Investigation of spore coat display of Bacillus subtilis β-galactosidase for developing of whole cell biocatalyst

The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses β-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101(ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of β-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction’s mixture.     

Virus-specific T-cell immunity correlates with control of GB virus B infection in marmosets

GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosets were assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-gamma ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.     

Proline-Functionalized Graphene Oxide Nanoparticles (GO–Pro NPs) Mitigate Salt-Induced Adverse Effects on Morpho-Physiological Traits and Essential Oils Constituents in Moldavian Balm (Dracocephalum moldavica L.)

Many methodologies have been established to lessen negative impacts of salinity on plants. Of those methodologies, nanoparticles (NPs) application has achieved great importance thanks to their unique physico-chemical properties. Consequently, formerly respecting encouraging impacts of graphene oxide (GO) and proline (Pro) on different plant processes under non-stress and stress conditions, proline-functionalized graphene oxide nanoparticles “GO–Pro NPs” were synthesized and characterized. Graphite powder, as starting material, was used to synthesize GO using modified Hummers method followed by functionalization of its surface by proline in basic media. Afterward, GO–Pro NPs, GO and Pro, each at 0, 50 and 100 mg L^?1 concentrations with three replications, were applied on Moldavian balm (Dracocephalum moldavica L.) plants to assay their effects under non-stress (0 mM) and salt stress (50 and 100 mM) conditions. GO–Pro NPs and Pro effectively alleviated negative effects of salinity through increasing morphological parameters, photosynthetic pigments, chlorophyll fluorescence parameters, chlorophyll index (SPAD), and membrane stability index (MSI) and decreasing hydrogen peroxide and malondialdehyde, as well. Also application of GO–Pro NPs enhanced proline, antioxidant enzymes activities, and most dominant constituents of essential oil. The highest MSI (48.87%) and proline content (15.36 µM g^?1 FW) were observed in plant treated with GO–Pro NPs (50 mg L^?1) under 100 mM NaCl salinity stress. The GO–Pro NPs treatment at lower dose (50 mg L^?1) could be introduced as the best preservative treatment for Moldavian balm under salt stress. GO application mostly had no effect on the measured parameters announcing it as carrier for Pro to enhance its efficiency. In conclusion, GO–Pro NPs application could promote Moldavian balm performance and essential oil under salinity presenting GO–Pro NPs as new treatment against stress conditions.

     

Hyperprolactinemia after laparoscopic ovarian drilling: An unknown phenomenon

Background  

The effects of ovarian drilling on the serum levels of gonadotropins and androgens have been studied previously. The aim of this study is to evaluate the effects of ovarian drilling on the serum prolactin levels and its relation to ovulation in women with polycystic ovary syndrome.     

High-performance liquid chromatographic determination of lamivudine in human serum using liquid-liquid extraction; application to pharmacokinetic studies

A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid - liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm x 6 mm i.d.) which was packed with 5 microm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5-2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively.     

Effects of omeprazole consumption on serum levels of trace elements

ProjectOmeprazole is one of the most frequently prescribed drugs in patients with gastroesophageal reflux disease (GERD). It increases the gastric pH and this in turn may change the intestinal absorption of trace elements. This study was conducted to assess the effects of omeprazole consumption on the serum level of trace elements.ProceduresThe studied subjects were selected from the list of patients referred to the gastroenterology department of 5 Azar hospital in Golestan province of Iran for whom omeprazole was prescribed by a gastroenterologist. Blood samples were obtained before (phase I) and after an eight-week period (phase II) of omeprazole consumption. Serum levels of trace elements were assessed by the photometric method.ResultsSixty seven patients were recruited of whom, 35.82% were males. There was no significant difference in serum levels of Fe, P, Ca and Cu between phases I and II. Serum concentration of Zn was significantly lower in phase II than I (P = 0.02). The proportion of male patients with low Zn levels was significantly higher in phase II (50%) than I (16.7%) (P = 0.01). We found no significant difference in the proportion of female patients with low Zn levels between phase I (37.2%) and phase II (27.9%).ConclusionsWe found no significant reduction in serum levels of Fe, P, Ca and Cu in phase II. However, our results showed that serum level of Zn was significantly lower after omeprazole consumption in males. So, nutritional supplement of Zn should be considered in male patients treated with omeprazole.     

Description of Crassolabium persicum sp. n. (Nematoda, Dorylaimida, Qudsianematidae), an interesting species from Iran

A new species of the genus Crassolabium, Crassolabium persicumsp. n., collected from Arasbaran rangelands of Iran, is described and illustrated. It is characterized by its body 1.92-2.40 mm long, lip region offset by constriction and 17-19 μm wide, odontostyle 16-19 μm long with aperture occupying less than one-third (27-30%) its length, neck 428-690 μm long, pharyngeal expansion 369-390 μm long or occupying 54-56% of total neck length, female genital system amphidelphic, uterus bipartite and 162-218 μm long or 2.3-3.5 times as long as body diameter, pars refringens vaginae well developed, V = 54-57.5, vulva longitudinal, prerectum bearing a blind sac, tail conical with rounded tip to conoid (25-36 μm, c=60-69, c'=0.5-0.9), spicules 68-72 μm long, precloacal pair of supplements far (22-27 μm) from cloacal aperture, and 13-17 shortly spaced ventromedian supplements with hiatus. The new taxon is compared in depth to its relatives in Crassolabium as well as other similar species of Aporcelaimellus and Amblydorylaimus.     

Comparative modeling of CCRL1, a key protein in masked immune diseases and virtual screening for finding inhibitor of this protein

Human CCRL1 belongs to the family of silent chemokine receptors. This transmembrane protein plays a role in blunting function of chemokines through binding to them. This will attenuate immune responses. Interaction between CCRL1 and CCL21 determines this immune extinction. Thus inhibiting the action of this atypical chemokine seems to stimulate immune responses especially in the case of suppressed and immune deficient conditions. In this study we predicted 3D structure of CCRL1 using comparative modeling and Hiddebn Markov Model algorithm. Final predicted model optimized by Modeller v9.8 and minimized regarding energy level using UCSF chimera candidate version1.5.3. ClasPro webserver was used to find interacting residues between CCRL1 and CCL21. Interacting residues were used as target for chemical inhibitors by simulated docking study. For finding potential inhibitors, library of KEGG compounds screened and 97 obtained chemicals docked against interacting residues between CCRL1- CCL21 and MolDock was used as docking scoring function. Results indicated that Hexadecanal is a potential inhibitor of CCRL1- CCL21 interaction. Inhibition of this interaction will increase intercellular level of CCl21 and interaction between CCL21 and CCR7 causes immune potentiaiton.     

High-performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: Application to a bioequivalence study

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 microg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 microg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.     

Determination of oseltamivir carboxylic acid in human serum by solid phase extraction and high performance liquid chromatography with UV detection

A new straightforward method based on cloud-point extraction (CPE) has been developed, optimized and validated for the determination of venlafaxine in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection. The non-ionic surfactant Triton X-114 (polyethylene glycol tert-octylphenyl ether) was chosen as the extract solvent. Separation was obtained using a reversed-phase Diamonsil column (C(18), 250mmx4.6mm I.D., 5mum) and a mobile phase composed of acetonitrile-phosphate buffer solution (pH 3.0)-triethylamine (33.5:66.5:0.4). Fluorescence detection was used (lambda(ex) 276nm, lambda(em) 598nm). Maprotiline was used as the internal standard. Under the optimum conditions, the linear range of venlafaxine in human plasma was 10-800ngmL(-1) (r(2)=0.9995). The limit of detection (LOD) was less than 2ngmL(-1) (S/N=3) and the limit of quantification (LOQ) was less than 10ngmL(-1) (S/N=10). The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine capsules in nine healthy volunteers.