Escherichia coli cytosine deaminase: Structural and biotechnological aspects.

Suicide gene therapy involves introducing viral or bacterial genes into tumor cells, which enables the conversion of a nontoxic prodrug into a toxic-lethal drug. The application of the bacterial cytosine deaminase (bCD)/5-fluorocytosine (5-FC) approach has been beneficial and progressive within the current field of cancer therapy because of the enhanced bystander effect. The basis of this method is the preferential deamination of 5-FC to 5-fluorouracil by cancer cells expressing cytosine deaminase (CD), which strongly inhibits DNA synthesis and RNA function, effectively targeting tumor cells. However, the poor binding affinity of toward 5-FC compared to the natural substrate cytosine and/or inappropriate thermostability limits the clinical applications of this gene therapy approach. Nowadays, many genetic engineering studies have been carried out to solve and improve the activity of this enzyme. In the current review, we intend to discuss the biotechnological aspects of Escherichia coli CD, including its structure, functions, molecular cloning, and protein engineering. We will also explore its relevance in cancer clinical trials. By examining these aspects, we hope to provide a thorough understanding of E. coli CD and its potential applications in cancer therapy.

Case reports of juvenile GM1 gangliosidosisis type II caused by mutation in GLB1 gene.

BACKGROUND: Type II or juvenile GM1-gangliosidosis is an autosomal recessive lysosomal storage disorder, which is clinically distinct from infantile form of the disease by the lack of characteristic cherry-red spot and hepatosplenomegaly. The disease is characterized by slowly progressive neurodegeneration and mild skeletal changes. Due to the later age of onset and uncharacteristic presentation, diagnosis is frequently puzzled with other ataxic and purely neurological disorders. Up to now, 3-4 types of GM1-gangliosidosis have been reported and among them type I is the most common phenotype with the age of onset around 6 months. Various forms of GM1-gangliosidosis are caused by GLB1 gene mutations but severity of the disease and age of onset are directly related to the position and the nature of deleterious mutations. However, due to its unique genetic cause and overlapping clinical features, some researchers believe that GM1 gangliosidosis represents an overlapped disease spectrum instead of four distinct types. CASE PRESENTATION: Here, we report a less frequent type of autosomal recessive GM1 gangliosidosis with perplexing clinical presentation in three families in the southwest part of Iran, who are unrelated but all from "Lurs" ethnic background. To identify disease-causing mutations, Whole Exome Sequencing (WES) utilizing next generation sequencing was performed. Four patients from three families were investigated with the age of onset around 3 years old. Clinical presentations were ataxia, gate disturbances and dystonia leading to wheelchair-dependent disability, regression of intellectual abilities, and general developmental regression. They all were born in consanguineous families with no previous documented similar disease in their parents. A homozygote missense mutation in GLB1 gene (c. 601 G > A, p.R201C) was found in all patients. Using Sanger sequencing this identified mutation was confirmed in the proband, their parents, grandparents, and extended family members, confirming its autosomal recessive pattern of inheritance. CONCLUSIONS: Our study identified a rare pathogenic missense mutation in GLB1 gene in patients with complex neurodevelopmental findings, which can extend the list of differential diagnoses for childhood ataxia in Iranian patients.

Designing of a Novel Fusion Protein Vaccine Candidate Against Human Visceral Leishmaniasis (VL) Using Immunoinformatics and Structural Approaches.

Leishmaniasis is caused by an obligate intracellular protozoan parasite. The clinical forms of leishmaniasis differ from cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis (VL) which depend on the parasite species and the host's immune responses. There are significant challenges to the available anti-leishmanial drug therapy, particularly in severe forms of disease, and the rise of drug resistance has made it more difficult. Currently, no licensed vaccines have been introduced to the market for the control and elimination of VL. A potential target for use in candidate vaccines against leishmaniasis has been shown to be leishmania Kinetoplastid membrane protein-11 (KMP-11) antigen. In this study, we chose KMP-11 antigen as target antigen in our vaccine construct. In addition, B-type flagellin (fliC) was used as an adjuvant for enhancing vaccine immunogenicity. The GSGSGSGSGSG linker was applied to link the KMP-11 antigen and fliC (KMP-11-fliC) to construct our fusion protein. Bioinformatics approaches such as; 3D homology modeling, CTL, B-cell, MHC class I and II epitopes prediction, allergenicity, antigenicity evaluations, molecular docking, fast simulations of flexibility of docked complex and in silico cloning were employed to analysis and evaluation of various properties of the designed fusion construct. Computational results showed that our engineered structure has the potential for proper stimulation of cellular and humoral immune responses against VL. Consequently, it could be proposed as a candidate vaccine against VL according to these data and after verifying the efficacy of the candidate vaccine through in vivo and in vitro immunological tests.

Synergetic Effect of Silver Nanoparticles and UVC Irradiation on H2AX Gene Expression in TK6 Cells.

OBJECTIVE: The use of nanoscale particles, for instance silver nanoparticles (Ag NPs) has considerably increased recently. Since Ag NPs can be transmuted into silver ions; the toxicity and genotoxicity of these NPs along with other external factors such as ultraviolet type C (UVC) irradiation must be evaluated. In the present study, the aim was to investigate the genotoxic effects Ag NPs and UVC co-exposure on human lymphoblastoid TK6 cells. MATERIALS AND METHODS: In this experimental study, Ag NPs (~20 nm) were purchased from US Research Nanomaterials Inc. and H2AX gene expression was evaluated using quantitative real time polymerase chain reaction (qRT-PCR), 1 and 24 hours post Ag NPs and UVC treatment. RESULTS: Results showed that treatment of TK6 cells with different Ag NP concentrations without exposure to UVC can reduce H2AX gene expression, but treatment of these cells with Ag NPs in combination UVC irradiation can reduce viability that leads to a synergistic increase in the amount of H2AX gene expression. CONCLUSION: According to our findings, Ag NPs can act to sensitize cells to UVC radiation when used for cancer treatment. So, combination of Ag NPs and UVC irradiation could be used in radiotherapy.

Co-exposure to an Aryl Hydrocarbon Receptor Endogenous Ligand, 6-Formylindolo[3,2-b]carbazole (FICZ), and Cadmium Induces Cardiovascular Developmental Abnormalities in Mice.

6-Formylindolo[3,2-b]carbazole (FICZ) is a signal substance and an endogenous activator of aryl hydrocarbon receptor (AHR). Cadmium (Cd) is an environmental pollutant that can activate both AHR and Wnt/ß-catenin signaling pathways. We aimed to determine how dysregulated signaling through AHR-Wnt/ß-catenin cross-talk can influence mice heart development. Mice fetuses were exposed to Cd alone or in combination with FICZ in gestation day (GD) 0. In GD18, fetuses were harvested and randomly divided into two parts for stereological and molecular studies. Stereological and tessellation results revealed that when fetuses were co-exposed with FICZ and Cd, abnormalities were synergistically raised. In the presence of FICZ, mRNA expression levels of Wnt/ß-catenin target genes significantly enhanced, especially when animals co-treated with FICZ and Cd. Based on these findings, we propose that chemical pollutants can interfere with the normal function of AHR that has a physiological role in regulating Wnt/ß-catenin during cardiogenesis.

Enhancing Stability of Destabilized Green Fluorescent Protein Using Chimeric mRNA Containing Human Beta-Globin 5' and 3' Untranslated Regions.

BACKGROUND: In spite of recent progress in mRNA technologies and their potential applications for treatment of human diseases, problems such as the transient nature of mRNA limit the stability of gene up-regulation and, thus, potentially reduce mRNA efficiency for gene therapy. Using human ß-globin 5' and 3' untranslated regions (UTRs), this study aimed to develop the different chimeric constructs of mRNAs to increase the stability of destabilized green fluorescent protein (EGFPd2) in HEK 293 cells. METHODS: Purified human ß-globin (HBG) 5'-3'UTRs, and the coding sequence of destabilized green fluorescent protein (EGFPd2) were amplified separately and ligated to each other using SOEing PCR method in a different format. As controls, the original construct of EGFPd2 under the control of T7 promoter was used. Following in vitro transcription, HEK 293 cells were then transfected with several constructs and incubated at 37°C in a CO2 incubator. They were monitored under a fluorescence microscope every four hours for the first 24 hr, then every 12 hr afterwards. The resulting fluorescence was measured as a surrogate for translation efficiency and duration. RESULTS: By monitoring the HEK cells over 48 hr, cells transfected with mRNA with various HBG UTRs showed significantly different fluorescence intensity and stability in comparison with the pEGFPd2 prototype (control transcript) overtime. Overall, the images show that replacement of the 3' UTR end of the prototype vector pGFPd2 with the 3' end of ß-globin mRNA increases the half-life of the chimeric mRNA for more than 32 hr. CONCLUSION: This result indicates that ß-globin 3' UTR would definitely increase the half-life of mRNA and may help to decrease the mRNA therapeutic dosage in the treatment of diseases associated with mRNA therapy.

The Synergistic Effects of Celecoxib and Sodium Valproate on Apoptosis and Invasiveness Behavior of Papillary Thyroid Cancer Cell Line In-vitro.

Metastasis to lymph nodes and distant organs is the main challenge in the treatment of papillary thyroid cancer. In the current investigation, we aimed to evaluate the synergistic effects of celecoxib (CX) and sodium valproate (VPA) against cell survival, invasiveness properties, and expression of metalloproteinase-2 and -9 (MMP-2 and MMP-9) in papillary thyroid cancer cell line, B-CPAP cells. The effect of CX and VPA on B-CPAP cells viability and apoptosis were investigated using MTT assay and annexin V/7-AAD flowcytometry, respectively. The effects of the drugs on invasiveness properties of B-CPAP cells and expression of MMP-2 and MMP-9 were evaluated using transwell assay and real time PCR, respectively. MTT assay showed that CX and VPA decreased viability of B-CPAP cells dose dependently (IC50 32.4µM and 6.8 mM, respectively). Combination of CX (5 µM) and VPA (2.5 and 5 mM) increased apoptosis, and reduced cell migration and invasion of B-CPAP cell, synergistically. Real time PCR results showed that both CX (5 µM) and VPA (2.5 and 5 mM) reduced MMP-2 expression (P < 0.05) but had no significant effects on the expression of MMP-9. Our findings suggest that CX and VPA synergistically increase apoptosis and suppress migration and invasion of B-CPAP cells through inhibition of MMP-2 expression.

Detection of Hepatitis E Virus Genotype 1 Among Blood Donors From Southwest of Iran.

BACKGROUND: Infection with hepatitis E virus (HEV) is endemic in developing countries and reveals significant regional differences. Several studies have reported virus transmission via blood transfusion. To date, however, no cases of HEV RNA detection in blood donors have been reported from Iran. OBJECTIVES: The aim of this study was to determine the presence of HEV RNA in plasma samples of blood donors referred to a blood transfusion center in Shiraz in the southwest of Iran. The HEV genotypes were also investigated using nucleotide sequencing. PATIENTS AND METHODS: Blood samples were collected from 700 blood donors who were referred to Fars blood transfusion organization from January to March 2014. Plasma samples were screened for the presence of HEV IgG and IgM antibodies by standard enzyme immunoassay. Samples seroreactive to anti-HEV were further tested for the presence of HEV RNA using nested polymerase chain reaction (PCR) with universal primers for detection of all four HEV genotypes. Positive PCR samples were then subjected to DNA sequencing for further analysis. RESULTS: Fifty (50, 7.1%) out of 700 plasma samples tested positive for anti-HEV antibodies. HEV RNA was detected in 7/50 (12%) of the antibody-positive samples, the majority of which were IgM positive. Sequence analysis of seven isolates of the HEV RNA ORF 2 gene region revealed > 80% similarity with genotype 1. CONCLUSIONS: The analysis indicates that the HEV isolated from blood donors in the southwest of Iran belongs to genotype 1. However, more samples from other geographic regions of Iran are needed to confirm these findings. Because transmission of HEV by administration of blood or blood components is likely to occur, it may be sensible to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection when the recipient is immunocompromised.