mis en evidence un foie de choc, une congestion polyviscerale ainsi qu’un point d’injection au dos d’une main. Ce dernier a ete preleve, ainsi que les matrices usuelles en vue d’une expertise toxicologique. Methodes Une toxicologie de reference et une estimation de l’etat glycemique au moment du deces ont ete realisees. Le dosage de l’IH et de ses analogues dans le serum a ete effectue par immunochimie de types ECLIA (ElectroChimiLuminescent ImmunoAssay)(Cobas E411) (LDD = 1.0 mU/L) et IRMA (Cisbio) (LDD = 0.2 mU/L) et par LC-HRMS (phase inverse) apres preparation associant precipitation (ACN/MeOH 50 :50 v/v), filtration et immunopurification par kit iso-insulin (Mercodia) (LDD = 28,8 mU/L). Resultats Le dosage d’ethanol etait negatif dans le sang peripherique (SP). Differentes molecules medicamenteuses psychotropes ont ete retrouvees dans le sang cardiaque (SC) : amitriptyline (52 ng/mL), nortriptyline (83 ng/mL), diazepam (30 ng/mL), nordazepam (53 ng/mL), oxazepam (262 ng/mL), venlafaxine (129 ng/mL) et zopiclone (511 ng/mL). Les parametres glycemiques etaient les suivants : Hemoglobine glyquee (HbA1c) = 5,3 %, glucose et lactates dans l’humeur vitree = 0,38 mmol/L et 9,0 mmol/L respectivement. Dans le SC, le peptide-C a ete dose a 0,047 nmol/L. Le dosage d’insuline etait negatif ( Conclusion Les kits immunochimiques disponibles en clinique presentent des limites qualitatives et quantitatives. Les reactions croisees avec les analogues propres a chaque kit peuvent mener a des resultats tres variables allant d’une quantification erronee jusqu’a la non detection. Au contraire, la spectrometrie de masse permet d’identifier les insulines et donc de realiser un dosage specifique. En revanche, elle necessite des analyseurs de haute performance et une preparation complexe de l’echantillon. Les auteurs preconisent l’analyse par 2 kits presentant des reactivites croisees differentes et par une technique chromatographique couplee a la spectrometrie de masse pour documenter les cas medicolegaux impliquant l’insuline ou ses analogues.

No recent study has focused on clinical features of subclinical hypothyroidism (SCH), especially in older patients. TSH measurement has remarkably evolved these last 20 years and thus reconsideration is needed. In our prospective multicenter study (2012-2014) including 807 subjects aged \textless60 years (\textless60y) and 531 subjects \textgreater/=60 years (\textgreater/=60y), we have monitored 11 hypothyroidism-related clinical signs (hCS) together with TSH, FT4, FT3 and anti-thyroperoxidase antibodies values. hCS expression has been compared in patients with SCH vs euthyroidism in each age group. The number of hCS above 60y of age were found to be more elevated in the euthyroid population (1.9 vs 1.6, p\textless0.01) than in the SCH population (2.3 vs 2.6, p=0.41) while increase in hCS is limited to SCH subjects in the \textless60y group (p\textless0.01). The percentage of subjects with at least 3 signs increased with SCH in the \textless60y group (42.6% vs 25.0%, p\textless0.01) but not \textgreater/=60y (34.4% vs 33.9%, p=0.96). In older individuals, only three hCS could be related to both SCH and a decreased T3/T4-ratio (0.26 vs 0.27, p\textless0.01), suggesting either a reduced activity of TSH, or an adaptive response with aging. While hCS are clearly associated with SCH in patients \textless60y, they are not so informative in older subjects. TSH measurements carried out on the basis of hCS need to be interpreted with caution in aged patients. A reassessment of the TSH reference range in older patients is clearly needed and should be associated to more appropriate monitoring of thyroid dysfunction.

AIMS/HYPOTHESIS: Recent studies have demonstrated that residual beta cells may be present in some people with long-standing type 1 diabetes, but little is known about the potential impact of this finding on alpha cell function and incretin levels. This study aimed to evaluate whether insulin microsecretion could modulate glucagon and glucagon-like peptide-1 (GLP-1) responses to a mixed meal tolerance test (MMTT). METHODS: Adults with type 1 diabetes onset after the age of 15 years (n = 29) underwent a liquid MMTT after an overnight fast. Insulin microsecretion was defined when peak C-peptide levels were \textgreater30 pmol/l using an ultrasensitive assay. Four individuals with recent-onset type 1 diabetes were included as controls. Glucagon and GLP-1 responses were analysed according to C-peptide patterns. RESULTS: We found comparable peak values, Delta0-max levels and AUCs of glucagon and GLP-1 responses in C-peptide-positive participants (n = 9) and C-peptide-negative participants (n = 16) with long-standing diabetes and in participants with recent-onset diabetes (n = 4). Mean glucagon levels, however, differed (p = 0.01). Mean GLP-1 responses were significantly lower according to C-peptide positivity (p \textless 0.001, ANOVA). Interestingly, GLP-1 levels correlated to glucagon values in C-peptide-positive participants with long-standing diabetes (Pearson's r = 0.915, p = 0.004) and in participants with recent-onset diabetes (p \textless 0.001) but not in C-peptide-negative participants. CONCLUSIONS/INTERPRETATION: The glucagon response to an MMTT in people with long-standing type 1 diabetes is not reduced by the presence of residual beta cells. The reduction of GLP-1 responses according to residual C-peptide levels suggests specific regulatory pathways.

Glucotoxicity-induced beta cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and endoplasmic reticulum (ER). Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. The role of MAMs in beta cells is still largely unknown and their implication in glucotoxicity-associated beta cell dysfunction remains to be defined. Here, we report that acute glucose stimulation stimulated ER-mitochondria interactions and calcium (Ca(2+)) exchange in INS-1E cells, whereas disruption of MAMs altered glucose-stimulated insulin secretion (GSIS). Conversely, chronic incubations with high glucose of either INS-1E cells or human pancreatic islets altered GSIS, and concomitantly reduced ER Ca(2+) store, increased basal mitochondrial Ca(2+) and reduced ATP-stimulated ER-mitochondria Ca(2+) exchanges, despite an increase of organelle interactions. Furthermore, glucotoxicity-induced perturbations of Ca(2+) signaling are associated with ER stress, altered mitochondrial respiration and mitochondria fragmentation, and these organelle stresses may participate to increased organelle tethering, as a protective mechanism. Lastly, sustained induction of ER-mitochondria interactions using a linker reduced organelle Ca(2+) exchange, induced mitochondrial fission and altered GSIS. Therefore, dynamic organelle coupling participates to GSIS in beta cells and over-time disruption of organelle Ca(2+) exchange might be a novel mechanism contributing to glucotoxicity-induced beta cell dysfunction.

AIMS: Programmed cell death-1 and programmed death ligand 1 (PD-1/PD-L1) inhibitors restore antitumor immunity, but many autoimmune side-effects have been described. Diabetes mellitus is a rare complication, and little data concerning its pathophysiology and phenotype have been published. This study aimed to describe both pancreatic endocrine and exocrine functions, immunological features and change in pancreas volume in subjects with diabetes mellitus induced by PD-1 and PD-L1 inhibitors. METHODS: We analyzed the data of six subjects treated with immunotherapy who presented acute diabetes. RESULTS: There were five men and one woman. Median age was 67 years (range 55-83). Three subjects were treated with nivolumab, two with pembrolizumab and one with durvalumab. Median time to diabetes onset after immunotherapy initiation was 4 months (range 2-13). Four patients presented fulminant diabetes (FD); none of these had type 1 diabetes (T1D)-related autoantibodies, none of them had T1D or FD-very high-risk HLA class II profiles. The bi-hormonal endocrine and exocrine pancreatic failure previously reported for one FD patient was not found in other FD subjects, but glucagon response was blunted in another FD patient. Pancreas volume was decreased at diabetes onset in 2 FD patients, and all patients presented a subsequent decrease of pancreas volume during follow-up. CONCLUSIONS: In the patients presented herein, immunotherapy-induced diabetes was not associated with T1D-related autoantibodies. The hormonal and morphological analysis of the pancreatic glands of these six cases contributes to the understanding of the underlying and probably heterogeneous mechanisms. There is a need to find biomarkers to identify patients at risk to develop these new forms of diabetes at early stages of the process to prevent ketoacidosis and to evaluate preventive strategies.

Glucotoxicity-induced β-cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and the endoplasmic reticulum (ER). Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. The role of MAMs in β-cells is still largely unknown, and their implication in glucotoxicity-associated β-cell dysfunction remains to be defined. Here, we report that acute glucose treatment stimulated ER-mitochondria interactions and calcium (Ca

In this study, we propose Maghnite- $$\hbox {H}^{+}$$ , as an ecological, cost-effective and easily renewable catalyst, for the polymerization of decamethylcyclopentasiloxane (D5). The Maghnite is a clay consisting primarily of smectite minerals (montmorillonite group), which can be activated/reactivated through a simple process, by replacing interlayer cations by protons. Linear polymers with higher molecular mass and narrower molecular mass distribution (MMD) can be obtained with less cyclic by-products, using Maghnite- $$\hbox {H}^{+}$$ . The reaction was investigated in bulk, taking into account the effect of time, temperature and amount of catalyst on the monomer conversion and MMD. When the yield rises to 96%, the MMD is broadened, suggesting the formation of cyclic oligomers and/or crosslinking bonds, this was confirmed experimentally by infra-red spectroscopy, proton nuclear magnetic resonance ( $$^{1}\hbox {H}$$ NMR) and $$^{13}\hbox {C}$$ NMR analyses. The kinetics have been also studied; the reaction is first order in the monomer. The proposed reaction mechanism shows clearly the role of Maghnite- $$\hbox {H}^{+}$$ for D5 polymerization.

International audience; Physical activity has a major impact on bone density and on osteoporosis prevention. Sclerostin is produced by osteocytes and inhibits bone formation. The impact of exercise on sclerostin secretion has not been studied so far. This pilot study aimed to explore circulating sclerostin levels immediately after acute exercise. Healthy young women practicing physical activity less than 120 min per week were enrolled. The exercise was a 45-min, low-speed, treadmill running test. Blood samples were taken at rest before exercise and within 5 min after the end of exercise. We assessed serum creatinine, 25-OH vitamin D, alkaline phosphatase, C-telopeptide of type I collagen, bone-specific alkaline phosphatase, and sclerostin. Sclerostin stability at rest was also validated over the same period of time among women fulfilling the same inclusion criteria. The study included 23 participants (mean +/- SD age: 22.9 +/- 1.5 years) for the exercise test and 9 participants for the resting test (26.1 +/- 3.1 years). There was no difference in body mass index between the two groups. Sclerostin increased after exercise in comparison to baseline (mean +/- SEM: 410 +/- 27 vs. 290 +/- 19 pg/mL; p \textless 0.001) corresponding to an increase of +44.3 +/- 5.5%. In the resting test, sclerostin remained stable (303 +/- 20 vs. 294 +/- 20 pg/mL, p = 0.76). There was a substantial increase in serum sclerostin in untrained healthy young women immediately after physical activity. These results suggest the existence of an acute release of systemic sclerostin in response to physical activity.

Objectives To describe the use of antifungal medicines in English hospitals in adults with life-limiting illness and to investigate the association between socio-demographic variables and the use of high-cost formulations. Methods Pseudonymised patient-level information extracted from hospital pharmacy systems in 34 English acute general hospitals was linked to a National Health Service database of diagnoses and procedures. National Information Governance Board for England and Ethics approval was granted. The impact of socio-demographic variables on the use of high-cost formulations was assessed using stepwise logistic regression across 13 disease groups. Hospital guidelines on the use of antifungals were sourced and compared. Results People with haematological malignancies and unconfirmed infection formed the largest disease group (49.3%). Fungal infection was confirmed in an additional 12.6%. Guidelines focused on antifungal use in neutropenic patients. No guideline cited patient age, deprivation, gender or ethnicity as independent factors influencing treatment. Fluconazole dominated use (75% admissions). Significant associations were found between age, gender, deprivation and ethnicity and the use of high-cost antifungals. However, the direction of that association was not consistent across disease groups. Conclusions This study found widespread use of fluconazole, echoing results of earlier studies across Europe. It also found associations between patient factors and high-cost antifungal use that are not easily explained by disease, comorbidities, contraindications, guidelines or any systematic bias against particular groups of patients. It is clear that the drivers of antifungal therapy in hospital are complex and that antifungal stewardship poses a significant challenge for pharmacy.

Mesoporous MCM-41 materials containing aluminum with different Si/Al ratios (20, 40, and 80) were synthesized under hydrothermal condition. The materials were characterized using X-ray diffraction (XRD), nitrogen adsorption, Fourier-transform infrared (FTIR), scanning electron microscopy (SEM), energy-dispersive x-ray (EDX), transmission electron microscopy (TEM), and NH3 temperature programmed desorption (TPD) techniques. Incorporation of aluminum into MCM-41-type silica leads to creation of active and selective catalysts for condensation of pyrrole with 4-hydroxybenzaldehyde. The reaction was carried out at room temperature whilst stirring at 500 rpm. The catalyst Al-MCM-41 with Si/Al ratio of 20 produced a very high yield of [(pyrrole-2,5-diyl)-co-(benzylidene)]. The catalyst could be easily separated from the polymer product and regenerated by calcination.

The purpose of this study was to obtain polyamide-6 nanocomposites with national organically modified clay (termed 12-montmorillonite). The formation of polyamide-6 was confirmed by infrared spectroscopy (IR), the prepared nanocomposites were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA). The evolution of mechanical properties was also studied. The obtained results confirm the intercalation of molecules of salt in the clay layers, and a good interaction with the polymer, showing the formation of intercalated and/or partially exfoliated structures. The nanocomposites showed higher thermal stability compared to pure polymer, and the mechanical properties presented interesting and promising results.

Summary Diagnostic of thyroid dysfunction is largely based on TSH measurement in blood. Defining the upper reference limit of TSH values is of paramount importance to establish normal level of TSH. Ideally, accurate TSH measurement around the normal reference range should permit to determine which of the patients will undergo additional investigation and/or hormonal therapy. At present, there is a common agreement on the 0.4–4.0 mIU/L values but the upper limit range of TSH remains controversial. Work from the past 10 years has rather denied the proposal of lowering the threshold. TSH is a glycoprotein hormone for which the nature and circulating level may widely change depending on environmental (iodine intake and photoperiod) and physiological (age, sex, weight and ethnic origin) factors as well as on patient habits (smoking). In addition, the hormone is composed of multiple glycoforms, which display variable immunological behavior and thereby lead to altered TSH measurement. Therefore, this review aims at reinvestigating the biological significance of blood TSH values and at proposing how to reach improved accuracy in TSH testing.

International audience; We report the case of a patient with diabetes presenting a severe insulin-resistance syndrome due to the production of insulin autoantibodies by a lymphocytic lymphoma. We describe the various mechanisms leading to the production of insulin autoantibodies and insulin receptor autoantibodies and review the therapeutic possibilities.

ObjectivesInappropriate calcitonin (CT) release, a major feature of medullary thyroid cancer (MTC), may occur in neuroendocrine tumors (NETs). The aims of this retrospective study were to assess i) the characteristics and prognosis of CT-producing NETs, and ii) the value of CT monitoring during follow-up.MethodsAll patients with NETs in whom serum CT was assayed between 2010 and 2012 were included. MTCs were excluded. Clinical, biological, and histological characteristics were studied.ResultsTwenty-one (12%) of 176 patients in whom serum CT was systematically assayed had concentrations >100 ng/l, with tumours predominantly of bronchial or pancreatic origin (PP=0.0006). Poor prognosis was linked to high CT levels, poor differentiation, and grade 3. In a total group of 24 patients with serum CT >100 ng/l, symptoms potentially attributable to CT were recorded in eight, with occasional overlap with the carcinoid syndrome among other secretory syndromes. Immunohistochemistry could be performed in six tumor specimens, CT being detected in five. In 11 patients with five or more successive CT assays, hormone levels were fairly well correlated with clinical courses.ConclusionSerum CT levels may be raised in some patients with NETs, especially from foregut origin, and of high grade. The suggested value of CT monitoring during follow-up must be confirmed in further studies.

Niemann-Pick type C disease (NPC), a neurovisceral disorder characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. We report the diagnosis of six unrelated patients with NPC2, all with homozygous mutations. We further attempted functional characterization of the p.P120S, p.Q146X and IVS1 + 2 t>c mutations under native conditions. This was achieved by immunoblotting and immunocytofluorescence microscopy on cultured skin fibroblasts and in silico modeling. IVS1 + 2 t>c led to multiple transcripts, with only abnormally spliced cDNAs. Among the three NPC2 variants, only p.P120S led to detectable amounts of an immunoreactive protein. This protein showed a normal lysosomal localization. Our results suggest that the p.P120S mutation, the first naturally occurring missense mutation located in the cholesterol-binding Evolutionarily Constrained Regions D domain, results in reduced amounts of a protein capable to reach the lysosome, but unable to efficiently bind cholesterol. The patient had a juvenile neurological onset form of the disease. An update of the 22 families with mutations in the NPC2 gene, currently known to us, confirms the good genotype-phenotype correlations seen in this disorder. Characterization of more naturally occurring NPC2 mutations may help to dissect further the functional domains of the protein.

Thyroid-stimulating hormone-receptor autoantibodies normally causes hyperthyroidism. However, they might have blocking activity causing hypothyroidism. A 11-year-old girl followed due to type 1 diabetes mellitus, celiac disease and euthyroid lymphocytic thyroiditis at diagnosis. Two years after the initial evaluation, thyroid-stimulating hormone was suppressed with normal free T4; nine months later, a biochemical evolution to hypothyroidism with thyroid-stimulating hormone-receptor autoantibodies elevation was seen; the patient remained always asymptomatic. Chinese hamster ovary cells were transfected with the recombinant human thyroid-stimulating hormone -receptor, and then exposed to the patient's serum; it was estimated a 'moderate' blocking activity of these thyroid-stimulating hormone-receptor autoantibodies, and concomitantly excluded stimulating action. In this case, the acknowledgment of the blocking activity of the serum thyroid-stimulating hormone-receptor autoantibodies, supported the hypothesis of a multifactorial aetiology of the hypothyroidism, which in the absence of the in vitro tests, we would consider only as a consequence of the destructive process associated to lymphocytic thyroiditis.Os anticorpos anti-receptor da hormona estimulante da tiróide causam habitualmente hipertiroidismo. No entanto, a sua atividade pode ser bloqueadora, condicionando o hipotiroidismo. Apresenta-se o caso de uma doente do sexo feminino, 11 anos, com diabetes mellitus tipo 1, doença celíaca e tiroidite linfocítica em eutiroidismo ao diagnóstico. Cerca de dois anos após a avaliação inicial, a hormona estimulante da tiróide apresentava-se suprimida com T4 livre normal. Nove meses depois, a evolução foi para hipotiroidismo notandose concomitantemente elevação dos anticorpos anti-receptor da hormona estimulante da tiróide, mantendo-se a doente sempre assintomática. Células chinese hamster ovary foram transfetadas com o receptor de hormona estimulante da tiróide, e após incubação com o soro da doente, verificou-se uma atividade bloqueadora âmoderadaâ dos anticorpos anti-receptor da hormona estimulante da tiróide, excluindo-se ação estimuladora concomitante. Neste caso, o reconhecimento da ação bloqueadora dos anticorpos anti-receptor da hormona estimulante da tiróide detetados no soro suporta a hipótese de uma etiologia multifatorial para o hipotiroidismo que, na ausência dos testes in vitro, tenderíamos a interpretar unicamente como sequela do processo destrutivo associado à tiroidite linfocítica.

γ-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l -2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the γ glutamylaminobutyrate formed is measured at m / z = 233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.

Niemann-Pick disease type C (NPC), a severe neurovisceral lysosomal disorder, is due to mutations on the NPC1 gene or, in a minority of families, the NPC2 gene. Few investigations have been devoted to the NPC2 protein, for which only 13 different disease-causing mutations (including three novel ones in this report) have been described. Among the currently known NPC2 mutant alleles, six resulted in a premature stop codon. Only five missense mutations, c.115G>A (p.V39M), c.140G>T (p.C47F), c.199T>C (p.S67P), c.278G>T (p.C93F), and (this report) c.295T>C (p.C99R) were identified. In the present study, we generated cDNA constructs harboring each of these missense mutations and, upon overexpression in human fibroblasts with a nonsense NPC2 mutation, characterized the mutated proteins by immunoblotting, immunocytofluorescence microscopy, and complementation. Mutation p.V39M, described in the homozygous state in two patients with an adult-onset neurological disease, resulted in the synthesis of apparently functional recombinant proteins correctly targeted to lysosomes. Although a mild functional impact could possibly be overlooked in our overexpression system, comparative studies with NPC1 mutants indicated that mild mutations might not necessarily affect localization of the protein or its quantity in the native state. Conversely, mutations p.C47F, p.C93R, p.C99R but also, less predictably, p.S67P, led to the synthesis of misfolded recombinant proteins that colocalized with an endoplasmic reticulum marker. The four latter proteins were normally secreted but were unable to correct cholesterol storage in NPC2−/− cells. Functional characterization of the mutant proteins showed an excellent genotype–phenotype correlation in the three cases for whom a clinical history was available. Hum Mutat 26(1), 20–28, 2005. © 2005 Wiley-Liss, Inc.

Background The pre-analytical stabilities of vitamins A, E, K, B1, B2, B6, B12, C, carotenoids and folates in whole blood were studied. The aim of this work was to provide clear and workable pre-analytical procedures specifying optimal delay before freezing for laboratories which perform themselves such analyses or which receive and transfer such specimens to referral laboratories. Methods The stability of vitamins was studied in whole blood at room temperature after light exposure up to 24 h (vitamin C), 48 h (vitamins A, E, B1, B2, B6 and carotenoids) and 72 h (vitamins K, B12, red blood cell (RBC) and serum folates). Vitamin C stability after baseline acidification was analysed up to 48 h. Changes observed were compared to a clinical cut-off defined as total change limit based on a combination of analytical performance and within-subject variation. Results Clinically and statistically significant changes occurred only in vitamins C (−22.5%), B6 (+9.9%) and serum folates (−16.8%) concentrations after 6, 24 and 48 h, respectively. Vitamins A, E, K, B1, B2, B12, RBC folates and carotenoids showed good stability up to 48 or 72 h. Vitamin C in acidified serum conserved at room temperature appeared unstable. The optimal condition for acidified vitamin C conservation was at less than −20℃. Conclusion The majority of vitamins remain stable for up to 48 h. Vitamin C quantification requires serum acidification followed by freezing as soon as possible. Freezing, respectively, within 12 h and 24 h for determination of plasma vitamin B6 and serum folates concentrations is recommended.

In this study, a new and easy strategy was discussed for the purpose of synthesizing of a polymer of phenylmethylcyclosiloxane type (PPMS). The cationic ring opening polymerization of triphenyltrimethylcyclotrisiloxane (D3Ph,Me) was initiated by a solid, efficient and environmentally-friendly catalyst called Maghnite-H+. Maghnite-H+ is a natural clay composed essentially of montmorillonite, it is activated with an acid treatment by replacing the interlayer ions by protons, that results to the increase of the basal space, this may be confirmed by XRD analysis. The reaction was carried out without solvent at different temperatures and for different periods of time, using also several catalyst contents. Subsequently, the operating conditions were opted in order to obtain a maximum yield of the linear polymer and a high average molecular mass as well. The structure of polymers obtained was confirmed by IR analysis. 1H NMR and 13C NMR analyzes were used to follow the crosslinking of polymer chains over time. The thermal behavior was investigated by DSC analysis. The average molecular mass and the polydispersity indices were determined by GPC.

Aims/hypothesis Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route. Methods Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA). Results HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues. Conclusions/interpretation Exosomes act as ‘paracrine-like’ signals and modify muscle homeostasis during high-fat diets. Electronic supplementary material The online version of this article (doi:10.1007/s00125-014-3337-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

ContextHyperthyroidism occurs in 1% of neonates born to mothers with active or past Graves' disease (GD). Current guidelines for the management of GD during pregnancy were based on studies conducted with first-generation thyroid-binding inhibitory immunoglobulin (TBII) assays.ObjectiveThis retrospective study was conducted in order to specify the second-generation TBII threshold predictive of fetal and neonatal hyperthyroidism, and to identify other factors that may be helpful in predicting neonatal hyperthyroidism.MethodsWe included 47 neonates born in the Lyon area to 42 mothers harboring measurable levels of TBII during pregnancy. TBII measurements were carried out in all mothers; bioassays were carried out in 20 cases.ResultsNine neonates were born with hyperthyroidism, including five with severe hyperthyroidism requiring treatment. Three neonates were born with hypothyroidism. All hyperthyroid neonates were born to mothers with TBII levels >5 IU/l in the second trimester (sensitivity, 100% and specificity, 43%). No mother with TSH receptor-stimulating antibodies (TSAb measured by bioassay) below 400% gave birth to a hyperthyroid neonate. Among mothers of hyperthyroid neonates, who required antithyroid drugs during pregnancy, none could stop treatment before delivery. Analysis of TBII evolution showed six unexpected cases of increasing TBII values during pregnancy.ConclusionMaternal TBII value over 5 IU/l indicates a risk of neonatal hyperthyroidism. Among these mothers, a TSAb measurement contributes to identify more specifically those who require a close fetal thyroid ultrasound follow-up. These results should be confirmed in a larger series.

International organizations require from medical laboratories a quantitative statement of the uncertainty in measurement (UM) to help interpret patient results. The French accreditation body (COFRAC) recommends an approach (SH GTA 14 IQC/EQA method) using both internal quality control (IQC) and external quality assessment (EQA) data. The aim of this work was to validate an alternative way to quantify UM using only EQA results without any need for IQC data. This simple and practical method, which has already been described as the long-term evaluation of the UM (LTUM), is based on linear regression between data obtained by participants in EQA schemes and target values. We used it for 43 routine analytes covering biochemistry, immunoassay, and hemostasis fields.Data from 50 laboratories participating in ProBioQual (PBQ) EQA schemes over 25 months were used to obtain estimates of the median and 90th percentile LTUM and to compare them to the usual analytical goals. Then, the two UM estimation methods were compared using data from 20 laboratories participating in both IQC and EQA schemes.Median LTUMs ranged from 2.9% (sodium) to 16.3% (bicarbonates) for biochemistry analytes, from 12.6% (prothrombin time) to 18.4% (factor V) for hemostasis analytes when using the mean of all participants, and were around 10% for immunoassays when using the peer-group mean. Median LTUMs were, in most cases, slightly lower than those obtained with the SH GTA 14 method, whatever the concentration level.LTUM is a simple and convenient method that gives UM estimates that are reliable and comparable to those of recommended methods. Therefore, proficiency testing (PT) organizers are allowed to provide participants with an additional UM estimate using only EQA data and which could be updated at the end of each survey.

International audience; OBJECTIVES: Differentiated thyroid cancer (DTC) requires long-term follow-up by serum thyroglobulin assay and cervical ultrasound, due to the risk of recurrence. Guidelines recommend basal assay under hormone therapy at 3 months, repeated at 6-12 months post-surgery, with or without associated isotopic ablation, after stimulation by recombinant human TSH to improve assay sensitivity. It was hypothesized that a new-generation assay kit with lower limits of detection and quantification would improve the sensitivity of the basal assay, enhance detection of premature recurrence and decrease the rate of false-negatives, thereby avoiding the need for the complementary stimulation test. MATERIAL AND METHODS: A validation study of the second-generation thyroglobulin serum assay was performed in the laboratory of the Lyon Sud Hospital Centre (Lyon, France), with comparison to stimulation test results. Low-concentration serum pools were constituted, including patients followed for stage I to III DTC for whom basal and post-stimulation samples were available in the serum bank. RESULTS: The new assay proved robust and reliable, with good correlation with the technique presently used in the Lyon hospitals. None of the 54 patients showed false-negative results, which was the objective of our choice of threshold, and 5 were false-positive, for thyroglobulin thresholds of 0.1mug/L at baseline and 1.0mug/L post-stimulation. Positive and negative predictive values were 100% and 87.8% respectively. CONCLUSION: These results allow an improvement in the follow-up algorithm for DTC, replacing the stimulation test by the new-generation thyroglobulin assay in post-therapeutic assessment.

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the “variant” biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination–codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and “classic” (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the “classic”/“variant” biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the “variant” phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.

International audience; It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as "endocrine signals" during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube-exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.

International audience; Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.