Modification in Media Composition to Obtain Secretory Production of STxB-based Vaccines using Escherichia coli

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl2). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.

Analytical methodologies for determination of methotrexate and its metabolites in pharmaceutical,biological and environmental samples

Methotrexate(MTX)is a folate antagonist drug used for several diseases,such as cancers,various malignancies,rheumatoid arthritis(RA)and inflammatory bowel disease.Due to its structural features,including the presence of two carboxylic acid groups and its low native fluorescence,there are some challenges to develop analytical methods for its determination.MTX is metabolized to 7-hydroxymethotrexate(7-OH-MTX),2,4-diamino-N10-methylpteroic acid(DAMPA),and the active MTX polyglutamates(MTXPGs)in the liver,intestine,and red blood cells(RBCs),respectively.Additionally,the drug has a narrow therapeutic range;hence,its therapeutic drug monitoring(TDM)is necessary to regulate the pharmacokinetics of the drug and to decrease the risk of toxicity.Due to environmental toxicity of MTX;its sensitive,fast and low cost determination in workplace environments is of great interest.A large number of methodologies including high performance liquid chromatography equipped with UVevisible,fluorescence,or electrochemical detection,liquid chromatography-mass spectroscopy,capillary electrophoresis,UVevisible spectrophotometry,and electrochemical methods have been developed for the quantitation of MTX and its metabolites in pharmaceutical,biological,and environmental samples.This paper will attempt to review several published methodologies and the instrumental conditions,which have been applied to measure MTX and its metabolites within the last decade.

A simple multiplex PCR for assessing prevalence of extended-spectrumβ-lactamases producing Klebsiella pneumoniae in Intensive Care Units of a referral hospital in Shiraz,Iran

Objective:To identify three common genes(bla_(TEM),bla_(SHV)and bla_(CTX-M)responsible for ESBL production in Klebsiella pneumnniae(K.pneumnniae)isolated from Intensive Care Units of Namazi Hospital,Shiraz,Iran.Methods:A total of 60 non-repetitive nosocomial isolates from 60patients were selected during 2009-2010.The phenotypic identification of ESBL production was confirmed by Double Disk Synergy Test(DDST)according to CLSI guidelines.The ESBL's genotype was then analyzed by multiplex PCR of bla_(TEM),bla_(SHV)and bla_(CTX-M)genes and DNA sequencing.Results:The primary susceptibility tests of K.pneumoniae showed that among 10 examined antibiotics,the most resistant and susceptible antibiotics identified in this study were ampicillin and imipenem,respectively.The phenotypic determination of ESBL by DDST showed that 60%(n=36)of isolates produced ESBL.Multiplex PCR of genes among K.pneuunoniae isolates showed that 39%(n=18)of them have TEM,39%in=18)of then have both CTX-M and TEM and 13%(n=8)of them have TEM,SHV,CTX-M.Conclusions:Our findings reveal the high prevalence(60%)of ESBL prcxducing K.pneumnniae from ICL patients along with a new pattern of bla_(TEM)distribution differ from other countries.

Comparative Study on Characteristics and Cytotoxicity of Bifunctional Magnetic-Silver Nanostructures: Synthesized Using Three Different Reducing Agents

Magnetic-silver nanostructures were synthesized via optimized chemical conditions, and their characteristics and cytotoxicity were compared as candidates for the magnetic delivery of silver nanoparticles toward cancer cells. Magnetic-silver nanostructures were prepared through the reduction of silver ions in the presence of iron oxide nanoparticles using three different reducing agents （glucose, maltose and sodium citrate）. Their physicochemical characteristics were determined using ultraviolet-visible spectroscopy, X-ray diffraction analysis, transmission electron microscopy, selected area electron diffraction analysis, Fourier transformed infrared spectroscopy, atomic absorption spectroscopy, vibrating sample magnetometry and differential scanning calorimetry. Cytotoxic activities were evaluated against a human liver hepatocellular carcinoma cell line. Fabricated nanostructures, which exhibit differences in size, silver content, magnetic saturation value and cytotoxicity, represent sufficient superparamagnetic properties and considerable cytotoxicity to be suggested as effective tools in magnetic targeting of silver nanoparticles as an approach to cancer therapy.