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KCa3.1 (also known as SK4 or IK1) is a mammalian intermediate-conductance potassium channel that plays a critical role in the activation of T cells, B cells, and mast cells, effluxing potassium ions to maintain a negative membrane potential for influxing calcium ions. KCa3.1 shares primary sequence similarity with three other (low-conductance) potassium channels: KCa2.1, KCa2.2, and KCa2.3 (also known as SK1–3). These four homotetrameric channels bind calmodulin (CaM) in the cytoplasmic region, and calcium binding to CaM triggers channel activation. Unique to KCa3.1, activation also requires phosphorylation of a single histidine residue, His358, in the cytoplasmic region, which relieves copper-mediated inhibition of the channel. Near the cytoplasmic C-terminus of KCa3.1 (and KCa2.1–2.3), secondary-structure analysis predicts the presence of a coiled-coil/heptad repeat. Here, we report the crystal structure of the C-terminal coiled-coil region of KCa3.1, which forms a parallel four-helix bundle, consistent with the tetrameric nature of the channel. Interestingly, the four copies of a histidine residue, His389, in an ‘a’ position within the heptad repeat, are observed to bind a copper ion along the four-fold axis of the bundle. These results suggest that His358, the inhibitory histidine in KCa3.1, might coordinate a copper ion through a similar binding mode. [ABSTRACT FROM AUTHOR]

Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk. [ABSTRACT FROM AUTHOR]

Background: A decline in estimated glomerular filtration rate (eGFR) is reportedly associated with increased prevalence rates of cognitive impairment. However, data concerning the association between the cerebral saturation of oxygen (rSO2) and cognitive function of patients with chronic kidney disease (CKD) is limited. This study aimed to (i) elucidate the clinical factors associating with cerebral rSO2 and (ii) investigate the association between cerebral rSO2 and cognitive assessment in CKD patients. Methods: A total of 40 CKD patients not requiring dialysis (26 men and 14 women; mean age, 61.0 ± 2.7 years) were recruited. The numbers of patients at each CKD stage were as follows: G1, 5; G2, 8; G3a, 6; G3b, 5; G4, 11; and G5, 5. Cerebral rSO2 was monitored at the forehead using the oxygen saturation monitor INVOS 5100C. The cognitive function of each patient was confirmed using the Mini-Mental State Examination (MMSE). Results: Cerebral rSO2 levels were significantly higher in CKD patients than in hemodialysis patients (63.8 ± 1.5% vs. 44.9 ± 2.2%, p < 0.001). Multiple regression analysis showed that cerebral rSO2 was independently associated with eGFR (standardized coefficient: 0.530), serum albumin concentration (standardized coefficient: 0.365), and serum sodium concentration (standardized coefficient: 0.224). Furthermore, MMSE showed a significantly positive correlation with cerebral rSO2 (r = 0.624, p < 0.001). Conclusions: Cerebral rSO2 was affected by eGFR and serum albumin and sodium concentrations in CKD patients. Furthermore, cerebral rSO2 monitoring, which reflected MMSE scores, might be a useful method for assessing cognitive function in CKD patients. [ABSTRACT FROM AUTHOR]

The use of pesticides exposes humans to numerous harmful molecules. Exposure in early-life may be responsible for adverse effects in later life. This study aimed to assess the metabolic modifications induced in pregnant rats and their offspring by a pesticide mixture representative of human exposure. Ten pregnant rats were exposed to a mixture of eight pesticides: acetochlor (246 μg/kg bw/d) + bromoxynil (12 μg/kg bw/d) + carbofuran (22.5 μg/kg bw/d) + chlormequat (35 μg/kg bw/d) + ethephon (22.5 μg/kg bw/d) + fenpropimorph (15.5 μg/kg bw/d) + glyphosate (12 μg/kg bw/d) + imidacloprid (12.5 μg/kg bw/d) representing the main environmental pesticide exposure in Brittany (France) in 2004. Another group of 10 pregnant rats served as controls. Females were fed ad libitum from early pregnancy, which is from gestational day (GD) 4 to GD 21. Urine samples were collected at GD 15. At the end of the exposure, mothers and pups were euthanized and blood, liver, and brain samples collected. 1H NMR-based metabolomics and GC-FID analyses were performed and PCA and PLS-DA used to discriminate between control and exposed groups. Metabolites for which the levels were significantly modified were then identified using the Kruskal-Wallis test, and p-values were adjusted for multiple testing correction using the False Discovery Rate. The metabolomics analysis revealed many differences between dams of the two groups, especially in the plasma, liver and brain. The modified metabolites are involved in TCA cycle, energy production and storage, lipid and carbohydrate metabolism, and amino-acid metabolism. These modifications suggest that the pesticide mixture may induce oxidative stress associated with mitochondrial dysfunction and the impairment of glucose and lipid metabolism. These observations may reflect liver dysfunction with increased relative liver weight and total lipid content. Similar findings were observed for glucose and energy metabolism in the liver of the offspring, and oxidative stress was also suggested in the brains of male offspring. [ABSTRACT FROM AUTHOR]

Background: Improving the knowledge of angiomyolipoma physiopathology might help in refining its pharmacological treatment. We investigated if angiomyolipoma cells have migratory properties, how their growth and motility can be influenced by the hormonal milieu, and if this can be related to a specific gender. Methods: Primary cells were isolated from angiomyolipomas surgically resected for therapeutical reasons in a female and in a male patient. The genetic control demonstrated no TSC2 deletion. Bi- (wound healing) and three-dimensional (transwell assay) migration were analyzed in vitro in basal conditions and under the influence of 17- β-estradiol and SDF-1α. Results: Treatment up to 72 hours with 17-β-estradiol (0.1–100 nM), tamoxifen (0.2–20 μM) or with both, does not modify angiomyolipoma cells proliferation. On the other hand, SDF-1α and 17-β-estradiol treatment induce a significant motility increase (both bi- and three-dimensional) which becomes evident already after 2 hours of incubation. Angiomyolipoma cells express mRNA coding for SDF-1α and 17-β-estradiol receptors and secrete both the metalloproteases principally involved in malignant phenotype acquisition, i.e. MMP-2 and MMP-9. Conclusion: Angiomyolipoma cells behave similarly, despite their different source. Primary angiomyolipoma cells migrate in response to hormonal milieu and soluble factors, and produce active metalloproteases, both aspects being consistent with the theory claiming they can migrate to the lungs (and/or other organs) and colonizing them. No main feature, among the aspects we analyzed, seems to be referable to the gender of origin. [ABSTRACT FROM AUTHOR]

Sthenoteuthis oualaniensis (SA) is an important squid species in the South China Sea. Based on SA samples collected in 2016, SA was divided into the “dwarf” form (DF) and “medium” form (MF). To understand the changes in gastrointestinal function in SA during sexual maturation, we undertook transcriptomic analyses of the stomach and intestine tissues of the mature and immature DF and MF of SA using the deep-sequencing platform Illumina HiSeq™. We exploited a high-throughput method to delineate differentially expressed genes (DEGs) in the DF and MF of SA. A total of 135464 unigenes (68627 unigenes of the DG and 66837 unigenes of the MF) were generated. We identified 7965 and 4051 relative DEGs in the intestine and stomach tissues of the mature DF of SA compared with those of the immature DF of SA; and 22138 and 18460 DEGs in the intestine and stomach of the mature MF of SA compared with those of the immature MF of SA. Gastrointestinal function related to the metabolism of lipids, amino acids, glucose, and energy were changed in SA during sexual maturation. This work is the first to identify a set of genes associated with gastrointestinal function during sexual maturation in SA. [ABSTRACT FROM AUTHOR]

Background: Nonalcoholic fatty liver disease (NAFLD) has been reported to have a negative effect on bone mineral density (BMD) in Asian populations. Whether such an association exists in Western populations is less clear. Methods: This cross-sectional analysis of data from NHANES III, a United States national health survey conducted from 1988 to 1994, included 6089 participants aged 40–75 years, selected after excluding people with hepatitis virus serology, elevated alcohol consumption, decreased renal function, or steroid use, and pregnant females. The main outcome, BMD at the femoral neck, was measured using dual-energy X-ray absorptiometry. The primary exposure, NAFLD, was defined as moderate or severe hepatic steatosis diagnosed using abdominal ultrasonography. Result: After controlling for gender and menopausal status, race/ethnicity, age and body mass index, NAFLD was not significantly associated with BMD (beta coefficient: −0.006, 95%CI: −0.016, 0.003). A secondary analysis categorized participants with NAFLD according to their serum alanine aminotransferase (ALT) levels into high and normal ALT NAFLD groups, and compared these with the non-NAFLD group. NAFLD with higher levels of ALT was associated with lower levels of BMD (beta coefficient: −0.023, 95% CI: −0.044, −0.002). Conclusion: This study showed a relationship between NAFLD with high ALT and lower BMD in the general U.S. population. [ABSTRACT FROM AUTHOR]

Immunoproteomic analysis was performed to identify unknown, pathology-related molecules in patients with seronegative (SN) obstetric antiphospholipid syndrome (APS) who clinically satisfied the diagnostic criteria for APS, but not the serological criteria. We collected peripheral blood from 13 SN-APS outpatients with known thrombotic predisposition, 13 with no known thrombotic predisposition, and four multiparous women with no history of miscarriage (control). Plasma proteins from volunteers were purified and used as plasma protein antigens. Two-dimensional immunoblotting was performed using pooled control or SN-APS serum samples as the primary antibodies. Mass spectrometry of reactive spots specific to SN-APS serum led to the identification of complement molecule C9. Western blotting using commercial purified alkylated C9 was performed to detect autoantibodies. Examination of individual patient serum identified reactivity in one patient with, and in two patients without known thrombotic predisposition. This study suggests that SN-APS pathologies were associated with autoantibodies that react to specific C9 epitopes. [ABSTRACT FROM AUTHOR]

We have developed a very simple method, termed absorbance summation (AS), for comparing protein concentrations between samples in ELISA assays without a standard. This method sums the observed absorbance values from all dilutions to obtain one data point for each sample to be used for comparison. AS is less computationally intensive than fitting sigmoidal curves, and it avoids the difficulty of parameter estimation for samples with absorbance values lying primarily at the lower tail of the curve. Our simulation studies showed that it performs much better than the sigmoidal curve fitting method and the conventional endpoint titer method. The power of this simple method is as high as the formal curve fitting followed by the estimation of area under the curve (AUC). [ABSTRACT FROM AUTHOR]

Summary: In this report we introduce a weak-model approach for examination of the intrinsic time-varying properties of the hemoglobin signal, with the aim of advancing the application of functional near infrared spectroscopy (fNIRS) for the detection of breast cancer, among other potential uses. The developed methodology integrates concepts from stochastic network theory with known modulatory features of the vascular bed, and in doing so provides access to a previously unrecognized dense feature space that is shown to have promising diagnostic potential. Notable features of the methodology include access to this information solely from measures acquired in the resting state, and analysis of these by treating the various components of the hemoglobin (Hb) signal as a co-varying interacting system. Approach: The principal data-transform kernel projects Hb state-space trajectories onto a coordinate system that constitutes a finite-state representation of covariations among the principal elements of the Hb signal (i.e., its oxygenated (ΔoxyHb) and deoxygenated (ΔdeoxyHb) forms and the associated dependent quantities: total hemoglobin (ΔtotalHb = ΔoxyHb + ΔdeoxyHb), hemoglobin oxygen saturation (ΔHbO2Sat = 100Δ(oxyHb/totalHb)), and tissue-hemoglobin oxygen exchange (ΔHbO2Exc = ΔdeoxyHb—ΔoxyHb)). The resulting ten-state representation treats the evolution of this signal as a one-space, spatiotemporal network that undergoes transitions from one state to another. States of the network are defined by the algebraic signs of the amplitudes of the time-varying components of the Hb signal relative to their temporal mean values. This assignment produces several classes of coefficient arrays, most with a dimension of 10×10. Biological motivation: Motivating our approach is the understanding that effector mechanisms that modulate blood delivery to tissue operate on macroscopic scales, in a spatially and temporally varying manner. Also recognized is that this behavior is sensitive to nonlinear actions of these effectors, which include the binding properties of hemoglobin. Accessible phenomenology includes measures of the kinetics and probabilities of network dynamics, which we treat as surrogates for the actions of feedback mechanisms that modulate tissue-vascular coupling. Findings: Qualitative and quantitative features of this space, and their potential to serve as markers of disease, have been explored by examining continuous-wave fNIRS 3D tomographic time series obtained from the breasts of women who do and do not have breast cancer. Inspection of the coefficient arrays reveals that they are governed predominantly by first-order rate processes, and that each array class exhibits preferred structure that is mainly independent of the others. Discussed are strategies that may serve to extend evaluation of the accessible feature space and how the character of this information holds potential for development of novel clinical and preclinical uses. [ABSTRACT FROM AUTHOR]

Intrinsic and acquired resistance to current HER2 targeted therapies remains a challenge in clinics. We have developed a therapeutic HER2 siRNA delivered using mesoporous silica nanoparticles modified with polymers and conjugated with HER2 targeting antibodies. Our previous studies have shown that our HER2 siRNA nanoparticles could overcome intrinsic and acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancers. In this study, we investigated the effect of long-term (7 months) treatment using our therapeutic HER2 siRNA. Even after the removal of HER2 siRNA, the long-term treated cells grew much slower (67% increase in doubling time) than cells that have not received any treatment. The treated cells did not undergo epithelial-mesenchymal transition or showed enrichment of tumor initiating cells. Unlike trastuzumab and lapatinib, which induced resistance in BT474 cells after 6 months of treatment, HER2 siRNA did not induce resistance to HER2 siRNA, trastuzumab, or lapatinib. HER2 ablation with HER2 siRNA prevented reactivation of HER2 signaling that was observed in cells resistant to lapatinib. Altogether, our results indicate that a HER2 siRNA based therapeutic provides a more durable inhibition of HER2 signaling in vitro and can potentially be more effective than the existing therapeutic monoclonal antibodies and small molecule inhibitors. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the study was to investigate the components of day-to-day variability of repeated phase contrast mapping (PCM) magnetic resonance imaging measurements of global cerebral blood flow (gCBF). Materials and methods: Two dataset were analyzed. In Dataset 1 duplicated PCM measurements of total brain flow were performed in 11 healthy young volunteers on two separate days applying a strictly standardized setup. For comparison PCM measurements obtained from a previously published study (Dataset 2) were analyzed in order to assess long-term variability in an aged population in a less strictly controlled setup. Global CBF was calculated by normalizing total brain flow to brain volume. On each day measurements of hemoglobin, caffeine and glucose were obtained. Linear mixed models were applied to estimate coefficients of variation (CV) of total (CVt), between-subject (CVb), within-subject day-to-day (CVw), and intra-session residual variability (CVr). Results: In Dataset 1 CVt, CVb, CVw and CVr were estimated to be 11%, 9.4%, 4% and 4.2%, respectively, and to 8.8%, 7.2%, 2.7% and 4.3%, respectively, when adjusting for hemoglobin and plasma caffeine. In Dataset 2 CVt, CVb and CVw were estimated to be 25.4%, 19.2%, and 15.0%, respectively, and decreased to 16.6%, 8.2% and 12.5%, respectively, when adjusting for the same covariates. Discussion: Our results suggest that short-term day-to-day variability of gCBF is relatively low compared to between-subject variability when studied in standardized conditions, whereas long-term variability in an aged population appears to be much larger when studied in less a standardized setup. The results further showed that from 20% to 35% of the total variability in gCBF can be attributed to the effects of hemoglobin and caffeine. [ABSTRACT FROM AUTHOR]

Prenatal detection of the fetal RHD status can be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. Hemolytic disease causes morbidity and mortality of the fetus in the neonatal period. The routine use of antenatal and postnatal anti-D prophylaxis has reduced the incidence of hemolytic disease of the fetus and newborn. This study describe the detection of fetal RhD antigens in blood of RhD negative pregnant women using a nanopolymer coated electrochemical biosensor for medical diagnosis. Cell free fetal DNA in maternal plasma was also used to genotyping fetal RHD status using multiplex real-time PCR. Twenty-six RhD negative pregnant women in different gestational ages were included in the study. RhD positive fetal antibodies detected with a developed biosensor in maternal blood of RhD negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat, and corundum ceramic based screen printed gold electrode combined with the reference Ag/AgCl electrode, and the auxiliary Au/Pd (98/2%) electrode. Fetal RHD genotyping performed using fluorescence-based multiplex real-time PCR exons 5 and 7 of the RHD gene. The fetal RHD status of 26 RhD negative cases were detected 21 as RhD positive and 5 as RhD negative with electrochemical biosensor. Fetal RHD status confirmed with extracted fetal DNA in maternal plasma using multiplex real-time PCR RHD genotyping and by serological test after delivery. The new method for fetal RhD detection in early pregnancy is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to noninvasive fetal RHD genotyping from maternal plasma with real-time PCR-based techniques. We suggest the biosensor techniques could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility. [ABSTRACT FROM AUTHOR]

To study sodium homeostasis, we performed a genome-wide screen for deletion strains that show resistance to NaCl. We identified 34 NaCl-resistant strains. Among them, the largest group that consists of 10 genes related to membrane trafficking and 7 out of 10 genes are ESCRT proteins which are involved in cargo transportation into luminal vesicles within the multivesicular body. All of the ESCRT related mutants which showed sodium resistance also showed defects in vacuole fusion. To further understand the role of the ESCRT pathway in various ion homeostasis, we examined sensitivity of these ESCRT mutants to various cation salts other than NaCl, including KCl, LiCl, CaCl2, CoCl2, MgCl2, NiSO4 and MnCl2. While these ESCRT mutants showed resistance to LiCl, CoCl2 and MgCl2, they showed sensitivity to KCl, CaCl2, NiSO4 and MnCl2. Then we examined sensitivity of these ESCRT mutants to various drugs which are known to inhibit the growth of fission yeast cells. While these ESCRT mutants were more or equally sensitive to most of the drugs tested as compared to the wild-type cells, they showed resistance to some drugs such as tamoxifen, fluorouracil and amiodarone. These results suggest that the ESCRT pathway plays important roles in drug/ion resistance of fission yeast. [ABSTRACT FROM AUTHOR]

Porphyria cutanea tarda (PCT), the most common of the human porphyrias, arises from a deficiency of uroporphyrinogen decarboxylase. Studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with PCT. While these observations implicate HCV infection as a risk factor for PCT pathogenesis, the mechanism of interaction between the virus and porphyrin metabolism is unknown. This study aimed to assess the effect of HCV core protein on intracellular porphyrin metabolism to elucidate the link between HCV infection and PCT. The accumulation and excretion of porphyrins after treatment with 5-aminolevulinic acid, a porphyrin precursor, were compared between cells stably expressing HCV core protein and controls. Cells expressing HCV core protein had lower amounts of intracellular protoporphyrin IX and heme and had higher amounts of excreted coproporphyrin III, the oxidized form of coproporphyrinogen III, compared with controls. These observations suggest that HCV core protein affects porphyrin metabolism and facilitates the export of excess coproporphyrinogen III and/or coproporphyrin III, possibly via porphyrin transporters. Real-time PCR analysis revealed that the presence of HCV core protein increased the mRNA expression of porphyrin exporters ABCG2 and FLVCR1. Western blot analysis showed a higher expression level of FLVCR1, but not ABCG2, as well as a higher expression level of mature ALAS1, which is the rate-limiting enzyme in the heme synthesis pathway, in HCV core protein-expressing cells compared with controls. The data indicate that HCV core protein induced abnormal intracellular porphyrin metabolism, with an over-excretion of coproporphyrin III. These findings may partially account for the susceptibility of HCV-infected individuals to PCT development. [ABSTRACT FROM AUTHOR]

We have developed our original tissue engineering technology “cell sheet engineering” utilizing temperature-responsive culture dishes. The cells are confluently grown on a temperature-responsive culture dish and can be harvested as a cell sheet by lowering temperature without enzymatic digestion. Cell sheets are high-cell-density tissues similar to actual living tissues, maintaining their structure and function. Based on this “cell sheet engineering”, we are trying to create functional cardiac tissues from human induced pluripotent stem cells, for regenerative therapy and in vitro drug testing. Toward this purpose, it is necessary to evaluate the contractility of engineered cardiac cell sheets. Therefore, in the present study, we developed a contractile force measurement system and evaluated the contractility of human iPSC-derived cardiac cell sheet-tissues. By attaching the cardiac cell sheets on fibrin gel sheets, we created dynamically beating cardiac cell sheet-tissues. They were mounted to the force measurement system and the contractile force was measured stably and clearly. The absolute values of contractile force were around 1 mN, and the mean force value per cross-sectional area was 3.3 mN/mm2. These values are equivalent to or larger than many previously reported values, indicating the functionality of our engineered cardiac cell sheets. We also confirmed that both the contractile force and beating rate were significantly increased by the administration of adrenaline, which are the physiologically relevant responses for cardiac tissues. In conclusion, the force measurement system developed in the present study is valuable for the evaluation of engineered cardiac cell sheet-tissues, and for in vitro drug testing as well. [ABSTRACT FROM AUTHOR]

Background: Increased hepatic insulin clearance (HIC) is important in the pathophysiology of type 2 diabetes mellitus (T2DM). The aim of this study is to analyze an effective insulin resistance (IR) index that is minimally affected by HIC. Methods: Our study involved 20 participants with T2DM and 21 healthy participants without diabetes (Non-DM). Participants underwent a meal tolerance test from which plasma glucose, insulin and serum C-peptide immunoreactivity (CPR) were measured, and HOMA-IR and HIC were calculated. Participants then underwent a hyperinsulinemic-euglycemic clamp from which the glucose disposal rate (GDR) was measured. Results: The index CPR-IR = 20/(fasting CPR × fasting plasma glucose) was correlated more strongly with GDR, than was HOMA-IR, and CPR-IR could be used to estimate GDR. In T2DM participants with HIC below the median, HOMA-IR and CPR-IR were equally well correlated with GDR. In T2DM with high HIC, CPR-IR correlated with GDR while HOMA-IR did not. In Non-DM, CPR-IR and HOMA-IR were equally well correlated with GDR regardless of HIC. The mean HIC value in T2DM was significantly higher than that of Non-DM. Conclusions: CPR-IR could be a simple and effective index of insulin resistance for patients with type 2 diabetes that is minimally affected by HIC. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation permeable ion channels expressed primarily in neurons. Here we employed site-directed mutagenesis and electrophysiology to investigate the mechanism of inhibition of ASIC1a by diminazene. This compound inhibits mouse ASIC1a with a half-maximal inhibitory concentration (IC50) of 2.4 μM. At first, we examined whether neutralizing mutations of Glu79 and Glu416 alter diminazene block. These residues form a hexagonal array in the lower palm domain that was previously shown to contribute to pore opening in response to extracellular acidification. Significantly, single Gln substitutions at positions 79 and 416 in ASIC1a reduced diminazene apparent affinity by 6–7 fold. This result suggests that diminazene inhibits ASIC1a in part by limiting conformational rearrangement in the lower palm domain. Because diminazene is charged at physiological pHs, we assessed whether it inhibits ASIC1a by blocking the ion channel pore. Consistent with the notion that diminazene binds to a site within the membrane electric field, diminazene block showed a strong dependence with the membrane potential. Moreover, a Gly to Ala mutation at position 438, in the ion conduction pathway of ASIC1a, increased diminazene IC50 by one order of magnitude and eliminated the voltage dependence of block. Taken together, our results indicate that the inhibition of ASIC1a by diminazene involves both allosteric modulation and blocking of ion flow through the conduction pathway. Our findings provide a foundation for the development of more selective and potent ASIC pore blockers. [ABSTRACT FROM AUTHOR]

Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic β-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic β-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug’s therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion. [ABSTRACT FROM AUTHOR]

Prepartum supplementation with conjugated linoleic acid (CLA) may influence lipolysis and hyperketonemia in dairy cows. The objective of this study was to examine the effect of prepartum CLA supplementation on lactation performance and serum fatty acids (FA) and β-hydroxybutyrate (BHB) in early lactation dairy cows, and secondarily on reproductive performance. Multiparous cows were enrolled in the study at 18 days prior to expected calving date, and randomly assigned 100 g/day of Lutrell Pure (BASF, Ludwigshafen, Germany; 75% FA), providing 10 g/day of each CLA isomer (trans-10 cis-12 and cis-9 trans-11 CLA) or equivalent amount of rumen inert fatty acids as control (78 g/day of Energy Booster 100; Milk Specialties Global, Eden Prairie, MN). Treatments were top dressed daily to individual cows from enrollment to calving and all cows were offered the same ration. Blood samples were collected on the first day of supplementation, 10 days prepartum, and 1, 7, 14, and 30 days postpartum. Hyperketonemia was defined as serum BHB ≥ 1.2 mM. Milk yield was recorded daily until 60 days postpartum and averaged weekly. Milk samples were obtained weekly for component analysis. Prepartum CLA supplementation tended to increase serum concentration of cis-9, trans-11 CLA and increased trans-10, cis-12 CLA prepartum. Cows supplemented with CLA had increased milk protein yield and tended to have increased milk fat yield and milk yield, which together resulted in greater energy content of milk. Cows supplemented with CLA had lower serum FA on day 1 and 7 postpartum and overall lower serum BHB postpartum, which resulted in decreased prevalence of hyperketonemia on day 14 postpartum. There were no differences in body condition score change, other health disorders, or reproductive outcomes by treatment. Together, these findings indicate that prepartum CLA supplementation may be a plausible strategy to positively influence postpartum performance. [ABSTRACT FROM AUTHOR]

Two-pore domain K+ channels (K2P) are responsible for background K+ currents and regulate the resting membrane potential and cellular excitability. Their activity is controlled by a large variety of physicochemical factors and intracellular signaling pathways. The majority of these effects converge on the intracellular C-terminus of the channels, resulting in the modification of the gating at the selectivity filter. Another gating mechanism, the activation gate at the helix bundle crossing is also well documented in other K+ channel families, however, it remains uncertain whether this type of gating is functional in K2P channels. The regulation of TWIK-related spinal cord K+ channel (TRESK) is different from the other K2P channels. Regulatory factors acting via the C-terminus are not known, instead channel activity is modified by the phosphorylation/dephosphorylation of the unusually long intracellular loop between the 2nd and 3rd transmembrane segments. These unique structural elements of the regulation lead us to examine channel gating at the bundle crossing region. Ba2+ was applied to the intracellular side of excised membrane patches and the characteristics of the channel block were determined. We compared the kinetics of the development of Ba2+ block when the channels were phosphorylated (inhibited) or dephosphorylated (activated) and also in different mutants mimicking the two functional states. Neither the phosphorylation/dephosphorylation nor the point mutations influenced the development of Ba2+ block, suggesting that the conformational changes of the bundle crossing region do not contribute to the phosphorylation-dependent gating of TRESK. [ABSTRACT FROM AUTHOR]

The Chloride Channel Accessory (CLCA) protein family was first characterized as regulators of calcium-activated chloride channel (CaCC) currents (ICaCC), but the mechanism has not been fully established. We hypothesized that CLCAs might regulate ICaCC by modulating intracellular calcium levels. In cells stably expressing human CLCA2 or vector, we found by calcium imaging that CLCA2 moderately enhanced intracellular-store release but dramatically increased store-operated entry of calcium upon cytosolic depletion. Moreover, another family member, CLCA1, produced similar effects on intracellular calcium mobilization. Co-immunoprecipitation revealed that CLCA2 interacted with the plasma membrane store-operated calcium channel ORAI-1 and the ER calcium sensor STIM-1. The effect of CLCA2 on ICaCC was tested in HEK293 stably expressing calcium-activated chloride channel TMEM16A. Co-expression of CLCA2 nearly doubled ICaCC in response to a calcium ionophore. These results unveil a new mechanism by which CLCA family members activate ICaCC and suggest a broader role in calcium-dependent processes. [ABSTRACT FROM AUTHOR]

Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1–3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins. [ABSTRACT FROM AUTHOR]

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples. [ABSTRACT FROM AUTHOR]

Age is a major risk factor for diseases caused by ischemic hypoxia, such as stroke and coronary artery disease. Endothelial progenitor cells (EPCs) are the major cells respond to ischemic hypoxia through angiogenesis and vascular remodeling. However, the effect of aging on EPCs and their responses to hypoxia are not well understood. CD34+ EPCs were isolated from healthy volunteers and aged by replicative senescence, which was to passage cells until their doubling time was twice as long as the original cells. Young and aged CD34+ EPCs were exposed to a hypoxic environment (1% oxygen for 48hrs) and their gene expression profiles were evaluated using gene expression array. Gene array results were confirmed using quantitative polymerase chain reaction, Western blotting, and BALB/c female athymic nude mice hindlimb ischemia model. We identified 115 differentially expressed genes in young CD34+ EPCs, 54 differentially expressed genes in aged CD34+ EPCs, and 25 common genes between normoxia and hypoxia groups. Among them, the expression of solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) increased the most by hypoxia in young cells. Gene set enrichment analysis indicated the pathways affected by aging and hypoxia most, including genes “response to oxygen levels” in young EPCs and genes involved “chondroitin sulfate metabolic process” in aged cells. Our study results indicate the key factors that contribute to the effects of aging on response to hypoxia in CD34+ EPCs. With the potential applications of EPCs in cardiovascular and other diseases, our study also provides insight on the impact of ex vivo expansion might have on EPCs. [ABSTRACT FROM AUTHOR]

Combining different metabolomics platforms can contribute significantly to the discovery of complementary processes expressed under different conditions. However, analysing the fused data might be hampered by the difference in their quality. In metabolomics data, one often observes that measurement errors increase with increasing measurement level and that different platforms have different measurement error variance. In this paper we compare three different approaches to correct for the measurement error heterogeneity, by transformation of the raw data, by weighted filtering before modelling and by a modelling approach using a weighted sum of residuals. For an illustration of these different approaches we analyse data from healthy obese and diabetic obese individuals, obtained from two metabolomics platforms. Concluding, the filtering and modelling approaches that both estimate a model of the measurement error did not outperform the data transformation approaches for this application. This is probably due to the limited difference in measurement error and the fact that estimation of measurement error models is unstable due to the small number of repeats available. A transformation of the data improves the classification of the two groups. [ABSTRACT FROM AUTHOR]

Soluble aggregates of α-synuclein, so-called oligomers, are hypothesized to act as neurotoxic species in Parkinson’s disease, Lewy body dementia and multiple systems atrophy, but specific tools to detect these aggregated species are only slowly appearing. We have developed an α-synuclein oligomer ELISA that allows us to detect and compare α-synuclein oligomer levels in different in vivo and in vitro experiments. The ELISA is based on commercially available antibodies and the epitope of the capture antibody MJF14-6-4-2 is folding- and aggregate-dependent and not present on monomers. [ABSTRACT FROM AUTHOR]

Tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase 1 (NtCDPK1) is involved in feedback regulation of the plant hormone gibberellin through the phosphorylation of the transcription factor, REPRESSION OF SHOOT GROWTH (RSG). Previously, Ser-6 and Thr-21 were identified as autophosphorylation sites in NtCDPK1. Autophosphorylation of Ser-6 and Thr-21 not only decreases the binding affinity of NtCDPK1 for RSG, but also inhibits the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreases the phosphorylation efficiency of RSG. We demonstrated that Ser-6 and Thr-21 of NtCDPK1 are phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhances the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These findings suggested that autophosphorylation of Ser-6 and Thr-21 prevents excessive phosphorylation of RSG. In this study, we attempted to determine which autophosphorylation site is responsible for the functional regulation of NtCDPK1. Ser-6 was autophosphorylated within 1 min, whereas Thr-21 required over 5 min to be completely autophosphorylated. Furthermore, we found that Ser-6 and Thr-21 were autophosphorylated by inter- and intramolecular mechanisms, respectively, which may be reflected in the faster autophosphorylation of Ser-6. Although both autophosphorylation sites were involved in the reduction of the binding affinity of NtCDPK1 for RSG and the inhibition of NtCDPK1 homodimerization, autophosphorylation of Ser-6 alone was sufficient to decrease the kinase activity of NtCDPK1 for RSG. These results suggest that autophosphorylation of Ser-6 is important for the rapid reduction of NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role. [ABSTRACT FROM AUTHOR]

Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood. Antibody variants with optimal pH-dependent binding to C5 exhibited little to no TMDD in mice in the presence of human C5. To further enhance the efficiency of FcRn-mediated recycling of the antibody, two additional substitutions were introduced to increase affinity for human FcRn. These substitutions yielded an additional doubling of the t½ of surrogate anti-mouse C5 antibodies with reduced TMDD in transgenic mice expressing the human FcRn. In conclusion, ALXN1210 is a promising new therapeutic candidate currently in clinical development for treatment of patients with PNH and atypical hemolytic uremic syndrome. [ABSTRACT FROM AUTHOR]

Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation. [ABSTRACT FROM AUTHOR]

Purpose: Intravenous immunoglobulin (IVIg) products contain various amounts of dimeric IgG complexes. Current insights into the possible biological activities of these dimers remain controversial, and both immunemodulating and immune-activating effects have been reported. Here, we analyzed the putative immune-activating effects of dimers isolated from IVIg. Methods: Dimers isolated from IVIg were purified by high-performance size-exclusion chromatography (HP-SEC) and tested for the ability to induce neutrophil degranulation in vitro. Results: Dimers isolated from IVIg were found to be incapable of inducing in vitro neutrophil degranulation or complement activation, even at concentrations exceeding those expected to be reached upon administration in patients. These results depend on the removal of artefactual activation by using 0.1 micron filtration and the use of poloxamer to prevent adsorption of IgG onto the solid phase. Conclusions: The data suggest dimeric IgG found in IVIg may bind to Fc-receptors without causing activation. [ABSTRACT FROM AUTHOR]

Background and aims: The feature of blood glucose dynamics in patients with chronic liver disease (CLD) is marked blood glucose fluctuations. However, the detail of blood glucose dynamics is not well known. The aim of the present study was to evaluate glycemic fluctuations by continuous glucose monitoring (CGM). Materials and methods: A total of 105 CLD patients with type 2 diabetes mellitus (T2DM) were enrolled in this study. Various parameters of glycemic variability were evaluated. The association of these parameters with liver functional reserve was examined. The parameters were also evaluated according to glycated hemoglobin A1c (HbA1c) levels. Results and discussion: Data of all patients showed that mean blood glucose (MBG) levels and the difference between highest and lowest blood glucose (ΔBG) increased significantly with worsening of liver functional reserve (P < 0.001 and P = 0.005, respectively). Although many of the cases were being treated for diabetes, postprandial hyperglycemia was seen in 92% of patients. Nocturnal hypoglycemia was seen in 22% of patients. In non-anemic patients with HbA1c levels of < 7.0%, the percentage of patients with mean amplitude of glycemic excursion (MAGE) of ≥ 77.4 mg/dL and that of MBG levels of > 145 mg/dL were higher in liver cirrhosis (LC) patients than in chronic hepatitis (CH) patients. In them, homeostasis model assessment for insulin resistance (HOMA-IR) of > 2.5 and LC were significantly associated with the increase in MAGE. LC was also significantly associated with increased MBG levels. Conclusion: The CGM systems were useful in finding hidden abnormalities of blood glucose fluctuations in CLD patients with T2DM, especially in non-anemic CLD patients with HbA1c levels of < 7.0%. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is an autoimmune condition characterised by pancreatic beta cell destruction and absolute insulin deficiency. The strongest predictor of diabetes complications is glycaemic control and achieving HbA1c ≤ 7.0% is the primary management target. However, standard treatment appears to be lacking and adjunctive strategies require consideration. A systematic review was conducted to examine the effect of low-carbohydrate diets on type 1 diabetes management. Four databases were searched from inception until 28 March 2017: MEDLINE; CINAHL; Cochrane Library; and EMBASE. All primary studies containing a methods section (excluding cross-sectional) were included. Reports had to quantitatively measure the effect(s) of a dietary intervention or observed intake over at least two weeks where carbohydrate is below 45% total energy in adults and/or children with type 1 diabetes. The primary outcome was HbA1c and secondary outcomes were severe hypoglycaemia, total daily insulin, BMI, quality of life and mean daily glucose. Seventy-nine full-text articles were assessed for eligibility and nine were included (two randomised controlled trials, four pre-post interventions, two case-series, one case-report). Eight studies reported a mean change in HbA1c with a low-carbohydrate diet. Of these, four reported a non-significant change (P ≥ 0.05) and three reported statistically significant reductions (P < 0.05). Two studies reported severe hypoglycaemia, five reported total insulin, three reported BMI, and one reported blood glucose. Due to the significant heterogeneity of included studies, an overall effect could not be determined. This review presents all available evidence on low-carbohydrate diets for type 1 diabetes and suggests an urgent need for more primary studies. [ABSTRACT FROM AUTHOR]

Background: Serum anti-glycan antibodies are a promising tool for differential diagnosis, disease stratification and prediction of Crohn’s disease (CD). To investigate possible heritability of the markers we assessed the presence of serum anti-glycan antibodies in affected and unaffected relatives of patients with CD. Methods: Serum samples of 169 IBD patients of the German inflammatory bowel disease (IBD) network (140 CD & 29 Ulcerative colitis (UC)), 349 relatives of CD patients, 63 relatives of UC patients and 46 healthy controls were tested for the presence of anti-glycan antibodies by ELISA in a blinded fashion. Clinical data of the IBD patients and controls were available. Results: A higher proportion of non-affected CD relatives was positive for anti-glycan antibodies compared to healthy subjects. No inheritance of a specific pattern of anti-glycan antibodies could be detected. No difference in marker expression depending on the degree of relationship in the non-affected relatives was noted and the presence of family history did not lead to a difference in marker levels in the affected CD subjects. Conclusions: Non-affected CD relatives had a higher frequency of anti-glycan antibodies compared to healthy subjects. This difference was mild and was found to be true for the overall reactivity to glycan antigens, but not for specific patterns. This may indicate an inherited mechanism resulting in a non-specific increased reactivity to microbial antigens in IBD. [ABSTRACT FROM AUTHOR]

Encapsulation of primary bovine adrenocortical cells in alginate is an efficacious model of a bioartificial adrenal cortex. Such a bioartificial adrenal cortex can be used for the restoration of lost adrenal function in vivo as well as for in vitro modeling of the adrenal microenvironment and for investigation of cell–cell interactions in the adrenals. The aim of this work was the optimization of a bioartificial adrenal cortex, that is the generation of a highly productive, self-regenerating, long-term functioning and immune tolerant bioartificial organ. To achieve this, it is necessary that adrenocortical stem and progenitor cells are present in the bioartificial gland, as these undifferentiated cells play important roles in the function of the mature gland. Here, we verified the presence of adrenocortical progenitors in cultures of bovine adrenocortical cells, studied the dynamics of their appearance and growth and determined the optimal time point for cell encapsulation. These procedures increased the functional life span and reduced the immunogenicity of the bioartificial adrenal cortex. This model allows the use of the luteinizing hormone-releasing hormone (LHRH) agonist triptorelin, the neuropeptide bombesin, and retinoic acid to alter cell number and the release of cortisol over long periods of time. [ABSTRACT FROM AUTHOR]

Bloodstream infections are associated with high morbidity and mortality with rates varying from 10–25% and higher. Appropriate and timely onset of antibiotic therapy influences the prognosis of these patients. It requires the diagnostic accuracy which is not afforded by current gold standards such as blood culture. Moreover, the time from blood sampling to blood culture results is a key determinant of reducing mortality. No established biomarkers exist which can differentiate bloodstream infections from other systemic inflammatory conditions. This calls for studies on biomarkers potential of molecular profiling of plasma as it is affected most by the molecular changes accompanying bloodstream infections. N-glycosylation is a post-translational modification which is very sensitive to changes in physiology. Here we have performed targeted quantitative N-glycoproteomics from plasma samples of patients with confirmed positive blood culture together with age and sex matched febrile controls with negative blood culture reports. Three hundred and sixty eight potential N-glycopeptides were quantified by mass spectrometry and 149 were further selected for identification. Twenty four N-glycopeptides were identified with high confidence together with elucidation of the peptide sequence, N-glycosylation site, glycan composition and proposed glycan structures. Principal component analysis, orthogonal projections to latent structures-discriminant analysis (S-Plot) and self-organizing maps clustering among other statistical methods were employed to analyze the data. These methods gave us clear separation of the two patient classes. We propose high-confidence N-glycopeptides which have the power to separate the bloodstream infections from blood culture negative febrile patients and shed light on host response during bacteremia. Data are available via ProteomeXchange with identifier PXD009048. [ABSTRACT FROM AUTHOR]

17β-estradiol treatment has shown benefit against schizophrenia symptoms, however long-term use may be associated with negative side-effects. Selective estrogen receptor modulators, such as raloxifene and tamoxifen, have been proposed as suitable alternatives to 17β-estradiol. An isomer of 17β-estradiol, 17α-estradiol, is considered less carcinogenic, and non-feminising in males, however little is known about its potential as a treatment for schizophrenia. Moreover, the mechanism underlying the therapeutic action of estrogens remains unclear. We aimed to investigate the ability of these estrogenic compounds to attenuate psychosis-like behaviour in rats. We used two acute pharmacologically-induced assays of psychosis-like behaviour: psychotomimetic drug-induced hyperlocomotion and disruption of prepulse inhibition (PPI). Female Long Evans rats were either intact, ovariectomised (OVX), or OVX and chronically treated with 17β-estradiol, 17α-estradiol, raloxifene or tamoxifen. Only 17β-estradiol treatment attenuated locomotor hyperactivity induced by the indirect dopamine receptor agonist, methamphetamine. 17β-estradiol- and tamoxifen-treated rats showed attenuated methamphetamine- and apomorphine (dopamine D1/D2 receptor agonist)-induced disruption of PPI. Raloxifene-treated rats showed attenuated apomorphine-induced PPI disruption only. Baseline PPI was significantly reduced following OVX, and this deficit was reversed by all estrogenic compounds. Further, PPI in OVX rats was increased following administration of apomorphine. This study confirms a protective effect of 17β-estradiol in two established animal models of psychosis, while tamoxifen showed beneficial effects against PPI disruption. In contrast, 17α-estradiol and raloxifene showed little effect on dopamine receptor-mediated psychosis-like behaviours. This study highlights the utility of some estrogenic compounds to attenuate psychosis-like behaviour in rats, supporting the notion that estrogens have therapeutic potential for psychotic disorders. [ABSTRACT FROM AUTHOR]

The Aedes aegypti mosquito is the principal vector of arboviruses such as dengue, chikungunya, yellow fever, and Zika virus. These arboviruses are transmitted during adult female mosquito bloodfeeding. While these viruses must transverse the midgut to replicate, the blood meal must also reach the midgut to be digested, absorbed, or excreted, as aggregation of blood meal metabolites can be toxic to the female mosquito midgut. The midgut peritrophic matrix (PM), a semipermeable extracellular layer comprised of chitin fibrils, glycoproteins, and proteoglycans, is one such mechanism of protection for the mosquito midgut. However, this structure has not been characterized for adult female Ae. aegypti. We conducted a mass spectrometry based proteomic analysis to identify proteins that comprise or are associated with the adult female Ae. aegypti early midgut PM. Altogether, 474 unique proteins were identified, with 115 predicted as secreted. GO-term enrichment analysis revealed an abundance of serine-type proteases and several known and novel intestinal mucins. In addition, approximately 10% of the peptides identified corresponded to known salivary proteins, indicating Ae. aegypti mosquitoes extensively swallow their own salivary secretions. However, the physiological relevance of this remains unclear, and further studies are needed to determine PM proteins integral for midgut protection from blood meal derived toxicity and pathogen protection. Finally, we describe substantial discordance between previously described transcriptionally changes observed in the midgut in response to a bloodmeal and the presence of the corresponding protein in the PM. Data are available via ProteomeXchange with identifier PXD007627. [ABSTRACT FROM AUTHOR]

Amyloid aggregates of the protein α-synuclein (αS) called Lewy Bodies (LB) and Lewy Neurites (LN) are the pathological hallmark of Parkinson’s disease (PD) and other synucleinopathies. We have previously shown that high extracellular αS concentrations can be toxic to cells and that neurons take up αS. Here we aimed to get more insight into the toxicity mechanism associated with high extracellular αS concentrations (50–100 μM). High extracellular αS concentrations resulted in a reduction of the firing rate of the neuronal network by disrupting synaptic transmission, while the neuronal ability to fire action potentials was still intact. Furthermore, many cells developed αS deposits larger than 500 nm within five days, but otherwise appeared healthy. Synaptic dysfunction clearly occurred before the establishment of large intracellular deposits and neuronal death, suggesting that an excessive extracellular αS concentration caused synaptic failure and which later possibly contributed to neuronal death. [ABSTRACT FROM AUTHOR]

Rationale: Aspartame (L-aspartyl phenylalanine methyl ester) is a non-nutritive sweetener (NNS) approved for use in more than 6000 dietary products and pharmaceuticals consumed by the general public including adults and children, pregnant and nursing mothers. However a recent prospective study reported a doubling of the risk of being overweight amongst 1-year old children whose mothers consumed NNS-sweetened beverages daily during pregnancy. We have previously shown that chronic aspartame (ASP) exposure commencing in utero may detrimentally affect adulthood adiposity status, glucose metabolism and aspects of behavior and spatial cognition, and that this can be modulated by developmental N-methyl-D-aspartate receptor (NMDAR) blockade with the competitive antagonist CGP 39551 (CGP). Since glucose homeostasis and certain aspects of behavior and locomotion are regulated in part by the NMDAR-rich hypothalamus, which is part of the hypothalamic-pituitary-adrenal- (HPA) axis, we have elected to examine changes in hypothalamic and adrenal gene expression in response to ASP exposure in the presence or absence of developmental NMDAR antagonism with CGP, using Affymetrix microarray analysis. Results: Using 2-factor ANOVA we identified 189 ASP-responsive differentially expressed genes (DEGs) in the adult male hypothalamus and 2188 in the adrenals, and a further 23 hypothalamic and 232 adrenal genes significantly regulated by developmental treatment with CGP alone. ASP exposure robustly elevated the expression of a network of genes involved in hypothalamic neurosteroidogenesis, together with cell stress and inflammatory genes, consistent with previous reports of aspartame-induced CNS stress and oxidative damage. These genes were not differentially expressed in ASP mice with CGP antagonism. In the adrenal glands of ASP-exposed mice, GABA and Glutamate receptor subunit genes were amongst those most highly upregulated. Developmental NMDAR antagonism alone had less effect on adulthood gene expression and affected mainly hypothalamic neurogenesis and adrenal steroid metabolism. Combined ASP + CGP treatment mainly upregulated genes involved in adrenal drug and cholesterol metabolism. Conclusion: ASP exposure increased the expression of functional networks of genes involved in hypothalamic neurosteroidogenesis and adrenal catecholamine synthesis, patterns of expression which were not present in ASP-exposed mice with developmental NMDAR antagonism. [ABSTRACT FROM AUTHOR]

A disproportional increase of circulating GAD65 within hours from an intraportal islet allotransplantation has been validated as biomarker of beta cell loss and poor functional outcome. More sensitive assays are, however, needed to allow detection of episodes of subtle beta cell loss during late-stage graft rejection or in the peri-onset period of type 1 diabetes. We applied the same sandwich monoclonal antibody couple reactive towards the C- and N-terminus of GAD65 on three advanced immunoassay platforms—the Cytometric Bead Array (CBA, Becton, Dickinson and Company), ElectroChemiLuminescence ImmunoAssay (ECLIA, Meso Scale Discovery) and digital ELISA technology (Single Molecule Array—SIMOA, Quanterix. We then compared analytical performance (linearity, imprecision, limit of detection and functional sensitivity), correlation of results, and practicality. All evaluated techniques showed linearity up to at least 500 ng/dL (76.9 pmol/L). SIMOA achieved the lowest imprecision. The 3 platforms correlate well with each other and could all detect subpicomolar concentrations of GAD65 in plasma, but only SIMOA and CBA could quantify down to that range. SIMOA can achieve the highest sample throughput. The three methods tested allow sensitive detection of GAD65, but SIMOA appears best suited for automated quantification of subpicomolar concentrations. [ABSTRACT FROM AUTHOR]

Detecting life-threatening common dyshemoglobins such as carboxyhemoglobin (COHb, resulting from carbon monoxide poisoning) or methemoglobin (MetHb, caused by exposure to nitrates) typically requires a laboratory CO-oximeter. Because of cost, these spectrophotometer-based instrument are often inaccessible in resource-poor settings. The aim of this study was to determine if an inexpensive pocket infrared spectrometer and smartphone (SCiO®Pocket Molecular Sensor, Consumer Physics Ltd., Israel) accurately detects COHb and MetHb in single drops of blood. COHb was created by adding carbon monoxide gas to syringes of heparinized blood human or cow blood. In separate syringes, MetHb was produced by addition of sodium nitrite solution. After incubation and mixing, fractional concentrations of COHb or MetHb were measured using a Radiometer ABL-90 Flex® CO-oximeter. Fifty microliters of the sample were then placed on a microscope slide, a cover slip applied and scanned with the SCiO spectrometer. The spectrograms were used to create simple linear models predicting [COHb] or [MetHb] based on spectrogram maxima, minima and isobestic wavelengths. Our model predicted clinically significant carbon monoxide poisoning (COHb ≥15%) with a sensitivity of 93% and specificity of 88% (regression r2 = 0.63, slope P<0.0001), with a mean bias of 0.11% and an RMS error of 21%. Methemoglobinemia severe enough to cause symptoms (>20% MetHb) was detected with a sensitivity of 100% and specificity of 71% (regression r2 = 0.92, slope P<0.001) mean bias 2.7% and RMS error 21%. Although not as precise as a laboratory CO-oximeter, an inexpensive pocket-sized infrared scanner/smartphone detects >15% COHb or >20% MetHb on a single drop of blood with enough accuracy to be useful as an initial clinical screening. The SCiO and similar relatively low cost spectrometers could be developed as inexpensive diagnostic tools for developing countries. [ABSTRACT FROM AUTHOR]

Small therapeutic peptides represent a promising field for the treatment of pathologies such as cardiac diseases. However, the lack of proper target-selective carriers hampers their translation towards a potential clinical application. Aptamers are cell-specific carriers that bind with high affinity to their specific target. However, some limitations on their conjugation to small peptides and the functionality of the resulting aptamer-peptide chimera exist. Here, we generated a novel aptamer-peptide chimera through conjugation of the PDGFRβ-targeting Gint4.T aptamer to MP, a small mimetic peptide that via targeting of the Cavβ2 subunit of the L-type calcium channel (LTCC) can recover myocardial function in pathological heart conditions associated with defective LTCC function. The conjugation reaction was performed by click chemistry in the presence of N,N,N’,N’,N”-pentamethyldiethylenetriamine as a Cu (I) stabilizing agent in a DMSO-free aqueous buffer. When administered to cardiac cells, the Gint4.T-MP aptamer-peptide chimera was successfully internalized in cells, allowing the functional targeting of MP to LTCC. This approach represents the first example of the use of an internalizing aptamer for selective delivery of a small therapeutic peptide to cardiac cells. [ABSTRACT FROM AUTHOR]

The role of vitamin D receptor (VDR) in immune responses has been broadly studied and it has been shown that activated VDR alters the levels of some interleukins (ILs). In this study, we studied the opposite, i.e. whether 13 selected pro-inflammatory and anti-inflammatory ILs influence the transcriptional activity of human VDR. The experimental models of choice were two human stably transfected gene reporter cell lines IZ-VDRE and IZ-CYP24, which were designed to evaluate the transcriptional activity of VDR. The gene reporter assays revealed inhibition of calcitriol-induced luciferase activity by IL-4 and IL-13, when 1 ng/mL of these two compounds decreased the effect of calcitriol down to 60% of the control value. Consistently, calcitriol-induced expression of CYP24A1 mRNA was also significantly decreased by IL-4 and IL-13. The expression of VDR and CYP27B1 mRNAs was not influenced by any of the 13 tested ILs. These data suggest possible cross-talk between the VDR signalling pathway and IL-4- and IL-13-mediated cell signalling. [ABSTRACT FROM AUTHOR]

The fine structures of Fc N-glycan modulate the biological functions and physicochemical properties of antibodies. By remodeling N-glycan to obtain a homogeneous glycoform or chemically modified glycan, antibody characteristics can be controlled or modified. Such remodeling can be achieved by transglycosylation reactions using a mutant of endoglycosidase from Streptococcus pyogenes (Endo-S) and glycan oxazoline. In this study, we generated improved mutants of Endo-S by introducing additional mutations to the D233Q mutant. Notably, Endo-S D233Q/Q303L, D233Q/E350Q, and several other mutations resulted in transglycosylation efficiencies exceeding 90%, with a single-digit donor-to-substrate ratio of five, and D233Q/Y402F/D405A and several other mutations resulted in slightly reduced transglycosylation efficiencies accompanied by no detectable hydrolysis activity for 48 h. We further demonstrated that the combined use of mutants of Endo-S with Endo-M or Endo-CC, endoglycosidases from Mucor hiemalis and Coprinopsis cinerea, enables one-pot transglycosylation from sialoglycopeptide to antibodies. This novel reaction enables glycosylation remodeling of antibodies, without the chemical synthesis of oxazoline in advance or in situ. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30–40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is associated with sensory abnormalities, including exacerbated responses to painful (hyperalgesia) or non-painful (allodynia) stimuli. These abnormalities are symptoms of diabetic peripheral neuropathy (DPN), which is the most common complication that affects approximately 50% of diabetic patients. Yet, the underlying mechanisms linking hyperglycemia and symptoms of DPN remain poorly understood. The transient receptor potential vanilloid 1 (TRPV1) channel plays a central role in such sensory abnormalities and shows elevated expression levels in animal models of diabetes. Here, we investigated the function of TRPV1 channels in sensory neurons cultured from the dorsal root ganglion (DRG) of neonatal mice, under control (5mM) and high glucose (25mM) conditions. After maintaining DRG neurons in high glucose for 1 week, we observed a significant increase in capsaicin (CAP)-evoked currents and CAP-evoked depolarizations, independent of TRPV1 channel expression. These functional changes were largely dependent on the expression of the receptor for Advanced Glycation End-products (RAGE), calcium influx, cytoplasmic ROS accumulation, PKC, and Src kinase activity. Like cultured neurons from neonates, mature neurons from adult mice also displayed a similar potentiation of CAP-evoked currents in the high glucose condition. Taken together, our data demonstrate that under the diabetic condition, DRG neurons are directly affected by elevated levels of glucose, independent of vascular or glial signals, and dependent on RAGE expression. These early cellular and molecular changes to sensory neurons in vitro are potential mechanisms that might contribute to sensory abnormalities that can occur in the very early stages of diabetes. [ABSTRACT FROM AUTHOR]

It has been estimated for dengue infection that the global population at risk is 3.5 billion people, which makes dengue an important public health problem. The causative agents of dengue are dengue viruses. For dengue virus replication, the dengue virus NS5 protein is of special importance as it has several enzyme activities important for viral replication. Previous reports of phosphorylation and SUMOylation of dengue NS5 have shown these protein modifications have important consequences for NS5 functions. In this report we identify glutathionylation, another reversible post translation modification that impacts on NS5 enzyme activity. Using dengue virus infected cells we employed specific antibodies and mass spectrometry to identify 3 cysteine residues of NS5 protein as being glutathionylated. Glutathionylation is a post translational protein modification where glutathione is covalently attached to a cysteine residue. We showed glutathionylation occurs on 3 conserved cysteine residues of dengue NS5. Then we generated two flavivirus recombinant full length proteins, dengue NS5 and Zika NS5, to characterize two of the NS5 enzyme activities, namely, guanylyltransferase and RNA-dependent RNA polymerase activities. We show glutathionylation of dengue and Zika NS5 affects enzyme activities of the two flavivirus proteins. The data suggests that glutathionylation is a general feature of the flavivirus NS5 protein and the modification has the potential to modulate several of the NS5 enzyme functions. [ABSTRACT FROM AUTHOR]

Background: There is growing evidence that the accumulation of protein- bound uremic retention solutes, such as indoxyl sulfate, p-cresyl sulfate and kynurenic acid, play a role in the accelerated cardiovascular disease seen in patients undergoing chronic hemodialysis. Protein-binding, presumably to albumin, renders these solutes poor-dialyzable. We previously observed that the free fraction of indoxyl sulfate was markedly reduced at the end of hemodialysis. We hypothesized that solute binding might be pH-dependent and attributed the changes in free solute concentration to the higher serum pH observed at the end of standard hemodialysis with dialysis buffer bicarbonate concentration greater than 35 mmol/L. We observed that acidification of uremic plasma to pH 6 in vitro greatly increased the proportion of freeIS. Methods: We tested our hypothesis by reducing the dialysate bicarbonate buffer concentration to 25 mmol/L for the initial half of the hemodialysis treatment (“isohydric dialysis”). Eight stable hemodialysis patients underwent “isohydric dialysis” for 90 minutes and then were switched to standard buffer (bicarbonate = 37mmol/L). A second dialysis, 2 days later, employed standard buffer throughout. Results: We found a clearcut separation of blood pH and bicarbonate concentrations after 90 minutes of “isohydric dialysis” (pH = 7.37, bicarbonate = 22.4 mmol/L) and standard dialysis (pH = 7.49, bicarbonate = 29.0 mmol/L). Binding affinity varied widely among the 10 uremic retention solutes analyzed. Kynurenic acid (0.05 free), p-cresyl sulfate (0.12 free) and indoxyl sulfate (0.13 free) demonstrated the greatest degree of binding. Three solutes (indoxyl glucuronide, p-cresyl glucuronide, and phenyl glucuronide) were virtually unbound. Analysis of free and bound concentrations of uremic retention solutes confirmed our prediction that binding of solute is affected by pH. However, in a mixed models analysis, we found that the reduction in total uremic solute concentration during dialysis accounted for a greater proportion of the variation in free concentration, presumably an effect of saturation binding to albumin, than did the relatively small change in pH produced by isohydric dialysis. The effect of pH on binding appeared to be restricted to those solutes most highly protein-bound. The solutes most tightly bound exhibited the lowest dialyzer clearances. An increase in dialyzer clearance during isohydric and standard dialyses was statistically significant only for kynurenic acid. Conclusion: These findings provide evidence that the binding of uremic retention solutes is influenced by pH. The effect of reducing buffer bicarbonate concentration (“isohydric dialysis:”), though significant, was small but may be taken to suggest that further modification of dialysis technique that would expose blood to a greater decrease in pH would lead to a greater increase the free fraction of solute and enhance the efficacy of hemodialysis in the removal of highly protein-bound uremic retention solutes. [ABSTRACT FROM AUTHOR]

Insulin-degrading enzyme (IDE) is an atypical zinc-metalloendopeptidase that hydrolyzes insulin and other intermediate-sized peptide hormones, many of which are implicated in skin health and wound healing. Pharmacological inhibitors of IDE administered internally have been shown to slow the breakdown of insulin and thereby potentiate insulin action. Given the importance of insulin and other IDE substrates for a variety of dermatological processes, pharmacological inhibitors of IDE suitable for topical applications would be expected to hold significant therapeutic and cosmetic potential. Existing IDE inhibitors, however, are prohibitively expensive, difficult to synthesize and of undetermined toxicity. Here we used phage display to discover novel peptidic inhibitors of IDE, which were subsequently characterized in vitro and in cell culture assays. Among several peptide sequences tested, a cyclic dodecapeptide dubbed P12-3A was found to potently inhibit the degradation of insulin (Ki = 2.5 ± 0.31 μM) and other substrates by IDE, while also being resistant to degradation, stable in biological milieu, and highly selective for IDE. In cell culture, P12-3A was shown to potentiate several insulin-induced processes, including the transcription, translation and secretion of alpha-1 type I collagen in primary murine skin fibroblasts, and the migration of keratinocytes in a scratch wound migration assay. By virtue of its potency, stability, specificity for IDE, low cost of synthesis, and demonstrated ability to potentiate insulin-induced processes involved in wound healing and skin health, P12-3A holds significant therapeutic and cosmetic potential for topical applications. [ABSTRACT FROM AUTHOR]