Development of a Semi-Quantitative Multiplex PCR Method for Detecting Residual Pichia Pastoris Host Cell DNA in Biopharmaceuticals

 The use of the methylotrophic yeast, Pichia pastoris, as one of the most effective and versatile systems for the expression of heterologous proteins in biopharmaceutical manufacturing has become increasingly popular in recent years. The impurity caused by residual host cell DNA is one of the major concerns in production of recombinant therapeutics. The aim of the present study was to develop a semi-quantitative, multiplex PCR method to determine the level of impurity in biopharmaceuticals produced in Pichia pastoris as the host. Primers were designed based on the rDNA repeat region and optimized to achieve the limit of detection in a multiplex PCR following by analyzing with MYImageAnalysis (Thermo Fisher Scientific, USA) software to quantify the concentration of Pichia pastoris genomic DNA in pertinent controls and drug samples. The multiplex PCR were able to detect up to 1 femtogram (fg) of genomic DNA under optimized conditions. Moreover, achieved concentration of DNA in controls and samples through relevant standard curve indicates the feasibility of this method in the presence of inhibitory effects. In comparison with other methods such as real-time PCR and Threshold assay, the assay shows acceptable sensitivity, precision and linearity along with ease of use, low equipment costs and analyte flexibility. We thus propose this method to be considered as a useful tool to estimate host cell residual DNA in biopharmaceuticals produced in Pichia pastoris.Highlights The impurity of residual host cell DNA is an important concern in production of biopharmaceuticals.Pichia pastoris is an effective and versatile system for the expression of recombinant proteinsQuantitative Polymerase Chain Reaction could be used for quantifying residual host-cell DNAWe designed a sensitive and valid PCR method for detection and quantification of Pichia residual DN

A minireview on the in vitro and in vivo experiments with anti-Escherichia coli O157 : H7 phages as potential biocontrol and phage therapy agents

Phage therapy is an old method of combating bacterial pathogens that has recently been taken into consideration due to the alarming spread of antibiotic resistance. Escherichia coli 0157:H7 is a foodborne pathogen that causes hemorrhagic colitis and life-threatening Hemolytic Uremic Syndrome (HUS). There are several studies on isolation of specific phages against E. coli 0157:H7 and more than 60 specific phase have been published so far. Although in vitro experiments have been successful in elimination or reduction of E. coli 0157:H7numbers, in vivo experiments have not been as promising. This may be due to escape of bacteria to locations where phages have difficulties to enter or due to the adverse conditions in the gastrointestinal tract that affect phage viability and proliferation. To get around the latter obstacle, an alternative phage delivery method such as polymer microen-capsulation should be tried. While the present time results are not very encouraging the work should be continued as more efficient phage treatment regimens might be found in future. (C)2016 Elsevier B.V. All rights reserved.Peer reviewe

Biosynthesis and recovery of rod-shaped tellurium nanoparticles and their bactericidal activities

In this study, a tellurium-transforming Bacillus sp. BZ was isolated from the Caspian Sea in northern Iran. The isolate was identified by various tests and 16S rDNA analysis, and then used to prepare elemental tellurium nanoparticles. The isolate was subsequently used for the intracellular biosynthesis of elemental tellurium nanoparticles. The biogenic nanoparticles were released by liquid nitrogen and purified by an n-octyl alcohol water extraction system. The shape, size, and composition of the extracted nanoparticles were characterized. The transmission electron micrograph showed rod-shaped nanoparticles with dimensions of about 20 nm � 180 nm. The energy dispersive X-ray and X-ray diffraction spectra respectively demonstrated that the extracted nanoparticles consisted of only tellurium and have a hexagonal crystal structure. This is the first study to demonstrate a biological method for synthesizing rod-shaped elemental tellurium by a Bacillus sp., its extraction and its antibacterial activity against different clinical isolates

A Recombinant Plasmodium vivax Apical Membrane Antigen-1 to Detect Human Infection in Iran

In Iran, Plasmodium vivax is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant P. vivax apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant His-tagged protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-P. vivax antibodies in the field was compared to light microscopy on 84 confirmed P. vivax patients and compared to 84 non-P. vivax infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with P. vivax infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis

A suggested new bacteriophage genus: “Viunalikevirus”

We suggest a bacteriophage genus, “Viunalikevirus”, as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-012-1360-5) contains supplementary material, which is available to authorized users

Secretory expression and purification of a soluble NADH cytochrome b5 reductase enzyme from Mucorracemosus in Pichiapastoris based on codon usage adaptation

Abstract The genome of Mucor racemosus was analyzed to determine the relative levels of codon usage. The codon bias differred from that of Escherichia coli. The active, soluble isoform of NADH cytochrome b5 reductase containing 228 amino acids was successfully overexpressed and secreted using alpha factor in Pichia pastoris under the control of the alcohol oxidase promoter and finally purified. The culture medium and incubation time were optimized, and the maximum expression level observed was about 23 U/ml using X-33

Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73%) with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli

Radioprotective Effect of Melatonin on The Cervical Spinal Cord in Irradiated Rats

Objective: It has been suggested that the vascular endothelial growth factor (VEGF) gene expression plays an important role in radiation-induced injury to the spinal cord. This study assesses the radioprotective effects of N-acetyl-5-methoxytryptamine (melatonin) through its modulation of VEGF expression after localized irradiation of the cervical spinal cord.Materials and Methods: In this experimental study, we divided 192 male rats into four groups: 1. control (n=48); 2. rats that received an intraperitoneal (IP) injection of melatonin (n=48); 3. rats that received an IP injection of melatonin 30 minutes prior to cervical spinal cord gamma irradiation [dose: 22 Gy; (n=48)]; and 4. rats that received an IP injection of vehicle prior to spinal cord irradiation (n=48). The changes in VEGF expression were assessed using real-time RT-PCR and enzyme-linked immunosorbent assays. Samples for light microscopy were stained with hematoxylin and eosin (H&E). The differences among the groups were analyzed using the analysis of variance (ANOVA) test followed by Tukey’s multiple comparisons test.Results: Up-regulation of VEGF expression was observed from 8 to 22 weeks after irradiation (p<0.05). Paralysis and other radiation-induced myelopathy manifestations developed within 22 weeks after irradiation. VEGF expression in the melatonin pre-treatment group significantly down-regulated in the 20th and 22nd weeks after irradiation compared to the radiation-only group.Conclusion: The results support the hypothesis that modulation of VEGF expression by melatonin administration may increase the survival rate of irradiated animals

Metal, Metalloid, and Oxide Nanoparticles for Therapeutic and Diagnostic Oncology

Nanoparticles have comprehensively affected various sights of human life. Through the wide range of claims about nanosized particles and their functions, biomedical applications are of much interest among health care researchers due to the nanoparticles' potential for use in the process of disease diagnosis, control and treatment. In this regard, inorganic nanoparticles, which have high potential in diagnostic and therapeutic systems, have recently received much attention in oncology. Although inorganic nanoparticles initially seemed an appropriate tool for cancer imaging and diagnosis, their ability to attack cancerous cells as anticancer drugs or carriers in other drugs has also demonstrated promising results. The present review primarily provides a brief survey of various studies in which metal nanoparticles such as gold, silver, iron oxide, and metalloid nanoparticles viz. tellurium and bismuth were exploited for therapeutic or diagnostic purposes in oncology. Then the application of selenium nanoparticles as a therapeutic agent against cancer in in vitro and in vivo studies is reviewed in detail. Although inorganic nanoparticles seem to be useful tools for cancer imaging and diagnosis, their potential for attacking cancerous cells as anticancer substances or even carriers for anticancer medications should not be underestimated

Gene expression profiling revealed overexpression of vesicle amine transport protein-1 (<i>VAT-1</i>) as a potential oncogene in gastric cancer

161-165Gastric cancer (GC) is one of the most common types of cancer worldwide. Owing to the distinct molecular pathology and the increasing progression rate of GC in Asia, suppression subtractive hybridization (SSH) was used as a high-throughput gene expression analysis method to find genes associated with GC pathogenesis. Total RNA was extracted from the clinical samples, and mRNA was isolated and used in SSH method. The subtracted library was subjected to cloning and the randomly selected clones were sequenced. qRT-PCR was used for expression analysis. The overexpression of vesicle amine transport protein-1 (VAT-1) gene was observed and its expression was analyzed by qRT-PCR in clinical tissue samples. According to the potential oncogenic activity of VAT-1 and its probable surface-occurrence in GC cells, it might be involved in GC pathogenesis and invasion, and it is suggested to be investigated in the diagnosis and therapeutics of GC