A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction

A capillary electrophoresis (CE) method for the characterization of recombinant NTPDases 1, 2, and 3, and for assaying NTPDase inhibitors has been developed performing the enzymatic reaction within the capillary. After hydrodynamic injection of plugs of substrate solution with or without inhibitor in reaction buffer, followed by a suspension of an enzyme-containing membrane preparation, and subsequent injection of another plug of substrate solution with or without inhibitor, the reaction took place close to the capillary inlet. After 5 min, the electrophoretic separation of the reaction products was initiated by applying a constant current of  μA. The method employing a polyacrylamide-coated capillary and reverse polarity mode provided baseline resolution of substrates and products within a short separation time of less than 7 min. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by their UV absorbance at 210 nm. The Michaelis–Menten constants (Km) for the recombinant rat NTPDases 1, 2, and 3 obtained with this method were consistent with previously reported data. The inhibition studies revealed pronounced differences in the potency of reactive blue 2, pyridoxalphosphate-6-azophenyl-2-4-disulfonic acid (PPADS), suramin, and N6-diethyl-β,γ-dibromomethylene-ATP (ARL67156) towards the NTPDase isoforms. Notably, ARL67156 does not inhibit all NTPDases, having only a minor inhibitory effect on NTPDase2. Dipyridamole is not an inhibitor of the NTPDase isoforms investigated. The new method is fast and accurate, it requires only tiny amounts of material (nanoliter scale), no sample pretreatment and can be fully automated; thus it is clearly superior to the current standard methods

Impact of physical activity level and dietary fat content on passive overconsumption of energy in non-obese adults

Background: Passive overconsumption is the increase in energy intake driven by the high-fat energy-dense food environment. This can be explained in part because dietary fat has a weaker effect on satiation (i.e. process that terminates feeding). Habitually active individuals show improved satiety (i.e. process involved in post-meal suppression of hunger) but any improvement in satiation is unknown. Here we examined whether habitual physical activity mitigates passive overconsumption through enhanced satiation in response to a high-fat meal. Methods: Twenty-one non-obese individuals with high levels of physical activity (HiPA) and 19 individuals with low levels of physical activity (LoPA) matched for body mass index (mean = 22.8 kg/m2) were recruited. Passive overconsumption was assessed by comparing ad libitum energy intake from covertly manipulated high-fat (HFAT; 50% fat) or high-carbohydrate (HCHO; 70% carbohydrate) meals in a randomized crossover design. Habitual physical activity was assessed using SenseWear accelerometers (SWA). Body composition, resting metabolic rate, eating behaviour traits, fasting appetite-related peptides and hedonic food reward were also measured. Results: In the whole sample, passive overconsumption was observed with greater energy intake at HFAT compared to HCHO (p  0.05). SWA confirmed that HiPA were more active than LoPA (p  0.05 for all). Conclusions: Non-obese individuals with high or low physical activity levels but matched for BMI showed similar susceptibility to passive overconsumption when consuming an ad libitum high-fat compared to a high-carbohydrate meal. This occurred despite increased total daily energy expenditure and improved body composition in HiPA. Greater differences in body composition and/or physical activity levels may be required to impact on satiation

A phase I study of bendamustine hydrochloride administered day 1+2 every 3 weeks in patients with solid tumours

The aim of the study was to determine the maximum tolerated dose (MTD), the dose limiting toxicity (DLT), and the pharmacokinetic profile (Pk) of bendamustine (BM) on a day 1 and 2 every 3 weeks schedule and to recommend a safe phase II dose for further testing. Patients with solid tumours beyond standard therapy were eligible. A 30-min intravenous infusion of BM was administered d1+d2 q 3 weeks. The starting dose was 120 mg m−2 per day and dose increments of 20 mg m−2 were used. Plasma and urine samples were analysed using validated high-performance liquid chromatography/fluorescence assays. Fifteen patients were enrolled. They received a median of two cycles (range 1–8). The MTD was reached at the fourth dose level. Thrombocytopaenia (grade 4) was dose limiting in two of three patients at 180 mg m−2. One patient also experienced febrile neutropaenia. Lymphocytopaenia (grade 4) was present in every patient. Nonhaematologic toxicity including cardiac toxicity was not dose limiting with this schedule. Mean plasma Pk values of BM were tmax 35 min, t1/2 49.1 min, Vd 18.3 l m−2, and clearance 265 ml min−1 m−2. The mean total amount of BM and its metabolites recovered in the first micturition was 8.3% (range 2.7–26%). The MTD of BM in the present dose schedule was 180 mg m−2 on day 1+2. Thrombocytopaenia was dose limiting. The recommended dose for future phase II trials with this schedule is 160 mg m−2 per day

How the serotonin transporter 5-HTTLPR polymorphism influences amygdala function: the roles of in vivo serotonin transporter expression and amygdala structure

The serotonin transporter-linked promoter region (5-HTTLPR) polymorphism of the serotonin transporter gene is associated with amygdala response during negative emotion. The aim of this study was to investigate whether this genotype effect on amygdala function is mediated by current serotonin transporter (5-HTT) levels or rather by genetically induced influences during neurodevelopment, shaping brain structure. A total of 54 healthy subjects underwent functional and structural magnetic resonance imaging, [11C]DASB positron emission tomography and 5-HTTLPR genotyping to analyze the interrelationships between amygdala activation during processing of unpleasant stimuli, 5-HTTLPR genotype, amygdala volumes and 5-HTT levels in the midbrain and in other brain regions. In line with previous research, carriers of the short allele (S) showed increased amygdala activation. Path analysis demonstrated that this genotype effect was not procured by current 5-HTT availability but by amygdala structure, with smaller amygdala volumes in the S than in the LL genotype, as well as smaller volumes being associated with increased amygdala activation. Our findings stress the role of genetic effects during neurodevelopment

Evidence for a wide extra-astrocytic distribution of S100B in human brain

BACKGROUND: S100B is considered an astrocytic in-situ marker and protein levels in cerebrospinal fluid (CSF) or serum are often used as biomarker for astrocytic damage or dysfunction. However, studies on S100B in the human brain are rare. Thus, the distribution of S100B was studied by immunohistochemistry in adult human brains to evaluate its cell-type specificity. RESULTS: Contrary to glial fibrillary acidic protein (GFAP), which selectively labels astrocytes and shows only faint ependymal immunopositivity, a less uniform staining pattern was seen in the case of S100B. Cells with astrocytic morphology were primarily stained by S100B in the human cortex, while only 20% (14–30%) or 14% (7–35%) of all immunopositive cells showed oligodendrocytic morphology in the dorsolateral prefrontal and temporal cortices, respectively. In the white matter, however, most immunostained cells resembled oligodendrocytes [frontal: 75% (57–85%); temporal: 73% (59–87%); parietal: 79% (62–89%); corpus callosum: 93% (86–97%)]. S100B was also found in ependymal cells, the choroid plexus epithelium, vascular endothelial cells, lymphocytes, and several neurones. Anti-myelin basic protein (MBP) immunolabelling showed an association of S100B with myelinated fibres, whereas GFAP double staining revealed a distinct subpopulation of cells with astrocytic morphology, which solely expressed S100B but not GFAP. Some of these cells showed co-localization of S100B and A2B5 and may be characterized as O2A glial progenitor cells. However, S100B was not detected in microglial cells, as revealed by double-immunolabelling with HLA-DR. CONCLUSION: S100B is localized in many neural cell-types and is less astrocyte-specific than GFAP. These are important results in order to avoid misinterpretation in the identification of normal and pathological cell types in situ and in clinical studies since S100B is continuously used as an astrocytic marker in animal models and various human diseases

Autoimmune Neuromuscular Disorders in Childhood

Autoimmune neuromuscular disorders in childhood include Guillain-Barré syndrome and its variants, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), juvenile myasthenia gravis (JMG), and juvenile dermatomyositis (JDM), along with other disorders rarely seen in childhood. In general, these diseases have not been studied as extensively as they have been in adults. Thus, treatment protocols for these diseases in pediatrics are often based on adult practice, but despite the similarities in disease processes, the most widely used treatments have different effects in children. For example, some of the side effects of chronic steroid use, including linear growth deceleration, bone demineralization, and chronic weight issues, are more consequential in children than in adults. Although steroids remain a cornerstone of therapy in JDM and are useful in many cases of CIDP and JMG, other immunomodulatory therapies with similar efficacy may be used more frequently in some children to avoid these long-term sequelae. Steroids are less expensive than most other therapies, but chronic steroid therapy in childhood may lead to significant and costly medical complications. Another example is plasma exchange. This treatment modality presents challenges in pediatrics, as younger children require central venous access for this therapy. However, in older children and adolescents, plasma exchange is often feasible via peripheral venous access, making this treatment more accessible than might be expected in this age group. Intravenous immunoglobulin also is beneficial in several of these disorders, but its high cost may present barriers to its use in the future. Newer steroid-sparing immunomodulatory agents, such as azathioprine, tacrolimus, mycophenolate mofetil, and rituximab, have not been studied extensively in children. They show promising results from case reports and retrospective cohort studies, but there is a need for comparative studies looking at their relative efficacy, tolerability, and long-term adverse effects (including secondary malignancy) in children

Association between diabetes mellitus and active tuberculosis: A systematic review and meta-analysis.

The burgeoning epidemic of diabetes mellitus (DM) is one of the major global health challenges. We systematically reviewed the published literature to provide a summary estimate of the association between DM and active tuberculosis (TB). We searched Medline and EMBASE databases for studies reporting adjusted estimates on the TB-DM association published before December 22, 2015, with no restrictions on region and language. In the meta-analysis, adjusted estimates were pooled using a DerSimonian-Laird random-effects model, according to study design. Risk of bias assessment and sensitivity analyses were conducted. 44 eligible studies were included, which consisted of 58,468,404 subjects from 16 countries. Compared with non-DM patients, DM patients had 3.59-fold (95% confidence interval (CI) 2.25-5.73), 1.55-fold (95% CI 1.39-1.72), and 2.09-fold (95% CI 1.71-2.55) increased risk of active TB in four prospective, 16 retrospective, and 17 case-control studies, respectively. Country income level (3.16-fold in low/middle-vs. 1.73-fold in high-income countries), background TB incidence (2.05-fold in countries with >50 vs. 1.89-fold in countries with ≤50 TB cases per 100,000 person-year), and geographical region (2.44-fold in Asia vs. 1.71-fold in Europe and 1.73-fold in USA/Canada) affected appreciably the estimated association, but potential risk of bias, type of population (general versus clinical), and potential for duplicate data, did not. Microbiological ascertainment for TB (3.03-fold) and/or blood testing for DM (3.10-fold), as well as uncontrolled DM (3.30-fold), resulted in stronger estimated association. DM is associated with a two- to four-fold increased risk of active TB. The association was stronger when ascertainment was based on biological testing rather than medical records or self-report. The burgeoning DM epidemic could impact upon the achievements of the WHO "End TB Strategy" for reducing TB incidence

Repertoire of microRNAs in Epithelial Ovarian Cancer as Determined by Next Generation Sequencing of Small RNA cDNA Libraries

MicroRNAs (miRNAs) are small regulatory RNAs that are implicated in cancer pathogenesis and have recently shown promise as blood-based biomarkers for cancer detection. Epithelial ovarian cancer is a deadly disease for which improved outcomes could be achieved by successful early detection and enhanced understanding of molecular pathogenesis that leads to improved therapies. A critical step toward these goals is to establish a comprehensive view of miRNAs expressed in epithelial ovarian cancer tissues as well as in normal ovarian surface epithelial cells.We used massively parallel pyrosequencing (i.e., "454 sequencing") to discover and characterize novel and known miRNAs expressed in primary cultures of normal human ovarian surface epithelium (HOSE) and in tissue from three of the most common histotypes of ovarian cancer. Deep sequencing of small RNA cDNA libraries derived from normal HOSE and ovarian cancer samples yielded a total of 738,710 high-quality sequence reads, generating comprehensive digital profiles of miRNA expression. Expression profiles for 498 previously annotated miRNAs were delineated and we discovered six novel miRNAs and 39 candidate miRNAs. A set of 124 miRNAs was differentially expressed in normal versus cancer samples and 38 miRNAs were differentially expressed across histologic subtypes of ovarian cancer. Taqman qRT-PCR performed on a subset of miRNAs confirmed results of the sequencing-based study.This report expands the body of miRNAs known to be expressed in epithelial ovarian cancer and provides a useful resource for future studies of the role of miRNAs in the pathogenesis and early detection of ovarian cancer