Hematite iron oxide nanoparticles: apoptosis of myoblast cancer cells and their arithmetical assessment.

Hematite (α-Fe2O3) forms iron oxide nanoparticles (NPs) which are thermally stable and have various electrochemical and optochemical applications. Due to their wide applicability, the present work was designed to form the hematite phase of iron oxide (αFe2O3NPs) NPs prepared via a solution process. Their cytological performance was checked with C2C12 cells. The crystalline property of the NPs was examined with X-ray diffraction patterns (XRD) and it was found that the size of the particles formed ranged from 12 to 15 nm. Structural information was also identified via field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM), which again confirmed that the size of each NP is about 12-15 nm. Surface topographical analysis was done via atomic force microscopy (AFM), which reveals that the size of the distance between two particles is in the range of 12 ± 3 nm. The C2C12 cells were cultured in a humidified environment with 5% CO2 and were checked via a microscope. The αFe2O3NPs were used for cytotoxic evaluation against C2C12 cells. A MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was utilized to check the viability of cells in a dose-dependent (100 ng mL-1, 500 ng mL-1 or 1000 ng mL-1) manner. The morphology of the cells under the influence of αFe2O3NPs for live and dead cells in a wet environment was confirmed via confocal laser scanning microscopy (CLSM). The apoptosis caused due to the αFe2O3NPs was evaluated in presence of caspases 3/7 with GAPDH genes, which confirmed the upregulation that is responsible in caspase 3/7 genes, with treatment of C2C12 at low (500 ng mL-1) and high (1000 ng mL-1) doses of αFe2O3NPs. Analytical studies were also performed to authenticate the obtained data for αFe2O3NPs using parameters such as precision, accuracy, linearity, limits of detection (LOD) and limit of quantitation (LOQ), quantitative recoveries and relative standard deviation (RSD). The analyses play a significant role in investigating the large effect of αFe2O3NPs on C2C12 cells.

Functional genomics assessment of narcotic and specific acting chemical pollutants using E. coli.

The knowledge of gene-chemical interaction can be used to derive toxicological mechanism of chemical pollutants, therefore, it might be useful to discriminate chemicals with different mechanisms. In this study, three narcotic chemicals (4-chlorophenol (4-CP), 3, 4-dichloroaniline (DCA) and 2, 2, 2-trichloroethanol (TCE)) and three specific acting chemicals (triclosan (TCS), clarithromycin (CLARY), sulfamethoxazole (SMX)) were assessed by Escherichia coli (E. coli) genome-wide knockout screening. 66, 97, 88, 144, 198 and 180 initial robust hits were identified by exposure to 4-CP, DCA, TCE, TCS, CLARY and SMX with two replicates at the concentration of IC50, respectively. The average fold change values of responsive mutants to the three narcotic chemicals were smaller than the three specific acting chemicals. The common gene ontology (GO) term of biological process enriched by the three narcotic chemicals was "response to external stimulus" (GO: 0009605). Other GO terms like "lipopolysaccharide biosynthetic process" (induced by 4-CP) and "purine nucleotide biosynthetic process" (induced by DCA) were also influenced by the narcotic chemicals. The toxic target of three known specific acting chemicals could be validated by GSEA of responsive genes. Four genes (flhC, fliN, fliH and flhD) might serve as potential biomarkers to distinguish narcotic chemicals and specific acting chemicals. The E. coli functional genomic approach presented here has shown great potential not only for the molecular mechanistic screening of chemicals, rather it can discriminate chemicals based on their mode-of-action.

Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods.

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.

Anticancer Activity of Chloroform Extract and Sub-fractions of Nepeta deflersiana on Human Breast and Lung Cancer Cells: An In vitro Cytotoxicity Assessment.

BACKGROUND: Cancer is one of the major causes of death worldwide. The plant-derived natural products have received considerable attention in recent years due to their diverse pharmacological properties including anticancer effects. Nepeta deflersiana (ND) is used in the folk medicine as antiseptic, carminative, antimicrobial, antioxidant, and for treating rheumatic disorders. However, the anticancer activity of ND chloroform extract has not been explored so far. OBJECTIVES: The present study was aimed to investigate the anticancer activities of chloroform Nepeta deflersiana extract and various sub-fractions (ND-1-ND-15) of ND against human breast cancer cells (MCF-7) and human lung cancer cells (A-549). MATERIALS AND METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays, and cellular morphological alterations using phase contrast light microscope were studied. Cells were exposed with 10-1000 µg/ml of sub-fractions of ND for 24 h. RESULTS: Results showed that selected sub-fractions of the chloroform extract significantly reduced the cell viability of MCF-7 and A-549 cells, and altered the cellular morphology in a concentration-dependent manner. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity compared to other fractions whereas, ND-1 did not cause any cytotoxicity even at higher concentrations. The A-549 cells were found to be more sensitive to growth inhibition by all the extracts as compared to the MCF-7 cells. CONCLUSION: The present study provides preliminary screening of anticancer activities of chloroform extract and sub-fractions of ND, which can be further used for the development of a potential therapeutic anticancer agent. SUMMARY: Nepeta deflersiana extract exhibit cytotoxicity and altered the cellular morphology. Sub-fractions of the chloroform extract of Nepeta deflersiana reduced the cell viability of MCF-7 and A-549 cells. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity. The A-549 cells were found to be more sensitive as compared to the MCF-7 cells. Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NRU: Neutral red uptake; DMEM: Dulbecco's modified eagle medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; DMSO: Dimethyl sulfoxide.

Use of ß-galactosidase (lacZ) gene α-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis.

The mutagenic potential of titanium dioxide nanoparticles (TiO(2)-NPs) of an average size 30.6nm was investigated using ß-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ(-)Escherichia coli DH5α system. Plasmid pUC19 was treated with varying concentrations of TiO(2)-NPs and allowed to transfect the CaCl(2)-induced competent DH5α cells. The data revealed loss in transformation efficiency of TiO(2)-NPs treated plasmids as compared to untreated plasmid DNA in DH5α host cells. Induction of multiple mutations in α-fragment of lacZ gene caused synthesis of non-functional ß-galactosidase enzyme, which resulted in a significant number of white (mutant) colonies of transformed E. coli cells. Screening of mutant transformants based on blue:white colony assay and DNA sequence analysis of lacZ gene fragment clearly demonstrated TiO(2)-NPs induced mutagenesis. Multiple alignment of selectable marker lacZ gene sequences from randomly selected mutants and control cells provided a gene specific map of TiO(2)-NPs induced mutations. Mutational analysis suggested that all nucleotide changes were point mutations, predominantly transversions (TVs) and transitions (TSs). A total of 32 TVs and 6 TSs mutations were mapped within 296 nucleotides (nt) long partial sequence of lacZ gene. The region between 102 and 147nt within lacZ gene sequence was found to be most susceptible to mutations with nine detectable point mutations (8 TVs and 1 TSs). Guanine base was determined to be more prone to TiO(2)-NPs induced mutations. This study suggested the pUC19/E. coli DH5αlacZ gene α-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials.

A validation study comparing the sensitivity and specificity of the new Dr. KSU H1N1 RT-PCR kit with real-time RT-PCR for diagnosing influenza A (H1N1).

BACKGROUND AND OBJECTIVES: A new test (Dr. KSU H1N1 RT-PCR kit) was recently developed to provide a less expensive alternative to real-time reverse transcriptase-polymerase chain reaction (RT-PCR). We report the findings of a validation study designed to assess the diagnostic accuracy, including sensitivity and specificity, of the new kit, as compared to real-time RT-PCR. DESIGN AND SETTING: Cross-sectional validation study conducted from 18-22 November 2009 at a primary care clinic for H1N1 at a tertiary care teaching hospital in Riyadh. PATIENTS AND METHODS: Nasopharyngeal swab samples and data on socio-demographic characteristics and symptoms were collected from 186 patients. Swab samples were sent to the laboratory for testing with both real-time RT-PCR and the new Dr. KSU H1N1 RT-PCR kit. We measured the sensitivity and specificity of the new test across the entire sample size and investigated how these values were affected by patient socio-demographic characteristics and symptoms. RESULTS: The outcomes of the two tests were highly correlated (kappa=0.85; P<.0001). The sensitivity and specificity of the new test were 99.11% and 83.78%, respectively. The sensitivity of the new test was affected only minimally (96%-100%) by patient characteristics and number of symptoms. On the other hand, the specificity of the new test varied depending on how soon patients were tested after onset of symptoms (100% specificity when swabs were taken on the first day of the symptoms, decreasing to 75% when swabs were taken on or after the third day). The specificity of the new test also increased with increasing body temperature. CONCLUSION: The new test seems to provide a cost-effective alternative to real-time RT-PCR for diagnosing H1N1 influenza. However, further testing may be needed to verify the efficacy of the test in different settings and communities.

Assessment of methyl thiophanate-Cu (II) induced DNA damage in human lymphocytes.

Dimethyl 4,4'-(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a category-III acute toxicant and suspected carcinogen to humans. Hence, the ability of this benzimidazole class of fungicide to engender DNA strand breaks was investigated using alkaline single cell gel electrophoresis (SCGE), alkaline unwinding and cytokinesis-blocked micronucleus (CBMN) assays. The SCGE of human lymphocytes treated with 1mM MT for 3h at 37 degrees C showed much higher Olive tail moment (OTM) value of 40.3+/-2.6 (p<0.001) vis-à-vis 3.3+/-0.09 in DMSO control. Treatment of cultured lymphocytes for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). Stoichiometric data revealed the intrinsic property of MT to bind with Cu (II) and its reduction to Cu (I), which is known to form reactive oxygen species (ROS). We have detected the intracellular ROS generation in MT treated lymphocytes and observed an elevated level of MT-induced strand breaks per unit of calf thymus DNA in presence of Cu (II). Overall the data suggested that the formation of MT-Cu (II)-DNA ternary complex and consequent ROS generation, owing to Cu (II)/Cu (I) redox cycling in DNA proximity, is responsible for MT-induced DNA damage.