In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes

Objectives

Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.

Methods

Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.

Results

The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of?~?63 and?~?72 kDa confirmed in western blotting, respectively.

Conclusion

The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

     

Salicylate poly(vinyl chloride) membrane electrode based on (2-[(E)-2-(4-nitrophenyl) hydrazono]-1-phenyl-2-(2-quinolyl)-1-ethanone) Cu(II)

A new salicylate-selective electrode based on the complex of (2-[(E)-2-(4-nitrophenyl)hydrazono]-1-phenyl-2-(2-quinolyl)-1-ethanone) Cu(II) as the membrane carrier was developed. The electrode exhibited a good Nernstian slope of -59.6+/-1.0 mV/decade and a linear range of 1.0 x 10(-6) to 1.0M for salicylate. The limit of detection was 5.0 x 10(-7) M. The electrode had a fast response time of 10 s and can be used for more than 3 months. The selective coefficients were determined by the fixed interference method and could be used in the pH range of 4.0 to 10.5. The electrode was employed as an indicator electrode for direct determination of salicylate in pharmaceutical and biological samples.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

Novel 2,2'-[1,2-ethanediylbis(nitriloethylidyne)]-bis-hydroquinone double-wall carbon nanotube paste electrode for simultaneous determination of epinephrine, uric acid and folic acid

The electro-oxidation of epinephrine (EP), uric acid (UA), folic acid (FA), and their mixture has been studied by modified carbon nanotube paste electrode of 2,2'-[1,2-ethanediylbis(nitriloethylidyne)]-bis-hydroquinone using cyclic voltammetry, chronoamperometry and differential pulse voltammetry. This modified electrode exhibited potent and persistent electron mediating behavior followed by well-separated oxidation peaks towards EP, UA and FA with activation overpotential. For the ternary mixture containing EP, UA and FA the three compounds can be well separated from each other at the scan rate of 20mVs(-1). The obtained catalytic peak current, was linearly dependent on the EP, UA and FA concentrations in the range of 0.7-1200muM, 25-750muM and 15-800muM and the detection limits for EP, UA and FA were 0.216+/-0.004, 8.8+/-0.2 and 11.0+/-0.3muM, respectively. The diffusion coefficient (D), and the kinetic parameters such as electron transfer coefficient, (alpha) and heterogeneous rate constant, (k') for EP were also determined using electrochemical approaches. The modified electrode showed good sensitivity, selectivity and stability, and was employed for the determination of EP, UA and FA in the real samples.     

Clinical and therapeutic implications of BRAF mutation heterogeneity in metastatic melanoma

Heterogeneity of BRAF mutation in melanoma has been a controversial subject. Quantitative data on BRAF allele frequency (AF) are sparse, and the potential relationship with response to BRAF inhibitors (BRAFi) in patients with metastatic melanoma is unknown. We quantitatively measured BRAF AF in a cohort of treatment naïve metastatic melanoma samples by pyrosequencing and correlated with survival data in patients treated with BRAFi as part of their clinical care. Fifty‐two samples from 50 patients were analysed. BRAF V600E mutations were detected in 71.1% of samples followed by V600K (25%) and V600R (3.9%). There was a wide range of AF from 3.9% to 80.3% (median 41.3%). In 33 patients treated with BRAFi, there was no difference in overall or progression‐free survival when the patients were categorized into high or low AF groups. There was no correlation between AF and degree of response, and no difference in survival based on genotype.     

A direct nitric oxide gas delivery system for bacterial and mammalian cell cultures.

Nitric oxide (NO) is the smallest known gaseous signaling molecule released by mammalian and plant cells. To investigate the pathophysiologic role of exogenous NO gas (gNO) in bacterial and mammalian cell cultures, a validated in vitro delivery method is required. The system should be able to deliver gNO directly to bacterial and/or cell cultures in a continuous, predictable, and reproducible manner over a long period of time (days). To accomplish this, a gas delivery system was designed to provide optimal growth conditions for bacteria and/or mammalian cells. Parameters for cell exposure, such as concentration of gNO, nitrogen dioxide (NO(2)), oxygen (O(2)), temperature, and relative humidity (RH) were continuously monitored and evaluated. Uptake of gNO into various media was monitored by measuring the nitrite concentration using the Griess reagent technique. A selection of standard growth media [saline, tryptic soy broth (TSB), Middlebrook 7H9 (MB 7H9), and Dulbecco's modified Eagle's medium (DMEM)] exposed to various concentrations of gNO revealed a steady and consistent transfer of gNO into the aqueous phase over a 48-h period. Validation of optimal growth conditions within the device, as compared to a conventional incubator, were accomplished by growing and observing viability of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and human fibroblast cultures in the absence of gNO. These results indicate that an optimal growth environment for the above tested cells was accomplished inside the proposed delivery system. Dose-dependent toxicological data revealed a significant bacteriostatic effect on P. aeruginosa and S. aureus with continuous exposure to 80 ppm gNO. No toxic effects were observed on dermal fibroblast proliferation at concentrations up to 400 ppm gNO for 48 h. In conclusion, the designed gNO exposure system is capable of supporting cellular viability for a representative range of prokaryote and eukaryotic cells. The exposure system is also capable of obtaining toxicological data. Therefore, the proposed device can be utilized to continuously expose cells to various levels of gNO for up to 72 h to study the in vitro effects of gNO therapy.     

The Effect of a Yeast Probiotic on Acute Diarrhea in Children

A probiotic is a living micro-organism administered to promote the health of the host by treating or preventing infections owing to strains of pathogens. Saccharomyces boulardii is a nonpathogen yeast that has a direct inhibitory effect on the growth of many pathogens, an anti-secretory effect and a trophic effect on enterocytes. The aim of this study was to determine the effect of S. boulardii on diarrhea in children. The children from 6 months to 6 years of age with acute watery diarrhea admitted in pediatric clinic in Kashan in 2012 were included in this trial. Exclusion criteria were high fever (T > 38.5 °C), severe dehydration, bloody diarrhea, severe malnutrition, using of antibiotics, anti-diarrheal or antifungal drugs and children with more than one complain. Two hundred patients were assigned into two groups: A total of 100 patients were treated with S. boulardii in addition to ORS (case group) and 100 patients were given placebo in addition to ORS (control group). The duration of diarrhea and frequency of stools were recorded by asking the mothers of the children every day. The results showed that the defecation frequency after second day of treatment in the case group was significantly less than the control group (P = 0.001) and the mean numbers of days of diarrhea was significantly lower in the case group (P = 0.001). The result of this study confirms that S. boulardii reduces the frequency of stool and duration of illness in children.     

Biodesulfurization of dibenzothiophene by Gordonia sp. AHV-01 and optimization by using of response surface design procedure

A novel desulfurizing bacterium has been isolated from oil-contaminated soils in Khuzestan. The ability for dibenzothiophene desulfurization and its biochemical pathway were investigated. The bacterium was identified as Gordonia sp. AHV-01 (Genbank Accession No HQ607780) by 16S rRNA gene sequencing. HPLC results and Gibb's assay were shown that dibenzothiophene desulfurized via 4S-pathway Maximum growth (0.426 g dry cells/L) and produced 2-hydroxybiphenyl (63.1 microM) were observed at 120 h of cultivation. By using of response surface design procedure the optimization of pH, temperature and rotary shaker round on the desulfurization reaction of isolate AHV-01 were performed. The optimum conditions were determined at pH of 7.0, temperature of 30 degrees C and rotary shaker round of 180 rpm. At these conditions, the dibenzothiophene desulfurization activity was increased and maximum 2-hydroxybiphenyl production was detected 70.29 microM at 96 h. According to these results, Isolate AHV-01 was capable to desulfurize dibenzothiophene via 4S-pathway and likely it can be useful to reduce organic sulfur contents of crude oil.     

Towards alpha- or beta-D-C-glycosyl compounds by tin-catalyzed addition of glycosyl radicals to acrylonitrile and vinylphosphonate,and flexible reduction of tetra-O-acetyl-alpha-D-glucopyranosyl bromide with cyanoborohydride

Photo-induced radical addition of acetylated alpha-D-glucopyranosyl bromide (1). to acrylonitrile or diethyl vinylphosphonate, in the presence of catalytic amounts of tri-n-butyltin chloride and sodium (or tetra-n-butylammonium) cyanoborohydride in excess, allowed efficient preparations of alpha-configurated nonononitrile and 2-(alpha-D-glucopyranosyl)-ethylphosphonate (79, 70% yields, respectively). These conditions led to 2-(alpha-D-manno-, and galactopyranosyl)-ethylphosphonates in 68 and 76% yields. Similarly, radical addition of acetylated 1-bromo-beta-D-glucopyranosyl chloride (2). to acrylonitrile or diethyl vinylphosphonate afforded mainly intermediate chlorides which, upon radical reduction with excess tri-n-butyltin hydride, afforded the corresponding beta anomers (40 and 38%, respectively) by sequential C-C and C-H bond formation. Stereocontrol relies on the alpha-stereoselective quenching of D-glycopyranos-1-yl radicals. We found also that UV light irradiation of 1 with excess NaBH(3)CN in tert-butanol afforded either 1,3,4,6-tetra-O-acetyl-2-deoxy-alpha-D-arabino-hexopyranose (65% after crystallization) or, when 10% mol thiophenol was added, 2,3,4,6-tetra-O-acetyl-1,5-anhydro-D-glucitol (79%). These are simple, tin-free, and easily controlled conditions, which compare well with known preparations of these reduced sugars.