The mobile phase consists of a mixture of 10 mM ammonium formate and 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). To quantify blonanserin, blonanserin C, and IS, multiple reaction monitoring (MRM) was performed in positive ESI mode. The calibration curve was linear in the concentration range of 0.012–5.78 ng·mL−1 for blonanserin and 0.023–11.57 ng·mL−1 for blonanserin C (r2 > 0.9990). The intra- and interday precision of three quality control (QC) levels in plasma were less than 7.5%. Finally, the current simple, sensitive, and accurate LC-MS/MS method was successfully applied to investigate the pharmacokinetics of blonanserin and blonanserin C in healthy Chinese volunteers.A highly sensitive, simple, and rapid liquid chromatography tandem mass spectrometry method to simultaneously determine blonanserin and blonanserin C in human plasma with AD-5332 as internal standard (IS) was established. A simple direct protein precipitation method was used for the sample pretreatment, and chromatographic separation was performed on a Waters XBridge C Blonanserin AD-5423) is a novel oral atypical antipsychotic agent with relatively selective potent activity of blocking dopamine D2 and serotonin 5-HT2A receptors, which is expected to have lower incidence of adverse events than other antipsychotics –3. Clini is a novBlonanserin has been sold in Japan and Korea and will be commercialized in China and some other countries in the world, indicated for the treatment of schizophrenia. With the development of clinical trials and clinical use on blonanserin throughout the world, it is necessary to establish a simple, sensitive, and accurate method for the quantification of blonanserin and its metabolite in human plasma. High-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-FLD) , GC-MS , HPLC-MSIn this paper, a sensitive HPLC-MS/MS method for the simultaneous determination of blonanserin and its major metabolite (blonanserin C) in human plasma was established, which has a simple direct protein precipitation method for the sample pretreatment and has a higher extraction recovery (>85%) and a lower LOQ than the existing studies. It has been successfully applied to a pharmacokinetic study in healthy Chinese volunteers.batch number 20110124, purity 99.8%), blonanserin C , and AD-5332   were supplied by the Medical Research Institute Co., Ltd., Jiangsu Stockhausen, China. Their chemical structures are shown in Blonanserin  coupled to an API 4000 triple-quadrupole mass spectrometer  via a electrospray ionization (ESI) interface was used for separation and detection. Data acquisition was performed with Analyst 1.5.2 software (AB Sciex).8  column. A guard column  was placed prior to the analytical column. A binary mobile phase consisting of a mixture of 10 mM ammonium formate and 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) was used at a flow rate of 0.6 mL·min−1. The temperature of column was 35°C, and the injection volume was 10 μL. Gradient elution was performed according to the following elution program: 0 min, 20% A/80% B; 2 min, 10% A/90% B; 4 min 30 sec, 10% A/90% B; 4 min 35 sec, 5% A/95% B; 5 min 30 sec, 5% A/95% B; 5 min 35 sec, 20% A/80% B.Separation was performed using a Waters XBridge CAll measurements were carried out with mass spectrometer operated under the positive ESI mode. The multiple reaction monitoring (MRM) transitions (Q1 and Q3) and declustering potential (DP), collision energy (CE), and collision exit potential (CXP) of blonanserin, blonanserin C, and IS are shown in −1 for blonanserin, 0.771 mg·mL−1 for blonanserin C, and 0.525 mg·mL−1 for IS. Working solutions used for spiking plasma were all freshly prepared by diluting the stock solutions with methanol to appropriate concentrations.Accurately weighed solid portions of standards  were dissolved in methanol to prepare stock solutions separately as follows: 1.156 mg·mL−1 for blonanserin and 0.023–11.57 ng·mL−1 for blonanserin C and processed as described in the preparation of sample solution  and 20 μL IS (5.25 ng·mL−1) solution. After a thorough vortex mixing for 30 s, and mixtures were precipitated with 400 μL acetonitrile, vortex-mixed for 30 s, centrifuged at 13000 rpm for 5 min, and then 10 μL of supernatant was injected into the HPLC-MS/MS system.After frozen human plasma samples were thawed at ambient temperature and adequately vortexed, a total of 200 −1 for blonanserin and 0.023 to 11.57 ng·mL−1 for blonanserin C. Calibration curves were constructed from the peak-area ratios of each analyte to IS versus plasma concentrations using a 1/x weighted least squares linear regression model.The selectivity, linearity, sensitivity, precision, and extraction recovery of the method and the stability of blonanserin and blonanserin C in plasma samples were assessed and investigated. To evaluate selectivity, drug-free plasma samples from 6 individuals were analyzed to check for the presence of any interfering peaks at the elution times of blonanserin, blonanserin C, and IS. The calibration curves were constructed using 7 standards ranging in concentration from 0.012 to 5.78 ng·mLn = 6): LLOQ (0.012 ng·mL−1 and 0.023 ng·mL−1), low (0.023 ng·mL−1 and 0.046 ng·mL−1), medium (0.23 ng·mL−1 and 0.46 ng·mL−1), and high (4.62 ng·mL−1 and 9.25 ng·mL−1) for blonanserin and blonanserin C, respectively. The relative standard deviation (RSD %) was calculated.The intraday and interday precisions were evaluated by assessing QC samples at the following concentrations  in a pharmacokinetic study, which has been approved by the ethics committee. The volunteers were given three dosages with single oral administration: low, 4 mg/volunteer, middle, 8 mg/volunteer, and high, 12 mg/volunteer, respectively. The study was conducted from low dosage to high dosage, with one week washout period between two dosages. Heparinized blood samples (5 mL) were collected at the following times: before (0.0 h) and at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, 16.0, 24.0, 36.0, 48.0, and 72.0 h after dosing. The blood samples were centrifuged at 4000 rpm for 10 min, and plasma samples were separated and stored at −80°C until analyzed.−1, 77 ng·mL−1, and 104 ng·mL−1 in HPLC solvent B for blonanserin, blonanserin C, and IS, resp.). ESI source with positive or negative ionization has been tested for the determination, and the results revealed that blonanserin, blonanserin C, and IS were more sensitive in positive ionization mode. Based on the Q1 and product ion scan  column was chosen in the present study for its good peak symmetry. Different mobile phases (acetonitrile-water and methanol water with different concentrations of modifiers: formic acid and ammonium formate) were examined to obtain efficient chromatography and relatively short run time for the analytes and IS. It was found that the addition of ammonium formate and formic acid could remarkably improve the peak symmetry and sensitivity of blonanserin, blonanserin C, and IS. When 0.1% formic acid in methanol was used as the organic phase, the sensitivity of blonanserin and blonanserin C was further improved. Therefore, the mobile phase was selected as 0.1% formic acid in methanol mixture of 10 mM ammonium formate and 0.1% formic acid in water to achieve higher sensitivity and less interference of other components in the plasma. The retention time for blonanserin, blonanserin C, and IS was 4.08, 4.09, and 4.22 min, respectively  least squares linear regression analysis with coefficient of determination r2 > 0.9990. Typical equations for the calibration curve were as follows: Y = (2.04 ± 0.18)X + (0.0121 ± 0.0039) and Y = (1.00 ± 0.05)X + (0.0057 ± 0.0032)  (n = 6) for blonanserin and blonanserin C, respectively, where X represents the plasma concentration of blonanserin or blonanserin C (ng/mL) and Y represents the ratio of blonanserin or blonanserin C peak area to that of IS.The standard calibration curves for spiked human plasma containing blonanserin and blonanserin C were linear over the range of 0.012~5.78 ng·mLS/N ratios were higher than 10. The LOQ of this is sufficient for the pharmacokinetic study of blonanserin and blonanserin C following an oral administration.The LOQ under the optimized conditions was 0.012 ng/mL and 0.023 ng/mL for blonanserin and blonanserin C, which were judged from the fact that the precision was less than 20% and the The RE and ME (%) of blonanserin and blonanserin C in human plasma of this method were determined in three concentration levels (six replicates per level). Results summarized in A” is the peak area obtained in neat solution standards; “B” is the corresponding peak area for standards spiked after extraction into plasma extracts; “C” is the peak area for standards spiked before extraction.“QC samples at three concentration levels were calculated over three validation runs (once a day). Six replicates of each QC level were determined in each run. The extraction recovery (>85%) and precision (<7.2%) for blonanserin and blonanserin C were all better than in references , 15, 17,The results of stability studies are shown in n = 6) plasma concentration versus time profiles for blonanserin and blonanserin C is depicted in The validated method was proved to be simple and sensitive enough to determine the concentration up to 72 h after dose of blonanserin tablets to 6 healthy Chinese volunteers. The precision and accuracy of the calibration and QC samples were well within the acceptable limits . The phaThe results from the pharmacokinetic trial in humans showed that after oral administration of the drug, the pharmacokinetics of blonanserin and blonanserin C within the used dosage range  were in accordance with linear pharmacokinetic characteristics. A simple and sensitivity direct protein precipitation-LC/MS/MS method for the quantification of blonanserin and blonanserin C in human plasma was developed. Method validation has been demonstrated by a variety of tests for selectivity, linearity, sensitivity, precision, recovery, and stability. This method is attractive for the pharmacokinetic analysis of blonanserin and blonanserin C, because of its simple sample processing steps, high analytical sensitivity, and accuracy.


Estrogenic functions in regulating behavioral states such as motivation, mood, anxiety, and cognition are relatively well documented in female humans and animals. In males, however, although the entire enzymatic machinery for producing estradiol and the corresponding receptors are present, estrogenic functions have been largely neglected. Therefore, and as a follow-up study to previous research, we sub-chronically applied a specific estrogen receptor α (ERα) antagonist in young male rats before and during a spatial learning task (holeboard). The male rats showed a dose-dependent increase in motivational, but not cognitive, behavior. The expression of hippocampal steroid receptor genes, such as glucocorticoid (GR), mineralocorticoid (MR), androgen (AR), and the estrogen receptor ERα but not ERβ was dose-dependently reduced. The expression of the aromatase but not the brain-derived neurotrophic factor (BDNF) encoding gene was also suppressed. Reduced gene expression and increased behavioral performance converged at an antagonist concentration of 7.4 µmol. The hippocampal and blood serum hormone levels  did not differ between the experimental groups and controls. We conclude that steroid receptors (and BDNF) act in a concerted, network-like manner to affect behavior and mutual gene expression. Therefore, the isolated view on single receptor types is probably insufficient to explain steroid effects on behavior. The steroid network may keep motivation in homeostasis by supporting and constraining the behavioral expression of motivation. In recent years, estrogen receptors have increasingly been identified as involved in modulating motivation and cognition in female human development, postmenopausal mood disorders, and corresponding animal models Hippocampal synthesis of estradiol in male rats is realized by the enzyme aromatase that converts testosterone into estradiol. Aromatase as well as synaptic and nuclear ERα have been identified in all subregions of the hippocampus and in the dentate gyrus. Estradiol can induce rapid upregulation of spine number and fast modulation of hippocampal synaptic plasticity In a previous study Therefore, while the MPP binds to cytosolic ER, we cannot rule out that membrane-bound receptors are blocked as well. The blood serum and hippocampal stress and sex hormone concentrations were also measured. In addition, aromatase and BDNF gene expression was measured. The latter was considered because rat hippocampal BDNF has been reported to interact with estrogens such that the administration of estradiol enhances hippocampal BDNF-mRNA Based on our previous data, we hypothesized reducing the motivation of males treated with an ERα antagonist, whereas the spatial cognitive aspect should be unaffected. The latter hypothesis was confirmed, whereas MPP treatment resulted in increased motivation. Possible reasons for these results are discussed, and advanced hypotheses are outlined.All experiments were performed in accordance with the European Communities Council Directive of 24th Nov. 1986 (86/609/EEC) and the German guidelines for the care and use of animals in laboratory research. The experimental protocols were approved by the ethics committee of Saxony-Anhalt. All efforts were made to reduce the number of rats used in this study and their suffering.ad libitum. After the implantation of a cannula when the rats were 7 weeks old, they were transferred to individual cages (40 cm×25 cm×18 cm).Wistar rats from the institute’s breeding colony were weaned at post-natal day 21 and then housed in groups of 5 males in standard cages (59 cm×38 cm×25 cm). They were maintained under a 12 h: 12 h light regimen with lights on at 6∶00 a.m.; the ground was covered with commercial bedding material  and food pellets , and tap water was given 5,80 = 1.44, p = 0.219), day 0 , day1,and day 2 .Seven-week-old rats were anesthetized with Nembutal . An intra-cerebroventricular (i.c.v.) cannula was stereotactically implanted in the lateral ventricle of the right hemisphere . The animals were allowed to recover from surgery for at least 3 days. After recovery, the animals were mildly food deprived for 4 days ; food deprivation persisted during the training procedure. Body weights between groups were not statistically different at all time points:day −3  with 16 regularly arranged holes, 7.5 cm in diameter and 7 cm deep. The board was surrounded with Plexiglas walls 50 cm high. The walls were covered with white paper on the outside and marked with a different black cue at each side. Four out of 16 holes were baited in a fixed pattern with standard food pellets .Path trajectories were recorded with the tracking system BiObserve Viewer software (Version 3.0.0.92), and behavioral parameters were measured. The head-dips of the animals were registered by photobeams at the middle of each hole. The photobeam signals were detected and counted by the holeboard-plugIn of the BiObserve Viewer software.Thus, the time to find all pellets , the average velocity , the number of pellets found, the hole dips/s, the total hole dips, the mean distance to the wall , the working memory errors , and the reference memory errors (visiting an unbaited hole) were recorded. We calculated an index of performance for both types of memory. This calculation helps to divide the animals that made no errors, because they did not move at all on the holeboard from those that made no errors because they performed the task correctly. The index was calculated as total visits/. Thus, an index of 0 indicates no hole visit at all (0 errors because of 0 attempts were set to 0), 0.5 the number of hole visits equals the number of errors, and 1 indicates no error (four dips coincides with four pellets found).Animals stayed in their home cages in the testing room during the entire experiment. Animals were transferred from the cage to the test arena for each training trial. A trial was automatically stopped after 2 min or when the animal had found all 4 pellets. All experimental animals were familiarized with the test set-up 1 day before training. Then they received spatial training on a fixed pattern of baited holes over 10 trials , with a 15 min inter-trial interval Animals were tested for 5 min in each test twice: open field at 9∶00 a.m. followed by the elevated plus test 1 h later. This procedure was repeated 24 h later. Animals stayed in the test room overnight. For these tests, we used non-food-deprived animals. The arenas were cleaned after each trial with 20% ethanol.The holeboard served also as an open-field arena, with the exception that the floor with the holes was covered with a black plastic plate. The arena was, via the tracking system, divided into different zones: four corner zones (25 cm×25 cm), four wall zones (25 cm×50 cm), and one center zone (50 cm×50 cm). The percentage of time spent in each zone, the mean velocity, and the track length were measured.The elevated plus maze consisted of two closed and two open arms of black plastic at a height of 80 cm above floor. The arms were 10 cm wide and 50 cm long. The closed arms were equipped with black walls (30 cm high). From a center arena (10 cm×10 cm), the animals started to explore the maze. The percentage of time spent in each arm, the mean velocity, and the track length were measured via the BiObserve Viewer software.The ERα antagonist MPP binds to extranuclear receptors with very high specificity Similar as in previous studies, we investigated the right hippocampus  as well as the hormone levels in blood serum.The rats used for the open-field and elevated plus maze tests were treated with 7.4 µmol MPP i.c.v. (the most effective dose in the holeboard task) or vehicle. In accordance with the holeboard procedure, daily injections started 5 days before the first test day and continued during the two test days.Animals were decapitated 15 min after the last trial, and trunk blood was collected in vials containing clot activator . Tissue from the right hippocampus was rapidly dissected and frozen (−20°C) until measurements. Samples were homogenized , diluted  to reach a final volume of 25 µl/mg tissue weight, and centrifuged . The supernatant was stored at −20°C. For the hormone assays, we used the enzyme-linked immunosorbent assay (ELISA). Briefly, samples were thawed and diluted . Samples and standards were applied in duplicate. OD values were measured at 450 nm in a micro-plate reader (Thermo Scientific MultiSkan FC ELISA Reader) and calculated via a standard four-parameter logistics plot. For the testosterone assay , the limit of detection (LOD) was 2.0 pg/ml, and the intra-assay and inter-assay coefficients of variation were 8.2% and 5.5%, respectively. The estradiol assay  had an LOD of 0.4 pg/ml. The intra-assay and inter-assay coefficients of variation were a maximum of 9.9% and 11.1%, respectively. For the corticosterone kit (Corticosterone ELISA by IBL Hamburg), the LOD was 1.631 nmol/L, and the intra-assay and inter-assay coefficients of variations were 2.77% and 6.14%, respectively. Randomly chosen subsets of the animals used in the behavioral experiments were analyzed.Tissue from the right hippocampus, added with an mRNA stabilizing agent , was stored at −80°C. For the analysis, mRNA was isolated  and transcribed to cDNA .AR: NM_012502.1 (Rn00560747_m1); ERα/ESR1: NM_012689.1 (Rn01430443_m1); ERβ/ESR2: NM_012754.1 (Rn00562610_m1); GR/NR3C1: NM_012576.2 (Rn01405584_m1); MR/NR3C2: NM_037263.1 (Rn00565562_m1); BDNF/BDNF: BC_087634.1 (Rn01484928_m1); Aromatase/Cyp19: NM_017085.2 (Rn01422547_m1); PGK/PGK: NM_053291.3 (Rn00821429_g1); HPRT/HPRT: NM_012583.2 (Rn01527840_m1);TaqMan Universal PCR Master Mix). Assays were performed in triplicate. As endogenous control, we used rat GAPDH (Vic/MGB Probe), while HPRT and PGK served as reference genes. All reagents were from Applied Biosystems. Differences in relative gene expression were calculated with the comparative cycle threshold method Gene expression analysis was performed with a StepOnePlus machine from Applied Biosystems. TaqMan Gene Expression assays with the classification “m” were used exclusively, whose primers (Life Technologies) span an exon junction and detect only genomic DNA, except PGK, where no assay with the classification “m” was available . The distribution of all data was tested with the Shapiro-Wilk-test. Differences in gene expression data and hormone concentrations were analyzed with the univariate general linear model (GLM) with the treatment as a factor followed (if significant) by post-hoc tests (Tukey). Data that were not normally distributed (reference and working memory errors) were analyzed with the Kruskal-Wallis test for k-group comparisons followed (if significant) by the Mann-Whitney-U-test for pairwise comparisons. Behavioral data over the trials were tested with a linear mixed model with trials, learning phases (days), treatment, and phase×treatment interaction as fixed factors and trials and learning phases as repeated measure variables. The linear mixed model, in contrast to the general linear model, compares the phases with different numbers of trials and handles missing values. Behavior in the open-field and elevated plus maze tests was analyzed with the GLM for repeated measures. All tests were two-tailed, and the level of significance was set at p≤0.05.We analyzed six behavioral parameters: the latency to find all pellets, the average velocity, the number of pellets found, the average dips per second, the total number of hole dips and the mean distance of the rats to the wall . The out2 = 9.75, df = 4, p = 0.045) but not working memory  indices in trial 9. However, single group comparisons revealed no significant differences in reference memory indices between any groups . In trial 10, no significant differences in the reference memory  or working memory  indices were identified.In addition to these behavioral parameters, we analyzed the working and reference memories. For investigating and comparing different memory states, we calculated the error indices for the end of the acquisition  and for retention trial 10 . We foun5,48 = 3.268, p = 0.014) levels, but not in the serum concentrations . The statistical analyses of the testosterone and estradiol concentrations  and serum samples .We found significant differences in the hippocampal corticosterone  and 12.6 µmol (p = 0.026) MPP.5,54 = 6.509, p<0.001; ERβ: F5,54 = 2.852, p = 0.024; AR: F5,54 = 7.567, p<0.001; MR: F5,54 = 28.802, p<0.001; GR: F5,54 = 11.714, p<0.001; BDNF: F5,54 = 11.805, p<0.001) except the aromatase gene Cyp19 .The gene expression data are given in The single group comparisons revealed that animals treated with 7.4 µmol MPP showed the highest suppression of relative gene expression of ERα compared with animals treated with 1.5 µmol (p = 0.008), 3.7 µmol (p = 0.017), and vehicle (p = 0.004) and untrained vehicle-treated control rats (p = 0.002). Furthermore, animals administered 12.6 µmol MPP had a significantly lower ERα-mRNA level than the vehicle-treated trained (p = 0.036) and untrained (p = 0.014) controls. The relative gene expression of ERβ, however, differed significantly only in the 7.4 µmol MPP-treated rats compared to the untrained vehicle-treated rats (p = 0.043).The expression pattern of the androgen receptor was similar to that of ERα . In addition, the 12.6 µmol MPP group showed a difference in AR expression compared with the 1.5 µmol MPP-treated animals (p = 0.017).The mineralocorticoid receptor also had the lowest gene expression in animals treated with 7.4 µmol MPP compared to all other groups . Moreover, the 3.7 µmol MPP group differed significantly from the 1.5 µmol MPP group (p = 0.030), as well as from the vehicle-treated trained (p = 0.005) and untrained (p = 0.028) control groups.For the BDNF and glucocorticoid receptor, we observed decreased gene expression in animals treated with 7.4 µmol compared to the 1.5 µmol (both: p<0.001), 3.7 µmol , 12.6 µmol (both: p<0.001), and vehicle-treated trained (both: p<0.001) and untrained (both: p<0.001) groups.To compare dose effects on behavior and gene expression, we calculated a dose-effect curve  at the i1,22 = 0.06, p = 0.815; F1,22 = 0.01, p = 0.932; time spent in corners F1,22 = 0.54, p = 0.471; F1,22 = 0.01, p = 0.911; time spent in wall zones F1,22 = 0.71, p = 0.407; F1,22 = 0.01, p = 0.941; velocity in the center F1,22 = 2.58, p = 0.122; F1,22 = 0.02, p = 0.894; velocity in the corners F1,22 = 1.37, p = 0.254; F1,22 = 0.39, p = 0.54; velocity in wall zones F1,22 = 0.51, p = 0.483; F1,22 = 0.261, p = 0.615; track length in the center F1,22 = 0.00, p = 0.995; F1,22 = 0.12, p = 0.734; track length in the corners F1,22 = 2.10, p = 0.161; F1,22 = 1.81, p = 0.192; track length in the wall zones F1,22 = 0.04, p = 0.839; F1,22 = 0.66, p = 0.425). Similarly, no treatment trial interaction and no treatment effect were determined in the elevated plus maze test .To test whether the effects observed in the holeboard merely resulted from unspecific effects on locomotor activity or differences in anxiety induced by the pharmacological treatment, separate groups of animals underwent open-field and elevated plus maze tests . These aWe found dose-dependent changes (up to 7.4 µmol MPP) in behavioral performance with most significant effects in animals treated with 7.4 µmol MPP. These animals needed less time to find all pellets, moved faster, had more hole dips, and found more pellets than the control animals. In contrast to our hypothesis, the MPP-treated animals performed better regarding motivation , whereas, in line with our hypothesis, the cognitive component of the task was not affected by the treatment. Treatment effects on the general locomotor activity and changes in anxiety, which could have contributed to the observed differences, can be ruled out by our open-field and plus maze tests. Regarding exploration and anxiety, the MPP-treated rats did not behave differently from the controls.The effects of the MPP treatment are particularly surprising, because we found previously that the upregulation of hippocampal ERα gene expression is a learning-related holeboard effect in males at the age tested here. We also found, although not significant, upregulation of ERα gene expression in vehicle-treated males compared to untrained animals. However, the MPP-treated animals showed significantly reduced levels of gene expression not only for ERα but also for MR, GR, AR as well as the BDNF gene, whereas ERβ and aromatase gene expression was unaffected in the trained animals. These results support two conclusions: i. “Specific” steroid receptor functions can be revealed only against the steroid network background, characterized by a stronger relation of ERα activity to its own gene expression and to that of glucocorticoid and testosterone binding androgen receptors than to ERβ, and an effect on the BDNF but not on the aromatase encoding gene. ii. The training-induced increased expression of the ERα encoding gene does not positively correlate with the receptor function, because the receptor blockade results in increased rather than impaired performance of motivation-indicating behavior. Activation of extranuclear ER induces hippocampal BDNF signaling, thus mediating its effects on neuroprotection and plasticity Thus, at least parts of the observed system of steroid receptors, and there are many others The network characterization is further supported by the fact that we found significant effects in behavior only when the expression of all genes (except the aromatase and ERβ encoding genes) was affected, although there were differences in the sensitivity to MPP treatment between genes, as suggested by the dose response curve. Second, changes in hormone concentrations, due to the treatment, were absent. Therefore, compensatory modulations of receptor expression resulting from altered hormonal states cannot explain the results. Last, the effects of MPP on gene expression are dose dependent (up to 7.4 µmol MPP), thus indicating not unspecific treatment-induced downregulation of gene expression. Further, not all genes were affected.We noted dose-dependent effects of the MPP treatment on all behavioral elements and only an interaction of dose and learning phase effects for the times to find all pellets. Thus, the differences remained stable over the training for most of the behavioral parameters. The partial but not completely similar behavioral results in animals treated with 3.7 and 7.4 µmol MPP  along with the differences in the effects on gene expression between the two dosages suggest complex effects of structural changes in the receptor network on motivation and behavior and may indicate a structural change in the receptor network over training. Training phase–specific changes in correlations of hippocampal steroid hormone concentrations with behavior has been described for the same learning procedure Surprisingly, the dose-dependent effects of MPP on behavioral performance disappeared when the animals were treated with a higher dose than 7.4 µmol MPP. Animals treated with 12.6 µmol MPP showed only a larger wall distance when compared to the vehicle-treated group. Notably, this group also showed similar MR and GR expression as the vehicle-treated animals and significantly higher expression of these receptor genes compared to the group treated with 7.4 µmol MPP. Thus, higher concentrations of MPP may result in unspecificity of the receptor blockade with a subsequent rescue of MR and GR expression and behavior.Due to the intra-cerebroventricular administration of MPP, resulting in brain-wide distribution, other brain regions such as the amygdala and hypothalamus, which contain estrogen receptors Video S1Representative example of the behavioral performance of a rat treated with 7.4 µmol MPP .(MPG)Click here for additional data file.Video S2Representative example of the behavioral performance of a rat treated with vehicle .(MPG)Click here for additional data file.


The mean (90% CI) values for the ratio of AUC∞0− and Cmax values of the test product over those of the reference product were 90.21 (83.69–97.24) and 108.68 (100.21–117.86), respectively . On the basis of pharmacokinetic parameters including AUC∞0−, AUCt0−, and Cmax values, both the formulations were bioequivalent.The objective of this study was to investigate the bioequivalence of two formulations of 40 mg pantoprazole sodium enteric-coated tablets: Tripepsa as the test and Pantocid as the reference. The two products were administered as a single oral dose according to a randomized two-phase crossover with a 1-month washout period in 25 healthy Indian volunteers. After drug administration, serial blood samples were collected over a period of 30 hours. Plasma pantoprazole concentrations were measured by high-performance liquid chromatography with UV detection. Pharmacokinetic parameters were analyzed based on noncompartmental analysis. The logarithmically transformed data of AUC Pantoprazole, a proton pump inhibitor (PPI), is indicated for the treatment of erosive esophagitis associated with gastroesophageal reflux disease (GERD). Pantoprazole is one of the highly prescribed PPI in the management of peptic ulcer diseases. It shows high specificity for the relevant binding sites on activated proton pumps with little propensity to cause unwanted systemic effects , 2.Cmax is approximately 2.5 mg/L with a Tmax of 2-3 h. Pantoprazole is extensively metabolized in the liver and has a total serum clearance of 0.1 l/h/kg, a serum elimination half-life of about 1.1 h, and an apparent volume of distribution of 0.15 L/kg. 98% of pantoprazole is bound to serum proteins. Elimination half-life, clearance, and volume of distribution are independent of the dose. Almost 80% of an oral or intravenous dose is excreted as metabolites in urine; the remainder is found in feces and originates from biliary secretion. The clearance of pantoprazole is only slightly affected by age, with its half-life being approximately 1.25 h in the elderly [Pantoprazole sodium, administered as a 40 mg enteric-coated tablet, is quantitatively absorbed. Its absolute bioavailability is 77% and does not change upon multiple dosing. Following a single oral dose of 40 mg, Cyclodextrins are nonreducing cyclic oligosaccharides, consisting of dextrose units. Cyclodextrins have a “doughnut” shape, with the interior of the molecule being relatively hydrophilic. Because of their unique chemical structure, cyclodextrins are capable of forming “inclusion complexes” with many drug molecules if the drug is capable of undergoing chemical degradation in solution. The drug molecule can be protected by inclusion complexation with a cyclodextrin , 6. AmonOf all the cyclodextrin derivatives available, hydroxypropyl-beta-cyclodextrin (HPBCD) is the safest, as it does not permeate the membranes. A literature survey shows that the toxicity of HPBCD has been extensively studied. HPBCD has been shown to have a reduced haemolytic potential, making it suitable for parenteral use as well as for oral and/or topical applications. HPBCD encapsulation technology is well known for its solubilizing power. HPBCD is well tolerated in most species, particularly if dosed orally, and shows limited toxicity, depending upon dose and route of administration . PreviouIn the present study, a generic preparation of pantoprazole with HPBCD has been developed for clinical use. Although the generic and the innovator preparations contain the same active ingredient, they differ from each other by manufacturing processes as well as content of excipients, which affect the rate and extent of absorption of active drug. Therefore, the bioequivalence testing is mandated to confirm the bioavailability between the two preparations in human subjects. In the present study, the objective was to determine the bioequivalence of two oral formulations of pantoprazole in human subjects.Twenty-six healthy Indian male subjects aged between 20 and 32 years old and the body mass index within 18–25 participated in this study. Subjects were in good health on the basis of medical history, physical examination, and routine blood test. Subjects with known contraindication or hypersensitivity to pantoprazole were excluded as well as those with history of drug abuse, heavy alcohol consumption or cigarette smoking. No drug was allowed 1 month before the study period. The study was approved by the Research Ethics Committee in Mumbai, India.The reference product was commercially available 40 mg pantoprazole sodium enteric-coated tablets Pantocid manufactured by Sun Pharma, Mumbai, India . Test product was formulated as 1 : 2 mixture of pantoprazole sodium with HPBCD enteric-coated tablets. Tripepsa manufactured by Akums Drugs & Pharmaceuticals Limited, India . The experimental design of two-way crossover and randomized study with 25 healthy male volunteers was adopted in the study. As per the randomization schedule, each subject received a single oral dose of 40 mg pantoprazole tablet (either Tripepsa or Pantocid) on the morning with 240 ± 2 mL drinking water at room temperature in sitting posture, under 10 hours overnight fasting condition. The fasting state continued for 04 hrs after dose. Water and lunch were served 2 hours and 4 hours after dose, respectively. The washout period between each treatment was 1 month. After a washout period, subjects were administered the different brand of pantoprazole in the same manner. An identical meal and fluid intake were served during the two study periods. Subjects were required to refrain from drinking caffeine containing beverages and alcohol. The blood samples for the analysis of pantoprazole in plasma and collected at the following time points. Predose blood sample (00.00 hr) was collected just after phlebotomy within 2.0 hours prior to drug administration and the postdose samples were collected at 0.30, 1.0, 1.30, 2.0, 2.20, 2.40, 3.0, 3.15, 3.30, 3.45, 4.0, 4.15, 4.30, 4.45, 5.0, 5.20, 5.40, 6.0, 7.0, 8.0, 9.0, 10.0, 12.0, 16.0, 24.0, and 30.0 hrs (each contains 1 × 5 mL) after dose, respectively. Within 30 minutes, the blood samples were centrifuged to separate the plasma. The plasma samples were immediately kept at −20°C until assay.μm, 200 mm × 4.6 mm ID, Beijing, China), using a mobile phase of methanol-water , which was delivered at a flow rate of 1.2 mL·min−1. Under the present chromatographic conditions, HPLC retention time of pantoprazole and the IS  was 6.3 min and 9.0 min, respectively. To a 500 μL aliquot of plasma sample, 50 μL of methanol-water  and 50 μL of the IS solution (betamethasons 400 μg·mL−1 in 50% methanol) were added. The mixed samples were then extracted with 3 mL of diethyl ether-acetic ether . The mixture was vortex-mixed for approximate 1 min, then shaken on a mechanical shaker for 10 min. After centrifugation at 2000 g for 5 min, the upper organic layer was removed and evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in 100 μL of the mobile phase, then vortex-mixed. A 20 μL aliquot of the resulting solution was injected onto the HPLC-UV system for analysis.Drug analysis of pantoprazole in plasma was performed by suitable analytical method developed and validated at the Analytical Department, Drug Monitoring Research Institute, according to the international guidelines. The assay was operated using a highperformance liquid chromatography (HPLC) with UV detector set at 288 nm. A highperformance liquid chromatography-ultraviolet detection (HPLC-UV) method was established to determine the concentration of pantoprazole in human plasma. The limit of quantification during sample analysis was concentration range for pantoprazole of 19.9 ng/mL to 5000.1 ng/mL. A Shimadzu LC-10ATvp pump  and a Shimadzu SPD-10Avp detector  were used. Chromatography was performed on a Diamonsil C18 column  was determined by least-square regression analysis of terminal logarithm-linear portions of the plasma concentration time profile (ke = −2.303 × slope). The elimination half-life (t1/2) was calculated as 0.693/ke. The AUCt0− from time zero to the last quantifiable point (Ct) was calculated using the trapezoidal rule, and extrapolated AUC from Ct to infinity (AUCt−∞) was determined as Ct/ke. Total AUC0−∞ was the sum of AUCt0− + AUCt−∞. Pharmacokinetics analysis was performed by means of a noncompartmental method. The parameters t-test for the crossover design with standard 90% confidence intervals (CI) of the test/reference ratio with logarithm-transformed data. The Tmax was analyzed by nonparametric test (Mann-Whitney test). The bioequivalence acceptance criteria required that the 90% CI for the test/reference ratios of the AUC and Cmax fell into 80%–125% for AUCt0− and AUC∞0− and 70%–143% for Cmax, respectively [Bioequivalence was evaluated by means of statistical analysis of variance (ANOVA) and Student's ectively . 2, respectively. All 25 patients completed the study as per the protocol. Their mean values of age, weight, height, and body mass index were 30.9 ± 3.2 year, 75.8 ± 12.8 kg, 1.73 ± 0.10 m, and 25.46 ± 4.27 kg/mCmax of the test formulation was seen greater than the reference formulation. Moreover, fairly rapid absorption of pantoprazole from the test formulation in the intestine showed linear increase in the Cmax within 2.56 hr. This might be suspected due to the increased solubility and absorption of pantoprazole by HPBCD. It has been previously reported that HPBCD increased the apparent solubility of pantoprazole by 36 times [Oral administrations of both 40 mg pantoprazole tablets were well tolerated. The mean plasma concentration time curves of test and reference were comparable . Taken t36 times . The main pharmacokinetic parameters for test and reference formulations are listed in Cmax and AUC0−∞ for test formulation were not significantly different from those of reference formulation (4057.04 ± 914.97 versus 3708 ± 720.75 ng/mL and 23907.75 ± 5745.31 versus 26369.31 ± 5965.38 ng·hr/mL). Bioequivalence analysis showed that 90% CI values for the test/reference ratios (%) of AUC0−∞ and Cmax were 90.21 (83.69–97.24) and 108.68 (100.21–117.86), respectively  of the ectively . The coeBased on the aforementioned results, the test formulation of pantoprazole sodium tablets (Tripepsa), formulated by Akums Drugs & Pharmaceuticals Limited, India, is considered bioequivalent with commercially available pantoprazole. The present randomized, two-way crossover design study indicated that two brands of pantoprazole sodium 40 mg preparations were bioequivalent. Hence, Tripepsa may have excellent therapeutic efficacy in patients with peptic acid disorders.


To the Editor: Drug-resistant tuberculosis (TB) has been identified as a major problem in the former Soviet Union, and was recently surveyed in the Aral Sea regions of Dashoguz (Turkmenistan) and Karakalpakstan (Uzbekistan) (ekistan) . Howeverekistan) ,3.2 with approximately 250,000 inhabitants, at the western end of Georgia on the Black Sea. The collapse of the Soviet Union lead to disruption of TB control activities in all Eastern bloc regions  drugs was determined by the proportion method on Middlebrook 7H10 agar. The critical concentrations used were streptomycin, 2 µg/mL; isoniazid, 0.2 µg/mL; rifampin, 1 µg/mL; ethambutol, 5 µg/mL; kanamycin, 5 µg/mL; ethionamide, 5 µg/mL; capreomycin, 10 µg/mL; p-aminosalicylic acid, 2 µg/mL; and ofloxacin, 2 µg/mL (Sputa were collected from all patients attending Guliripchi TB Hospital in Sukhumi, the capital of Abkhazia, from September 2000 to April 2004. Patients were either referred by their practitioners or came spontaneously because TB was suspected. Diagnosis, treatment, and hospitalization were provided free. Samples were treated as previously described . Of 489  2 µg/mL –8. CycloData on resistance to the first- and second-line drugs are given in the Few data have been reported on drug resistance to first- and second-line drugs in the former Soviet Union and in the Caucasian region –4. OveraThis is the first survey reporting drug susceptibility data for MTB within the Caucasus. It indicates that the prevalence of MDR strains is similar to that in other central Asia regions . Our res


Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by  Due to its structural complexity, several hydrolases are required for complete xylan degradation. The key enzyme in this process is endo-β--xylanase (EC 3.2.1.8), which cleaves the xylan backbone to xylooligosaccharides [Xylan is a major structural polysaccharide of plant-cell walls being the second most prevalent in nature after cellulose. It is a heterogeneous polymer constituted primarily by a linear β-,4-D-xyloase EC 3..1.8, whiInterest in xylanolytic enzymes has increased in recent years due to their potential application in biotechnology. Xylanolytic enzymes have applications in conversion of lignocellulosic materials to chemicals and fuels, animal feed digestion, food and textile industries, and as bleaching agents in the pulp and paper processing , 4.Industrial production of enzymes on large scale is associated mainly with the substrate. The use of agroindustrial wastes as low-cost substrates for the industrial enzymes production is a significant way to reduce production cost. Thus, lignocellulosic substrates have now received considerable interest because of their possible use in secondary fermentation process . VariousBrewer's spent grain (BSG), the main residue of brewing industry, is rich in cellulosic and noncellulosic polysaccharides. It is produced at large amounts during the year and represents around 85% of the total by-products generated . In BrazPenicillium glabrum is a filamentous fungus that is distributed worldwide, frequently involved in food contamination. Due to its ability to disperse a large number of spores in the environment, P. glabrum is frequently found in the food manufacturing industry [P. glabrum has been described in the literature [P. glabrum using different agroindustrial wastes, establish the best fungal growing conditions for xylanase production with the best substrate, and biochemically characterize the major enzyme purified.industry . Despiteterature . Since mP. glabrum used in the present work is available in the Culture Collection of Environmental Studies Center—CEA/UNESP, SP, Brazil. Conidia were obtained from cultures in Vogel solid medium [7 spores/mL and incubated at 30 °C for 5 days in stationary condition. d medium  containiThe agroindustrial wastes were obtained locally. The residues were prepared by exhaustive washing with distilled water, dried at 80 °C for 24–48 h, and milled (35 mesh).Cultures were harvested by filtration. The culture filtrate was assayed for extracellular activity and protein. The mycelium was washed with distilled and sterilized water, frozen, and ground with sand in 0.05 M sodium phosphate buffer pH 6.5. The slurry was centrifuged at 3.900 g at 4 °C, and the supernatant was used as intracellular protein source.μmol of reducing sugar per min, under assay conditions. Specific activity was expressed as unit per milligram of protein. All enzymatic assays were developed in triplicate, and the results are presented through mean values. Xylanase activity was determined at 50 °C using 1.0% (w/v) birchwood xylan  in McIlvaine buffer pH 6.5. This buffer is prepared by a mixture of 0.1 M citric acid and 0.2 M sodium monohydrogen phosphate. After 5 and 10 min of incubation, the reaction was interrupted by the addition of 3,5-dinitrosalicylic acid (DNS), and the reducing sugars released were quantified , using xTotal protein was determined by modified Bradford method , using bVogel's liquid medium was supplemented with various carbon sources at a concentration of 1% (w/v). The inoculated flasks were incubated at 28 °C under stationary condition, for five days. Xylanase activity was determined as described previously. P. glabrum was studied under standing culture and under shaking culture (120 rpm) for 8 days. The effect of initial pH on the enzyme production was assayed from 3.0 to 8.0, and the temperature influence was verified from 15 to 40 °C. The initial pH values were adjusted by the addition of 1.0 M sodium hydroxide or phosphoric acid solutions. The incubation period's influence on xylanase production by The crude enzyme (50 mL) was fractionated by ammonium sulfate precipitation . The supernatant of 90% ammonium sulfate saturation obtained after centrifugation  was extensively dialyzed against 0.05 M ammonium acetate buffer pH 6.8 before analyses. R2 = 0.993), by plotting log of the molecular weights of the standards against the ratio between elution volumes of the standards and the void volume of the column.The protein sample obtained in the step above was chromatographed on Sephadex G-75 column (2.6 × 64.0 cm), equilibrated, and eluted with 0.05 M ammonium acetate buffer, pH 6.8, flowing at 18 mL/h. Fractions (3 mL) whose protein content was estimated by reading absorbance at 280 nm and xylanase activity assayed as described previously. To determine xylanase molecular mass through gel filtration chromatography, the column was calibrated using blue dextran for the void volume determination and ribonuclease (15.4 kDa), chymotrypsin (25.0 kDa), ovalbumin (43.0 kDa) and bovine serum albumin (67.0 kDa) as standards. The molecular weight of xylanase was estimated from a regression curve  were used to plot the standard curve log of molecular weight against relative mobility in the gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a gradient of 8–18% (w/v) polyacrylamide according to Laemmli . The resTo determine the optimum pH, the purified xylanases were assayed at 50 °C, in different pH values using 0.05 M glycine-HCl buffer from pH 1.6 to 2.5 and McIlvaine buffer from 3.0 to 7.0. The optimum temperatures were determined by performing the reaction at temperatures ranging from 15 °C to 75 °C in McIlvaine buffer pH 3.0. For pH stability assays, the purified enzymes were diluted (1 : 2 v/v) in 0.05 M glycine-HCl buffer from pH 1.6 to 2.5 and McIlvaine buffer for pH range from 3.0 to 7.0. The samples were incubated at 4 °C for 24 h. After this period, the xylanase activity was assayed under optimal conditions. To evaluate the thermal stability, the purified enzyme was incubated at 50 °C, 55 °C, and 60 °C at the optimal pH determined above for different periods. The effect of metallic ions and other compounds on the xylanase activity was evaluated at concentrations of 2 mM and 10 mM. The residual activities were measured in relation to the control without substances by performing the enzyme assay at the optimal conditions. Specificity of xylanase against birchwood, beechwood, and oat spelt xylans, carboxymethyl cellulose, (CM-cellulose) and Avicel were assayed. Substrate solutions of 1% (w/v) were prepared in a buffer of optimum pH activity for the enzyme. Km) and maximum reaction velocity (Vmax) were estimated from the Lineweaver-Burk reciprocal plots, using “GraFit” 5.0 software. The enzyme was incubated with oat spelt, beechwood, and birchwood xylans, at concentrations between 4.0 and 30 mg/mL. The Michaelis-Menten constant . These values were higher than those obtained with oat spelt xylan. The production of xylanolytic enzymes by n et al. . HoweverP. glabrum growth, measured by the intracellular protein concentration, occurred on the fifth day in standing culture and on the third day in shake culture. Under shaking, as well as under standing condition, xylanases were expressed during the stationary phase, reaching the decline phase  was observed in the 90% ammonium sulfate saturation supernatant. The molecular exclusion chromatography elution profile resulted in one xylanase activity peak  at 60 °C and 55 °C were 15 and 32 min, respectively. This enzyme was stable at 50 °C with T1/2 of 150 min, retaining 70% of its activity over 60 min at this temperature. The P. glabrum xylanase is more thermostable than many fungal xylanases, such as those from Penicillium expansum [Aspergillus niger B03 [Talaromyces thermophilus [Thermal stability is an interesting enzyme property due to the great industrial importance. Then, enzyme stability analyses were carried out. The purified xylanase from nd 60 °C . The estexpansum  and Aspeiger B03 , howevermophilus . β-mercaptoethanol, at 2 mM and 10 mM concentrations , tetrasodium ethylenediaminetetraacetate (EDTA), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), and trations . In geneese ions , 31. Theund them . Xylanasxylanase .β-mercaptoethanol and DTT stimulated xylanase activity. The enzymatic activity stimulation in the presence of these thiol group-protecting agents can be explained by preventing the oxidation of sulfhydryl groups. Dutta et al. [Penicillium citrinum and Acrophialophora nainiana xylanases, respectively.EDTA, a metal chelator, decreased xylanase activity, indicating that the purified enzyme requires metal ions for their actions. Total loss of activity was observed in the presence of SDS, indicating that hydrophobic interactions must be important in maintaining xylanase structure. The reducing agents a et al.  and Carda et al.  also relP. glabrum xylanase did not hydrolyze Avicel or CM-cellulose but acted only on xylans. The purified xylanase exhibited typical Michaelis-Menten kinetics for oat spelt, birchwood, and beechwood xylans, allowing the corresponding kinetic constants to be calculated. P. glabrum xylanase showed Km values of 5.3, 3.1, and 1.2 mg mL−1 and Vmax values of 212.10, 194.21, and 393.17 μmol min−1 mg−1 of protein, for birchwood, beechwood, and oat spelt xylans, respectively. The Km and Vmax values exhibited by P. glabrum xylanase are in agreement with the values presented by other fungal xylanases which range from 0.09 to 40.9 mg mL−1 for Km and from 0.106 to 10.000 for Vmax [Km for these substrates indicated that this enzyme has higher affinity for oat spelt xylan. Similarly, the xylanases from A. nainiana [Fusarium oxysporum [Km for oat spelt xylan. Specificity studies indicated that the nainiana  and Fusaxysporum  showed hP. glabrum strain was able to produce high levels of xylanase using brewer's spent grain as substrate. Conventional purification methods were effective to purify the major xylanase from P. glabrum. The purification procedure resulted in higher overall yields as compared to others described in the literature [P. glabrum xylanase was active at very low pH, with optimum at 3.0, and it was stable in an acid pH range. These characteristics make it potentially useful in some biotechnological processes such as for animal feed, clarification, and maceration of juices and wines. Additionally, the use of brewer's spent grain as substrate for xylanase production can not only add value and decrease the amount of this waste but also reduce xylanase production cost.In this study, a terature , 26, 36.terature , 37. P.


Chung Hun Wha Dam Tang (CHWDT) herbal combination was reported to cease dizziness and phlegm. However, the effect of CHWDT in obesity has not yet been known mechanically. Therefore, we investigated whether this CHWDT could protect the cells from lipogenesis, gluconeogenesis, and inflammation in both in vivo and in vitro. CHWDT significantly decreased body weight, epididymal and perirenal fat content without affecting feed intake in high-fat diet-induced obese mice model. Additionally, CHWDT inhibited obesity-induced SREBP1, FAS, PGC1α, G6Pase, PEPCK and increased CPT1, ACO, and LCAD genes expression in vivo and in vitro. Proinflammatory cytokines like TNF-α and iNOS expression were reduced by CHWDT in both Raw264.7 macrophages and HepG2 cells. In addition, NO production was also significantly decreased by CHWDT in LPS-stimulated macrophages. Furthermore, AMPKα activation by CHWDT was involved in inhibition of obesity by reducing triglycerides production and increasing CPT1 expression. Based on all of the results, we suggest that CHWDT has inhibitory effects on obesity-induced lipogenesis, gluconeogenesis, and inflammation via AMPKα activation.The  Obesity is closely associated with life-style-related diseases such as atherosclerosis and noninsulin-dependent diabetes mellitus and with increased risk of coronary heart disease . In addiuropathy . Besidesuropathy . Also, Auropathy . Activaturopathy . TherefoPanax quinquefolium (American ginseng) and Panax ginseng (Asian ginseng) are proved for antihyperglycemia, insulin sensitization, islet protection, antiobesity, and antioxidation in many model systems [Up to now, many kinds of medicine have been developed for obese and diabetic patients of which mostly are chemical agents. However, considerable side effects always come with these agents . In the  systems . Traditi systems . MultiheChung Hun Wha Dam Tang (CHWDT) has been in widespread use for four hundred of years in traditional Korean medicine. It is known well that CHWDT relieves dizziness, phlegm, and inflammation [ammation –18. HoweLipopolysaccharide (LPS), sodium palmitate, Griess reagents, MTT , and metformin were from Sigma-Aldrich . Also, all other chemicals were obtained from Sigma-Aldrich.Herbal plants were purchased from Omniherb . In our study, we prepared CHWDT with combination of 16 different types of herbal plant . The mixture of herbs was subjected to freeze-drying to make powder. Further, 12.5 g from lyophilized powder of CHWDT was taken in 125 mL of distilled water and then mixed properly. 50 mL conical tubes containing sample were centrifuged at 3,000 ×g for 20 min. The extraction was repeated three times. The extracts were then filtered through a filer paper. The filtrates were collected and final concentration of the extract was calculated as 100 mg/mL. Prepared compound (CHWDT) was stored at −20°C. The HPLC profile of CHWDT was provided in supplemental Figure 1. ad libitum. The normal control group was being fed with standard laboratory chow and high fat diet  groups were being fed with high fat diet for 7 weeks. Body weight was recorded once in a week and feed intake was recorded twice in a week. As we expected, by the end of 7 weeks period, the body weight of high-fat-diet-fed mice were approximately 20% higher than the normal control group. Then, we started oral administration of CHWDT (800 mg/kg) in every other day basis for more 7 weeks [Seven-week-old male C57BL/6 mice were obtained from ORIENT . The mice were maintained under specific pathogen-free conditions at 18–24°C, 40–70% humidity, 12 h light/dark cycle, and access to drinking water  7 weeks . Experim2 until 75–80% confluence, where AML cells were cultured in DMEM/F12 medium with 0.005 mg/mL insulin, 0.005 mg/mL transferring, 5 ng/mL selenium, 40 ng/mL dexamethasone, and 10%FBS (ATCC CRL-2254). Raw264.7 and HepG2 cells were cultured in DMEM in addition with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin at 37°C in 5% CO4 cells/well. After one day, the old medium was discarded and fresh medium containing 50, 100, and 200 μg/mL of CHWDT were used. Twenty-four hours later, the medium was replaced with medium coupled 1 mg/mL of MTT (Sigma) as per the manufacturer's instructions. Samples were incubated with MTT reagent for 4 h at 37°C. After 4 h, samples were detected at 570 nm with an ELISA plate reader using spectrophotometer. The wavelength at 690 nm was deducted from the 570 nm.Cell viability was checked by MTT  detection was performed according to TG-S kit . In brief, after 24 h incubation 35 α, NOS2 , and beta-actin. The bands were visualized by using enhanced chemiluminescence Western blotting detection system . Anti-p-AMPK, AMPK, PGC1α antibodies were purchased from Cell Signaling Technology, Inc. . Anti-NOS2 , β-actin, and anti-goat antibody conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology .Cell extracts were prepared by using lysis buffer containing RIPA buffer, protease inhibitor, and phosphatase inhibitors. Protein concentration in the cells was measured by BCA assay . An equal amount of proteins for all samples were subjected to electrophoresis and proteins were transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) for 20 min and incubated at 4°C overnight with primary antibodies and followed by secondary antibodies conjugated with horseradish peroxidase for p-AMPK, AMPK, PGC1μg of total RNA was used to make cDNA by using M-MLV reverse transcriptase  and oligo (dT) 15 primer . The resulted cDNA was subjected to PCR for mouse GAPDH, 18s, SREBP1c, FAS, CPT1, ACO, LCAD, PGC1α, G6Pase, PEPCK, TNFα, and iNOS; and human GAPDH, SREB1c, FAS, CPT1, PGC1α, G6Pase, PEPCK, TNFα, and iNOS,  PCR was performed having the following conditions: denaturation temperature 94°C for 0.5 min, annealing temperature (according to respective primer) for 0.5 min, and extension temperature 72°C for 1 min, and PCR cycle was determined according to a kinetic profile. However, GAPDH and 18s were used as internal loading control to normalize all PCR products. Band intensities of the amplified DNAs were compared after visualization on an UV transilluminator.Total RNA was extracted using TRIzol reagent  according to manufacturer's instructions. In short, 2 μg/mL) was treated for 24 h. Nitrite production in the medium was assessed by measuring nitrite/nitrate, the stable degradation products of NO. The culture medium was incubated with coenzyme at 37°C for 15 min. Next, the reaction mixture was incubated with nitrate reductase (Sigma-Aldrich) at 37°C for 30 min. The Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in distilled water) was added to the reaction mixture. The absorbance of the mixture at 530 nm was determined using a microplate reader, and nitrite concentration was determined using a dilution of sodium nitrite as a standard [Raw264.7 cells were pretreated with CHWDT for 1 h and then LPS  (   (P < 0.05)  to mice every other day for 7 weeks. Interestingly, body weight of CHWDT administrated high-fat-diet-fed mice significantly decreased at 14 weeks ( < 0.01) . Feed in < 0.01) . Paralle Figures . Taken tμg/mL on cell viability as shown in Supplemental Figure 2, though 200 μg/mL had significant (P < 0.05) cytotoxic effect. Therefore, the CHWDT concentration was determined to be treated with 50 and 100 μg/mL of concentration to further study the mechanisms of antiobesity effect of CHWDT.In order to detect the effect of CHWDT on cell viability in HepG2 cells, MTT assay was performed on 24 hours after CHWDT treatment. There was no significant effect of CHWDT at 50 and 100 μg/mL) and palmitate (0.45 mM) was exposed for 12 h. Our formulated compound CHWDT significantly inhibited palmitate-induced expression of SREBP1c and FAS in both cell types  induced mRNA levels of PGC1α, G6Pase and PEPCK  for 24 h. As shown in Figures α mRNA and protein levels of iNOS were downregulated by CHWDT in LPS-stimulated Raw264.7 cells, respectively. Similarly, in HepG2 cells the iNOS and TNF-α mRNA levels (P < 0.001) (Obesity-induced type 2 diabetes is related to inflammation as well as increase of inflammatory markers . InhibitA levels  were dec< 0.001) . Taken tCHWDT was applied at a concentration of 100 μg/mL in a time-dependent manner  in HepG2 cells. Activation of AMPKα was found in a time-dependent manner (μg/mL for 9 h) of CHWDT also showed increase level of AMPKα activation in HepG2 cells , whereas compound C restored palmitate-induced TG levels in HepG2 cells  as a result of lipogenesis leads to increased level of lipogenic genes . Upregulα, G6Pase, and PEPCK [α is known to regulate thermogenesis, mitochondrial biogenesis, glucose uptake, and metabolism [α found to regulate glucose synthesis during gluconeogenesis [α, G6Pase, and PEPCK, ensuring noble antiobesity effects of CHWDT.Gluconeogenesis is a ubiquitous process mainly takes place in human liver in which glucose is produced from noncarbohydrate carbon substrates such as lactate, glycerol, and glucogenic amino acids.The main enzymes involved in gluconeogenesis are PGC1nd PEPCK , 31, 32.tabolism , PGC1α fogenesis , 35. Theogenesis . Differeogenesis . In factα play important roles in the regulation of the immune system. Further, enhanced level of iNOS was found in the liver of chronic HBV-infected patients [α, iNOS, and NO were suppressed by CHWDT in both HepG2 and Raw264.7 cells in vitro. Thus, CHWDT had anti-inflammatory potential in vitro.Inflammation is directly or indirectly involved with obesity as well as type 2 diabetes . Inflammpatients . Overpropatients  and it iα activation in a time- and dose- dependent manner. Further,   CHWDT-induced CPT1 expression was downregulated by compound C, indicating the role of AMPKα in CPT1 regulation. In addition, palmitate-induced TG accumulation was significantly reduced by CHWDT and CHWDT effect was abrogated by AMPK inhibitor, supporting role of AMPKα on lipid content as described in [α activation by CHWDT provided evidence for functions of CPT1, ACO, and LCAD involved with fatty acids oxidation [It is well known that AMPK regulates variety of signaling cascades, glucose transport, hepatic lipid metabolism, downregulating fatty acid synthesis by transcription of SREBP1c , 41. AMPribed in . Therefoxidation  as shownα activation and down regulation of SREBP1, FAS, PGC1α, PEPCK, G6Pase, iNOS, TNF-α, and NO production. These findings may provide molecular evidences for the use of CHWDT as a therapy for obesity-induced disorders.In conclusion, CHWDT had inhibitory effects on lipogenesis, gluconeogenesis, and inflammation in vitro and in vivo in high fat diet induced mice. It is suggested that antiobesity effects of CHWDT is mediated through CPT1 via AMPK-20°C.In our study, we prepared CHWDT with combination of 16 different types of herbal plant. The mixture of herbs was subjected to freeze-drying to make powder. Further, 12.5 g from lyophilized powder of CHWDT was taken in 125 mL of distilled water and then mixed properly. 50 mL conical tubes containing sample were centrifuged at 3,000×g for 20 min. The extraction was repeated three times. The extracts were then filtered through a filer paper. The filtrates were collected and final concentration of the extract was calculated as 100 mg/mL. Prepared compound (CHWDT) was stored at Click here for additional data file.Click here for additional data file.


Cu, Zn, and Pb did not reach levels of concern for human consumption, but the high Cd values determined in Mazatlán  indicate that this was the only metal of concern for human health because the daily individual consumption of fish muscle to reach the PTDI would be within 0.27 and 0.41 kg.To assess if they were within the safety limits for human consumption, the Cd, Cu, Pb, and Zn contents of fish muscles, bought from separate stalls of the fish markets of nine cities of NW Mexico, were determined by atomic absorption spectrophotometry. Considering all fish and markets, the mean contents were Zn: 23.23 ± 5.83, Cu: 1.72 ± 0.63, Cd: 0.27 ± 0.07, and Pb: 0.09 ± 0.04  Fish is an important protein and essential nutrient source but, because of metal accumulation , fish coAgriculture, mining, and food processing are the main industrial activities in the Mexican NW, and their effluents may increase the level of coastal pollution, which could be one of the reasons for the high Pb and possibly Cd levels found in aquatic organisms of Sinaloa State coastal waters .There is information on the metal content of some fish species of several lagoons and coastal areas of NW Mexico , 4, but Lutjanus spp., Mugil cephalus, Diapterus spp., and of a headless, gutted, and skinned small shark (SS), probably Rhizoprionodon longurio which is the most common small shark caught in local fisheries [The four species selected are all landed by the artisanal fishing fleets and may therefore be considered as originating from local fishing grounds. Specimens of isheries , were obIn each market, the fillets of three specimens  of each species, obtained at three separate stands, were placed in separate metal-free containers and transported in an ice box to the laboratory at 4°C. All specimens of the same species obtained in the same city were homogenized, freeze-dried, and ground in a teflon mortar to obtain a composite sample.3 : HCl 3 : 1 (v/v) acid mixture, digested at 130°C on a Mod block unit, transferred to clean polypropylene vials, and diluted to 25 mL with Milli-Q water [Subsamples (0.75 g) were placed in triplicate 30 mL Teflon vessels with 5 mL of trace metal grade HNO-Q water , and the-Q water .α = 0.05 [Since the three cities of each state share the same fish sources , the mean metal contents of each of the four species obtained from the threes markets of each state were compared with one-way ANOVA tests, parametric or nonparametric, depending on the results of Kolmogorov-Smirnov's and Bartlett's tests, and the different mean values were separated with Student-Newman-Keuls tests. In all cases, the level of significance was α = 0.05 .µg/g (dry weight). For all species, the highest Cd values were in Mazatlán , while the lowest were found in Tijuana , and Culiacán (Diapterus spp. and SS: 0.04 ± 0.05 and 0.02 ± 0.01 µg g−1) in the order.Considering all species and markets, the mean metal concentrations were Zn: 23.23 ± 5.83, Cu: 1.72 ± 0.63, Cd: 0.27 ± 0.07, and Pb: 0.09 ± 0.04 The highest mean values of Cd were determined in Sinaloa. This is in agreement with the high Cd levels found in the oysters of seven coastal lagoons of this state, which might be explained by the approximately 1.5 million ha of intensive agriculture of Sinaloa state, in view of the close association between Cd- and phosphate-based fertilizers .M. cephalus obtained in Mazatlán, Hermosillo, Mexicali, and Ensenada, Diapterus spp. of Mazatlán, Culiacán, Obregón, Hermosillo, Guaymas, and Mexicali, and of the SS samples of Mazatlán, Culiacán, Guaymas, Mexicali, and Ensenada were below the detection limit (0.01 µg g−1). The highest Pb values for M. cephalus and Lutjanus spp. (0.23 ± 0.09 and 0.23 ± 0.03 µg g−1) were determined in Guaymas, whereas those of SS (0.29 ± 0.09 µg g−1) and of Diapterus spp. (0.15 ± 0.12 µg g−1) were found in Ensenada and Tijuana, respectively.The Pb contents of Among the metals determined in this study, Pb had the lowest values, possibly because it has low potential for bioaccumulation and biomagnification in fish , and alsµg g−1 for M. cephalus, Diapterus spp., Lutjanus spp., and SS, respectively, and the ranges for Cu were 3.55–1.79, 1.85–0.20, 5.22–0.32, and 3.72–0.37 µg g−1 in the same order. The Cu content of Diapterus spp. was significantly lower in Sinaloa than in Sonora and BC  of eachThere was no evidence of biomagnification: the tendency to a higher Cd content in SS, which has the highest trophic level (troph > 4), was not significant, and the essential Cu and Zn tended to be higher in the species with the lowest troph value.M. cephalus obtained in the Sinaloa markets is higher than most data obtained in previous studies which used this species as monitoring organism , samples obtained off the New Jersey and the Catalonia coasts [µg g−1 dw, resp.), while those of Pb (0.08 and 0.52 µg g−1) were comparable or higher than those of this study.Similar studies in other geographic areas show wide discrepancies as well as similarities to our results: while the Cd and Zn contents of six fish species marketed in Kayseri, Turkey , rangeda coasts  had Cd cµg person−1 [µg person−1 [−1 [M. cephalus and possibly Diapterus spp.The provisional tolerable daily intakes (PTDI) of Cd and Cu are 58.3  and 245 person−1 . The resperson−1 . Accordion−1 [−1 , and forHowever, according to the latest national census, less than 23% of Mexican households consumed fish at least once in the preceding quarter, while fish consumption was more frequent in the 30% households of higher income, whose members would seem therefore the population at risk.The public faces conflicting reports on the advantages of fish consumption, which is considered advisable for a healthy diet and for cardiovascular health , 21, alt


Five peptides—PG-1, Bac7, SMAP-29, BMAP-27, and BMAP-28—proved to be active at very low concentrations (1 to 0.1 μg/mL), while LL-37 cathelicidin was ineffective even at a concentration of 100 μg/mL. In comparison to chlamydiae, S. negevensis proved to be more susceptible to the antimicrobial peptides tested.The  Simkania negevensis is an obligate intracellular Gram-negative bacterium belonging to the family of Simkaniaceae in the order Chlamydiales [Acanthamoeba poliphaga [mydiales , discovemydiales , and is oliphaga .S. negevensis and acute rejection in lung transplant recipients has been suggested [S. negevensis DNA has also been amplified from an aortic aneurysm [Epidemiologic studies have reported a human widespread exposure to this bacterium , 5, bothuggested . S. negeaneurysm , 14.S. negevensis in vitro susceptibility to azithromycin, minocycline, erithromycin, doxycycline, and ofloxacin like chlamydiae [Previous investigations reported lamydiae  and the lamydiae , 15, 16.in vitro antichlamydial activity of peptides, such as cathelicidins, stored by mammalian leucocytes and show an antimicrobial activity against bacteria, fungi, protozoa, and enveloped viruses. With regard to the mode of action of these peptides against chlamydiae, previous investigations on the activity of protegrins [Several studies –20 demonotegrins  suggesteotegrins  demonstrin vitro activity of six antimicrobial peptides against the reference strain Z of S. negevensis.In the present study, we investigate the S. negevensis (American Type Culture Collection VR-1471) was grown in LLC-MK2 cells [2HPO4, 13 mL 0.2 M NaH2PO4, 0.72 g L-glutamic acid), and frozen in aliquots at −80°C. The cathelicidin peptides—PG-1 from pig, Bac7 (1–35) from cattle, SMAP-29 from sheep, LL-37 from human, BMAP-27, and BMAP-28 from cattle—were chemically synthesized, purified, characterized, and provided as lyophilized peptide, as previously reported . The refK2 cells  in 6-larK2 cells , resuspeμL aliquots at −80°C until used. To determine the lowest peptide concentration required to achieve ≥50% reduction of inclusions with respect to untreated control, the various peptides were diluted tenfold from 200 to 0.02 μg/mL, in a volume of 100 μL with PBS in polypropylene tubes. An equal volume of 5 × 105 inclusion-forming units (IFU)/mL of purified S. negevensis EBs in SPG medium was then added, so obtaining a final cathelicidin concentration ranging from 100 to 0.01 μg/mL in a mixture of PBS and SPG at a final pH 7.4. EBs untreated with cathelicidins were used as a control. After incubation at room temperature for 2 h, a 200 μL aliquot was added to 800 μL chlamydial growth medium and inoculated onto LLC-MK2 cells grown in 24-well plates in a 5% CO2 atmosphere. After centrifugation at 800 × g for 1 h at 33°C and incubation at 35°C for 72 h, the cultures were fixed and stained for the presence of inclusions by immunofluorescence using an in-house rabbit polyclonal hyperimmune serum raised against S. negevensis. The number of IFU per coverslip was counted in 40 microscopic fields using a ZEISS UV microscope at a magnification of ×200.Stock solutions of each peptide at 1 g/liter were prepared in phosphate-buffered saline (PBS), pH 7.4, and stored frozen in 20-All tests were performed in triplicate and in three independent experiments. S. negevensis are reported in S. negevensis at a concentration of 0.1 μg/mL, whereas PG-1, BMAP-27 and BMAP-28 showed the same activity at a concentration of 1 μg/mL. All these five cathelicidins demonstrated a very slow decrease in their inhibitory effect while their concentration has been reducing from 100 to 1 or 0.1 μg/mL. LL-37 peptide did not exert any inhibitory effect, even at a concentration of 100 μg/mL. The activities of the six cathelicidin peptides against Chlamydia trachomatis strains, SMAP-29 being the most active peptide at a concentration of 10 μg/mL; on the contrary, LL-37 peptide was ineffective even at a concentration of 80 μg/mL. With regard to the other chlamydial species tested, only Chlamydophila pneumoniae and Chlamydophila felis were sensitive to 10 μg/mL of SMAP-29 and 80 μg/mL of SMAP-29/Bac7, respectively.In our previous study , the actS. negevensis. In comparison with chlamydiae, S. negevensis was not sensitive to LL-37 even at the concentration of 80 μg/mL, but very sensitive to all the other five cathelicidins that showed an inhibitory effect at very low concentrations from 1 to 0.1 μg/mL. To our knowledge no study on S. negevensis susceptibility to cathelicidins has been performed, until now. These preliminary results suggest a higher sensitivity of S. negevensis to cathelicidins respect to chlamydiae. A previous study showed differences in the susceptibility of different C. trachomatis serovars to antimicrobial peptides, suggesting that limited variations in the disulfide bonding in the membrane structure of these organisms may influence their susceptibility to peptides [S. negevensis probably differs significantly from that of other members of Chlamydiales, as monoclonal antibodies recognizing family-specific epitopes of chlamydial OMP-2, MOMP, and LPS did not bind S. negevensis antigens [S. negevensis and other Chlamydiales members were tested for cross-reactivity by Western blot using polyclonal antisera [S. negevensis to cathelicidins in comparison to chlamydiae could be referred, to a great extent, to its different polypeptides pattern.In the present investigation, the same six cathelicidins were tested against the reference strain Z of peptides . The memantigens ; in addiantisera . The hig


The fully collapsed air holes of the PCF at the spice regions allow the coupling of PCF core and cladding modes that makes a Mach-Zehnder interferometer. The transmission spectrum exhibits sinusoidal interference pattern which shifts differently when the cladding/core surface of the PCF is immersed with different RI of the surrounding medium. Experimental results using wavelength-shift interrogation for sensing different concentrations of sucrose solution show that a resolution of 1.62 × 10 The advantages of this type of sensor are their relatively simple construction, compactness, low cost, ease of use, immunity to electromagnetic interference, and high sensitivity to the external refractive index (RI) –10. ReceIn this paper, we present a PCF interferometer made by fusion splicing a piece of PCF between two standard single mode fibers with a fusion splicer. We report on the investigation and analysis of this class of novel PCF interferometers for refractive index sensing. The ability of the PCF interferometers, with five different sensing lengths , as a high sensitive RI sensor will be exploited. The unique sensing features of these spectral filters are particularly suitable for a wide variety of applications in sensor systems.2.2.1.λ can be expressed as follows [λ is the central wavelength of the light source; β1 and β2 are propagation constants of the modes involved in the interference, and L is the length of the interferometer. The fringe period is inversely proportional to propagation constant deviation Δβ = β1 − β2 and the interferometer length L.According to the interference theory, the value of the fringe period (Λ) of the Mach-Zehnder interferometer at any  follows ,14:(1)Λ2.2.The interferometer was fabricated by using a LMA-PCF fiber  which was designed to have a large effective mode field area (∼40 μm). It has an outside diameter of 125 μm, a core diameter of 10 μm, a hole diameter of 3.0 μm, and a hole pitch of 7.0 μm. 2.3.i.e., the length between two collapsed zones, L = 1.34, 2.67, 3.50, 4.40, and 5.00 cm). We fabricated three replicates for each length of interferometer and there are totally 15 interferometers in this study. The interference spectra contain the information of wavelength, transmission loss, and fringe period. The distance between any two consecutive peak fringes is determined as a fringe period. The fringe visibility was calculated for each PCF interferometer as follows [maxI and minI are the maximum and minimum intensity values of corresponding interference spectrum, respectively.The characteristic measurements were conducted for interferometers associated with five different sensing lengths ( follows :(2)ImaxThe RI measurements were performed using the above 15 interferometers and the refractive indices were in the range of 1.333 to 1.422. The interference spectra and the relationship between wavelength shift and refractive index were used to characterize the PCF Mach-Zehnder interferometers. During the course of data measurement, the magnetic clamps and a lift platform were used to keep the PCF interferometers straight lines without the bending effect see . We have3.3.1.L−1.0347 [R2 = 0.99695, see R2 = 0.996995). In addition, we compared the measured fringe period and the one from a sine curve fitting for each interferometer. y = oA + A sin (ωx - φ); where y is the transmission loss, x represents the wavelength, and other parameters are the regression coefficients. versus sensing length. A correlation was found that the calculated fringe period based on sine curve fitting was almost inversely proportional to the sensing length—Λ = 59.15×L−0.99255 (R2 = 0.99663). The absolute difference value between average measured fringe period and the one from sine curve fitting is calculated as follows:The transmission spectra of a PCF interferometer with different sensing lengths are shown in D value versus sensing length. The results show that the PCF interferometer has smaller D values, 0.48 nm and 0.14 nm, when sensing lengths are 4.40 cm and 5.00 cm, respectively.The box plot (or whisker graph) is a statistical way of graphically depicting groups of numerical data through their five-number summaries: maximum, minimum, median, upper quartile, and lower quartile. In addition, it could be even used to explore outliers . Figure 3.2.The ability of the PCF interferometer to detect changes in the surrounding RI was studied. The control of surrounding RI was through the use of sucrose solutions with various concentrations. 2 values were greater than 0.94. Thus, the relationship between wavelength shift and RI for PCF interferometers was assessed as a three-stage linear function and the boundary of the three-stage linear regression was determined. The graph of wavelength shift versus refractive index and three-stage linear regression models were plotted in L = 2.67 m, 3.50 cm, 4.40 cm, and 5.00 cm, respectively.Regarding versus air and RI for different concentrations of sucrose solution using a PCF interferometer. For example, 2 = 0.9971, 0.9891, and 0.9792) to the plot of the wavelength shift vs. RI of the sucrose solution. For the RI sensitivity of a 5.00-cm PCF interferometer was determined by the slopes of three-stage linear fits and it had 309 nm/RIU, 251 nm/RIU, and 326 nm/RIU in the RI range of 1.333–1.373, 1.373–1.403, and 1.403–1.422, respectively. We have collected and analyzed the data considering different sensing lengths in the PCF interferometer. σ/m, σ = standard deviation of sensor response in measuring the blank sample, m = slope) versus the sensing length (1.34–5.0 cm) for three-stage RI areas. Studies presented here demonstrate that these PCF interferometers  could provide a sensor resolution of 3.28 × 10−3–5.20 × 10−5 RIU for refractive indices in the range of 1.333 to 1.422. For example, the resolution of PCF interferometer was as low as in the range of 1.62 × 10−4–8.88 × 10−4 RIU or 1.02 × 10−4–9.04 × 10−4 RIU when sensing length is equal to 3.50 cm or 5.00 cm, respectively. Based on −4–8.88 × 10−4 RIU and 1.02 × 10−4–9.04 × 10−4 RIU, respectively. In addition, the 3.00-cm and 5.00-cm PCF interferometers exhibited relatively small sensitivity variation . However, if the 5.00-cm PCF interferometer is used, the bending effect and package technique should be noticed.A red shift in the resonance wavelength was observed with the increase of RI as shown in 4.−4–8.88 × 10−4 RIU or 1.02 × 10−4–9.04 × 10−4 RIU  can be achieved for refractive indices in the range of 1.333 to 1.422. The 3.00-cm and 5.00-cm PCF interferometers possessed comparable performance for the resolution in the range of 1.62 × 10−4–8.88 × 10−4 RIU and 1.02 × 10−4–9.04 × 10−4 RIU, respectively. Furthermore, the 3.00-cm and 5.00-cm PCF interferometers exhibited relatively small sensitivity variation . Such a highly sensitive PCF interferometer is attractive for chemical, biological, biochemical sensing with aqueous solutions, as well as for civil engineering and environmental monitoring applications.We have successfully demonstrated the feasibility of fabricating a class of highly sensitive refractive-index sensor based on the PCF interferometer that was realized by fusion splicing a short section of PCF between two standard single mode fibers (Corning SMF-28) using a Fitel-S175 splicer. The interferometer was completed by making two collapsing points in a PCF at localized regions with a fusion splicer. The high uniform interference fringes over the available spectral range  could be obtained. The realization of the sensor is through the measurement of the transmission spectrum of the PCF interferometer. A red shift in the resonance wavelength was observed with the increase of RI. We demonstrate the sensor resolution for different lengths of PCF interferometers by transmission spectrum interrogation, and the findings indicating that a resolution as low as 1.62 × 10


The designed generator produced up to 3500 ppm of ClO2 at up to 99% purity. Tilapia fillets were soaked in a 400 ppm ClO2 solution for 5, 10, and 25 min. Results show that total plate counts of tilapia, respectively, decreased by 5.72 to 3.23, 2.10, and 1.09 log CFU/g. In addition, a 200 ppm ClO2 solution eliminated coliform bacteria and Escherichia coli in 5 min with shaking treatment. Furthermore, ClO2 and trihalomethanes (THMs) residuals on tilapia fillets were analyzed by GC/MS and were nondetectable (GC-MS detection limit was 0.12 ppb). The results conform to Taiwan's environmental protection regulations and act governing food sanitation.This research aimed to develop an electrolysis method to generate high-concentration chlorine dioxide (ClO It is commonly used on drinking water and environmental disinfection. It was also recommended as a commercial sanitizer to replace electrolyzed oxidizing water [2), hypochlorous acid (HOCl), and hypochlorite (OCl−) [2 gas has been successfully used to disinfect strawberries, lettuce, cabbage, and cucumbers with continuous methods [2 was evaluated for cleaning tilapia fillets with different cleaning methods.Chlorine dioxide  –5. Contae (OCl−) –9. In Tae (OCl−) , 10–12.  methods , 13–17. 2 is commonly generated using chemical methods that react sodium chloride, sodium hypochlorite, or sodium chlorate with sulfuric acid or hydrochloride acid [2 needs a strong acid (pH 2~3), inhibits Cl2 hydrolysis, and takes a long time for activation. The yield of ClO2 depends on the purity of the raw materials, the catalyst, pH, reaction time, and temperature [2 [2 to disinfect with water, while simultaneously monitoring trihalomethane (THM) residuals on tilapia fillets.Commercial ClOide acid , 19. Theperature –24. Furtature [2 , 26. TheE. coli Petrifilm no. 6414 were purchased from Microbiology Products 3 M Health Care .Tilapia fillets were bought from a local traditional market in Pingtung, Taiwan. The microbiological media used in this study were peptone and tryptic soy agar (TSA) purchased from Difco Laboratories ; these were prepared according to the manufacturer's specifications. 3 M Coliform and 2 collecting tanks, and a cooling system  [2 collecting tank was maintained at 5~10°C by cooling water from the cooling system. NaCl was electrolyzed into NaClO2. The reaction equation is as follows:The self-designed electrolysis equipment consisted of a raw material tank, an electrolyzer, an air pump, two ClO0722557) .  Figure2 was further electrolyzed, the ClO2− was attracted by the cathode, and H2O was attracted by the anode to release H2  and pH of the ClO2 solutions were measured using an ORP/pH meter .The resultant ClO2 analysis: the concentration of ClO2 was analyzed using the iodine method [2 solution at 10 mL was diluted 200 times with pure water. It was adjusted to five pH levels and then titrated with a 0.01 N sodium thiosulfite solution. The titration volumes were A, B, C, D, and E. The following calculation formulas were used to calculate the concentrations of ClO2, Cl2, ClO2−, and ClO3−:ClOe method . The ClOHere, N is the concentration of the sodium thiosulfite solution.2 solutions were used to test the effect of tilapia disinfection. Tilapia fillets were inoculated with coliforms or E. coli at a concentration of 5~6 log CFU/g. At 99% purity, ClO2 solutions of 50, 100, and 200 ppm were used to wash the fillets by soaking or shaking treatment for 5, 15, and 25 min, and the total plate counts, coliforms, and E. coli of the fillets were determined.Tilapia fillets were incubated at 37°C until the total plate count reached 5~6 log CFU/g. The different purities (45%~99%) of 400 mg/L ClOE. coli assays followed Sasithorn and Sirirat [The total plate count assay followed the China National Standard (CNS 10890 N6186)  method:  Sirirat  using 1 μm phase film thickness) from Agilent Technologies. The initial temperature was 45°C for 3 min and then increased by 8°C/min to a final temperature of 220°C for 20.5 min. The injector temperature was set to 200°C. Nitrogen was used as the carrier gas at a flow rate of 38.5 mL/min.The analytical procedure was modified from Stack et al.'s  gas chroMS was operated in the electron ionization mode at 70 eV. The mass range was scanned at 40~350 m/z and for 0.60 seconds per scan for the full-scan mode. Temperatures for the trap, manifold, and transfer line were set to 250, 50, and 280°C, respectively. All data for quantification were collected in the selected ion monitoring mode at 83 and 85 m/z for chloroform, 127 and 129 m/z for dibromochloromethane, and 173 m/z for bromoform.P < 0.05).Three replicates were conducted, and each sample was assayed in duplicate. Data collected from the experiments were analyzed by an analysis of variance (ANOVA) and Duncan's multiple range test using the SAS 8.2 program . Signifi2 and 20% NaCl generated 4749 ppm of total chlorine and 99.8% pure ClO2, respectively. The pH was 2.18, and ORP was 1440 mV  of 99% ClOE. coli are shown in Tables 2 solutions with the shaking method for 5 min, the coliform counts, respectively, decreased to 1.78, 1.07 log CFU/g, and nondetectable  for 25 min, the waste solutions were analyzed for THMs using GC/MS. THMs include chloroform, dichloromethane, and methyl chloride. The results show that no THMs were detected in a used ClO2 solution after soaking (GC-MS detection limit was 0.12 ppb), as shown in 2 solution was no detected in solution (the instrument detection limit was 0.1 ppb).THM residuals are a problem for the safety of chlorine-treated food materials , 40. Aft2 using electrochemical technology. The maximum concentration and purity of ClO2 were obtained when using a mixture that blended 20% NaCl and 7%~10% NaClO2 together as the electrolytes. The concentration and purity of ClO2 were 3200~4700 ppm and 99.5~99.7%, respectively. Disinfection results indicate that a 200 ppm ClO2 solution reduced the total bacterial, coliform, and E. coli counts on tilapia fillets by 3.0~4.0 log CFU/g (P < 0.05). The soaking wash treatment was more effective than the shaking method. A GC-MS analysis also showed that when using a 200 ppm bactericide solution for 25 min, residual THMs of the ClO2 solution were nondetectable. Bactericidal treatment with a ClO2 solution for tilapia fillets also conforms to Taiwan's environmental protection regulations and act governing food sanitation. The ClO2 solution is indeed a safer method for treating seafood, and our novel electrolysis equipment can produce highly purified, low-cost ClO2 to disinfect with water, for immediate use for agricultural product and seafood treatment.The results demonstrated the feasibility of stably producing ClO