Construction of a sensitive and specific lead biosensor using a genetically engineered bacterial system with a luciferase gene reporter controlled by pbr and cadA promoters.

BACKGROUND: A bacterial biosensor refers to genetically engineered bacteria that produce an assessable signal in the presence of a physical or chemical agent in the environment. METHODS: We have designed and evaluated a bacterial biosensor expressing a luciferase reporter gene controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously termed Ralstonia metallidurans) containing the CH34 and pI258 plasmids of Staphylococcus aureus, respectively, and that can be used for the detection of heavy metals. In the present study, we have produced and evaluated biosensor plasmids designated pGL3-luc/pbr biosensor and pGL3-luc/cad biosensor, that were based on the expression of luc+ and under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans. RESULTS: We found that the pGL3-luc/pbr biosensor may be used to measure lead concentrations between 1-100 µM in the presence of other metals, including zinc, cadmium, tin and nickel. The latter metals did not result in any significant signal. The pGL3-luc/cad biosensor could detect lead concentrations between 10 nM to 10 µM. CONCLUSIONS: This biosensor was found to be specific for measuring lead ions in both environmental and biological samples.

Evaluation of anticancer effects of cerium oxide nanoparticles on mouse fibrosarcoma cell line.

Cerium oxide nanoparticles are associated with anticancer effects. While protecting normal cells, these nanoparticles exert their anticancer effects via oxidative stress and apoptosis in the cancer cells. In this study, the anticancer properties of nanoceria on fibrosarcoma cell line are evaluated. Cerium oxide nanoparticles were synthesized by the coprecipitation method and their anticancer effects on mouse fibrosarcoma tumor cells (WEHI164) were investigated. Viability assay was evaluated by MTT, and the DC-FDA assay performed for the detection of reactive oxygen species. For apoptosis assay, the annexin V/PI test was done as well as measuring the mRNA and protein expression levels of Bax and Bcl2 by real-time PCR and western blot method, respectively. Characterization of nanoceria reveals that synthesized nanoceria has cubic floruit structure with a size of about 30 nm. Toxicity assessment results show that nanoceria increases ROS levels and induced apoptosis in a dose-dependent manner in cancer cells (WEHI164), whereas low levels of toxicity were observed in normal cells (L929), even at the concentrations above 250 µg/ml in MTT assay. Real-time PCR and western blot assays showed that nanoceria could significantly increase the Bax expression in cancer cells. The results showed that nanoceria could act as a potential therapeutic agent for the treatment of fibrosarcoma.

Evaluation of antitumor effect of oxygen nanobubble water on breast cancer-bearing BALB/c mice.

Hypoxia is a condition of low oxygen level which poses a common feature of most cancers. In the current study, we investigated effect of water containing oxygen nanobubble (ONB) on tumor growth in breast cancer 4T1-bearing mice during 14-day treatment period. Tumor-bearing mice were randomly divided into three groups (six mice per group), including the ONB group drinking water containing ONB, the air nanobubble (ANB) group drinking water containing ANB, and control group drinking normal water. Tumor weight and size were measured in 2-day interval during 14-day treatment. mRNA expression of p53, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF), and cyclin D/Cdk2 genes were measured in the treated and control mice. After 8, 12, and 14 days of treatment, tumor size in ONB group was significantly decreased by 40.5%, 32.8%, and 28%, respectively, when compared with the control group. In addition, ANB group showed a significant reduction in tumor burden as well. The messenger RNA (mRNA) level of p53 in tumor cells of ONB and ANB group was found to be 36-fold (P = 0.0001) and 33-fold (P = 0.0001) higher than that in the control group, respectively. There was a ninefold increase in mRNA expression of VEGF gene in tumor cells of ANB mice than that in control mice; however, there was no significant changes in ONB group. Expression of HIF gene was significantly lower in tumor cells of ONB and ANB group than in the control group. It is concluded that drinking ONB water has potential to inhibit tumor growth, however more preclinical and proof-of-concept studies are needed to confirm its safety and therapeutic effect.

Current Status and Perspectives Regarding LNA-Anti-miR Oligonucleotides and microRNA miR-21 Inhibitors as a Potential Therapeutic Option in Treatment of Colorectal Cancer.

Colorectal cancer (CRC) is among the leading causes of cancer-related death, principally due to its metastatic spread and multifactorial chemoresistance. The therapeutic failure can also be explained by inter- or intra-tumor genetic heterogeneity and tumor stromal content. Thus, the identification of novel prognostic biomarkers and therapeutic options are warranted in the management of CRC patients. There are data showing that microRNA-21 is elevated in different types of cancer, particularly colon adenocarcinoma and that this is association with a poor prognosis. This suggests that microRNA-21 may be of value as a potential therapeutic target. Furthermore, locked nucleic acid (LNA)-modified oligonucleotides have recently emerged as a therapeutic option for targeting dysregulated miRNAs in cancer therapy, through antisense-based gene silencing. Further work is required to identify innovative anticancer drugs that improve the current therapy either through novel combinatorial approaches or with better efficacy than conventional drugs. We aimed to provide an overview of the preclinical and clinical studies targeting key dysregulated signaling pathways in CRC as well as the therapeutic application of LNA-modified oligonucleotides, and miR inhibitors in the treatment of CRC patients. J. Cell. Biochem. 118: 4129-4140, 2017. © 2017 Wiley Periodicals, Inc.

Cerium oxide nanoparticles: A promising tool for the treatment of fibrosarcoma in-vivo.

In this study, we used cerium oxide nanoparticles and evaluated their anti-cancer effects in a mouse model of fibrosarcoma. For evaluation of anti-cancer effects of nanoceria, tumor volume measurement, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, quantitative real-time PCR (qPCR) for Bax and Bcl2 genes, a panel of liver and kidney function tests and hematoxylin-eosin staining were done. Nanoceria dominantly accumulated in the tumor and it could significantly decrease tumor growth and volume in tumor-bearing mice that received nanoceria for four weeks. Cerium oxide nanoparticle showed potential anti-cancer properties against fibrosarcoma.

Polyethylenimine-associated cerium oxide nanoparticles: A novel promising gene delivery vector.

AIMS: The development of highly efficient and low toxic non-viral gene delivery vectors is the most challenging issues for successful application of gene therapy. A particular focus has been on understanding structure-activity relationships for transfection activity and toxicity of polyethylenimine (PEI). During the last decade, the use of cerium oxide nanoparticles (CeO2-NPs) in biomedicine has attracted much attention due to their pH-dependent antioxidant activity. CeO2-NPs provide protection normal cells from various forms of reactive oxygen species, but possess innate cytotoxicity and apoptosis to cancer cells. The purpose of this study was to design a new class of gene carriers by low molecular weight PEI (B-PEI 10 kDa) coordination onto CeO2-NPs. MAIN METHODS: B-PEI 10 kDa was conjugated to CeO2-NPs by Epichlorohydrin linker. Transfection efficiency, cytotoxic and apoptotic effects of pDNA-PEI-CeO2 NPs were evaluated on WEHI 164 cancer cells and normal L929 cells lines. KEY FINDINGS: PEI-CeO2 NPs was able to condense the pDNA at carrier/plasmid (C/P) weight ratios of 0.5. The size and zeta potential of pDNA-PEI-CeO2 NPs were 124 ±â€¯7 nm and 22 ±â€¯2 mV, respectively. The transfection efficacy of synthesized pDNA-PEI-CeO2 NPs improved and the cytotoxicity was decreased compared to pDNA-PEI. Moreover, pDNA-PEI-CeO2 NPs induced more apoptosis than unmodified PEI and CeO2-NPs control groups. pDNA-PEI-CeO2 NPs displayed more transfection, cytotoxicity, and apoptosis in WEHI 164 cancer cells than normal L929 cells. SIGNIFICANCE: In conclusion, PEI-CeO2 nanocarriers could act as a potential candidate for gene and drug delivery to cancerous and tumor cells.

Developmental regulation and lateralization of GABA receptors in the rat hippocampus.

GABA is the chief inhibitory neurotransmitter in the adult brain. However, in the developing brain it acts as an excitatory transmitter causing depolarization. Thereby, activates calcium-dependent processes that are crucial for brain development. Accordingly, GABA receptors have the great role in the brain development, especially in the area with persisting neurogenesis such as hippocampus. The present study investigated the development and lateralization of two important subunits of GABA receptors, GABAAα1 and GABAB1, in the developing rat hippocampus during the neurogenesis-active period, at the first two postnatal weeks. Real-time PCR, western blot and immunohistochemistry were used. We found that the mRNA and protein of these GABA receptor subunits have already been expressed at birth and significantly increased at postnatal day (P) 7, and also at P14. Also, regarding the optical densities of GABAAα1 and GABAB1 expressing hippocampal cells, we found a significant increase in the distribution pattern of these subunits in the all hippocampal subregions on day 14 after birth. The highest optical density of GABAAα1 was observed in the CA3, and GABAB1 in the CA2. Nevertheless, our results did not show a significant laterality differences in the expression of these subunits. Regarding the crucial role of GABA receptors in the hippocampus development; they probably have the same effects on development of the rat hippocampus on both sides.

Green synthesis of labeled CeO2 nanoparticles with 99mTc and its biodistribution evaluation in mice.

AIMS: The in vivo targeted diagnostic applications of biosynthetic Cerium oxide nanoparticles (CeO2-NPs), prepared by applying chitosan as a stabilizer, was explored by evaluating the cytotoxicity through MTT assay on WEHI 164 cell line, the Hemolytic activity of CeO2-NPs and biodistribution in rats. MAIN METHODS: The CeO2-NPs were characterized through the use of TGA/DTG, PXRD, FESEM, FTIR, and UV-Vis spectroscopy. The biodistribution of CeO2-NPs were determined by directly labeled nanoparticles with Technetium-99 m (99mTc) radioisotope (99mTc-CeO2-NPs). The labeling efficiency and stability of 99mTc-CeO2-NPs were also measured with Instant Thin Layer Chromatography (ITLC) method. The saturation study was investigated by 1 mCi of 99mTc-CeO2-NPs using different concentrations of WEHI 164 cells after 4 h of incubation. In vivo biodistribution study was performed by intravenous injection of 600 µCi/200 µL 99mTc-CeO2-NPs through rat's tail. KEY FINDINGS: CeO2-NPs seemed to have a low cytotoxic effect on WEHI 164 cell line and did not result in hemolysis. The biodistribution of CeO2-NPs has shown that a huge amount of 99mTc-CeO2-NPs was amassed in the living human organs, including liver, lung, spleen, stomach, and thyroid which shows the in vivo stability of the labeled conjugate. Herein, we have developed a facile, economical, and greener synthetic procedure applying Chitosan template. This green approach is comparable to conventional methods that utilize hazardous materials which are would be a suitable alternative to circumvent synthetic issues related to these materials. SIGNIFICANCE: The bio-applications of nano-sized CeO2-NPs were explored to find new horizon to use nanotechnology as the diagnostic tool.

Ultrasmall superparamagnetic Fe3O4 nanoparticles: honey-based green and facile synthesis and in vitro viability assay.

INTRODUCTION: In the present research, we report a quick and green synthesis of magnetite nanoparticles (Fe3O4-NPs) in aqueous solution using ferric and ferrous chloride, with different percentages of natural honey (0.5%, 1.0%, 3.0% and 5.0% w/v) as the precursors, stabilizer, reducing and capping agent, respectively. The effect of the stabilizer on the magnetic properties and size of Fe3O4-NPs was also studied. METHODS: The nanoparticles were characterized by X-ray diffraction (XRD) analysis, field emission scanning electron microscopy, energy dispersive X-ray fluorescence, transmission electron microscopy (TEM), vibrating sample magnetometry (VSM) and Fourier transform infrared spectroscopy. RESULTS: The XRD analysis indicated the presence of pure Fe3O4-NPs while the TEM images indicated that the Fe3O4-NPs are spherical with a diameter range between 3.21 and 2.22 nm. The VSM study demonstrated that the magnetic properties were enhanced with the decrease in the percentage of honey. In vitro viability evaluation of Fe3O4-NPs performed by using the MTT assay on the WEHI164 cells demonstrated no significant toxicity in higher concentration up to 140.0 ppm, which allows them to be used in some biological applications such as drug delivery. CONCLUSION: The presented synthesis method can be used for the controlled synthesis of Fe3O4-NPs, which could be found to be important in applications in biotechnology, biosensor and biomedicine, magnetic resonance imaging and catalysis.