Induction of apoptosis in leukemic cells by the alkaloid extract of garden cress (Lepidium sativum L.).

OBJECTIVE: Garden cress (Lepidium sativum L.) is an important herb in traditional medicine used to improve production of breast milk in women and semen in men. In the present research the authors evaluated its ability to destroy leukemic cancer (Jurkat E6-1) cells, using the alkaloid extract of this plant. METHODS: Constituents of the alkaloid extract were analyzed by gas chromatography-mass spectrometry (GC-MS) and their cytotoxicity in leukemic cancer cells and healthy peripheral blood mononuclear cells (PBMCs) was assessed. Cell death via apoptosis was confirmed by DNA laddering, caspase-3 activity, annexin V-fluorescein isothiocyanate and mitochondrial toxicity assays. The specific course of gene activation in treated cells was determined through quantitative polymerase chain reaction (qPCR). RESULTS: GC-MS analysis identified six alkaloids and proto-alkaloids, namely, benzyl isothiocyanate (1), 2-ethoxy-4H-3,1-benzoxazin-4-one (2), (4R)-2-(2-aminophenyl)-4-phenyloxazoline (3), 5-acetyl-1,2-dihydro-6-methyl-2-oxo-4-phenyl-3-pyridinecarbonitrile (4), benzo[b][1,8]-naphthyridin-5(10H)-one,2,4,7-trimethyl (5) and 1,4-diaminoanthraquinone (6), in the alkaloid extract of L. sativum. Of these, compound 1 was previously identified in the seeds of L. sativum. Exposure to the alkaloid extract caused death of Jurkat E6-1 cells, with median lethal concentration (LC50) of 75.25 µg/mL. However, the alkaloid extract also showed a nontoxic and proliferative (1.6-fold) effect in healthy PBMCs. Further experiments performed with Jurkat cells at LC50 and sub-LC50 doses demonstrated DNA fragmentation, activation of caspase-3 and time-dependant phosphatidylserine translocation (apoptosis) from inner to outer cell membranes. Cell toxicity and assessment of adenosine triphosphate level, together with using qPCR to evaluate expression profile of major apoptosis genes, revealed that apoptosis may be induced by disruption in the mitochondrial outer membrane potential, through activation of extrinsic and intrinsic apoptosis pathways in Jurkat cells. CONCLUSION: The ability of the alkaloid extract of L. sativum seeds to induce apoptosis indicates a potential pharmacological use in cancer chemotherapy. The separation of individual active compounds and further in-depth exploration of the molecular mechanism of apoptosis may lead to novel chemotherapeutic compounds in our future antineoplastic research.

Antioxidant efficacy of chitosan/graphene functionalized superparamagnetic iron oxide nanoparticles.

The antioxidant potential of superparamagnetic iron oxide nanoparticles functionalized with chitosan and graphene were examined in the present work. Coprecipitation technique was followed for the synthesis of iron oxide nanoparticles. Graphene-iron oxide nanocomposites were synthesized by mechanical mixing followed by the heat treatment at moderate temperature. The chitosan coated iron oxide nanoparticles were prepared by dispersing nanoparticles in chitosan solution. The nanoparticles/nanocomposites were characterized using XRD, SEM, TEM and HAADF-STEM for phase structure, morphology and elemental analysis. The superparamagnetic behavior of nanoparticles/nanocomposites were confirmed by magnetic measurements using vibrating sample magnetometry. Antioxidant efficacy of these nanoparticles/nanocomposites were investigated in terms of free radical scavenging and reducing potential using an array of in vitro assay system. Ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) were used for the antioxidant capacity. The investigation suggests that the graphene improves the antiradical response of iron oxide nanoparticles at higher concentration which is almost comparable to the ascorbic acid used as standard.

Metal Immiscibility Route to Synthesis of Ultrathin Carbides, Borides, and Nitrides.

Ultrathin ceramic coatings are of high interest as protective coatings from aviation to biomedical applications. Here, a generic approach of making scalable ultrathin transition metal-carbide/boride/nitride using immiscibility of two metals is demonstrated. Ultrathin tantalum carbide, nitride, and boride are grown using chemical vapor deposition by heating a tantalum-copper bilayer with corresponding precursor (C2 H2 , B powder, and NH3 ). The ultrathin crystals are found on the copper surface (opposite of the metal-metal junction). A detailed microscopy analysis followed by density functional theory based calculation demonstrates the migration mechanism, where Ta atoms prefer to stay in clusters in the Cu matrix. These ultrathin materials have good interface attachment with Cu, improving the scratch resistance and oxidation resistance of Cu. This metal-metal immiscibility system can be extended to other metals to synthesize metal carbide, boride, and nitride coatings.

Application of cell-based assays for toxicity characterization of complex wastewater matrices: Possible applications in wastewater recycle and reuse.

Exposure to pre-concentrated inlet or outlet STP wastewater extracts at different concentrations (0.001% to 1%) induced dose-dependent toxicity in MCF-7 cells, whereas drinking water extracts did not induce cytotoxicity in cells treated. GC-MS analysis revealed the occurrence of xenobiotic compounds (Benzene, Phthalate, etc.) in inlet/outlet wastewater extracts. Cells exposed to inlet/outlet extract showed elevated levels of reactive oxygen species (ROS: inlet: 186.58%, p<0.05, outlet, 147.8%, p<0.01) and loss of mitochondrial membrane potential (Δψm: inlet, 74.91%, p<0.01; outlet, 86.70%, p<0.05) compared to the control. These concentrations induced DNA damage (Tail length: inlet: 34.4%, p<0.05, outlet, 26.7%, p<0.05) in treated cells compared to the control (Tail length: 7.5%). Cell cycle analysis displayed drastic reduction in the G1 phase in treated cells (inlet, G1:45.0%; outlet, G1:58.3%) compared to the control (G1:67.3%). Treated cells showed 45.18% and 28.0% apoptosis compared to the control (1.2%). Drinking water extracts did not show any significant alterations with respect to ROS, Δψm, DNA damage, cell cycle and apoptosis compared to the control. Genes involved in cell cycle and apoptosis were found to be differentially expressed in cells exposed to inlet/outlet extracts. Herein, we propose cell-based toxicity assays to evaluate the efficacies of wastewater treatment and recycling processes.

Direct Visualization of the Hydration Layer on Alumina Nanoparticles with the Fluid Cell STEM in situ.

Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions. We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. Our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles.

Dynamics of effluent treatment plant during commissioning of activated sludge process unit.

Industrial effluent treatment plants (ETPs) are very important in protecting the environment and different life forms from harmful industrial waste. Hence, the efficiency of ETPs must be regularly monitored, particularly after major repair or replacement work. Present study evaluated the performance of an ETP over a period of 4 months, during which aeration tank (T1) of the activated sludge unit was replaced with a new one (T2). System had to be maintained operational during this transition, which warranted close monitoring of the system performance due to the daily load of hazardous industrial wastewater. Analysis showed that the raw wastewater was highly variable in composition and contained many hazardous organic and inorganic pollutants, such as heavy metals, bisphenol A and cyanoacetylurea. It showed significant toxicity against HepG2 cells in vitro. However, the ETP was found to successfully treat and detoxify the wastewater. Denaturing gradient gel electrophoresis (DGGE) analysis showed large temporal fluctuations in the ETP microbial community, which is consistent with the variable composition of wastewater. It indicated that functional stability of the ETP was not associated with stability of the microbial community, probably due to high microbial biodiversity and consequently high functional redundancy. In conclusion, the CETP showed consistent level of detoxification and microbial community dynamics after switching to T2, indicating successful development, acclimatization and commissioning of T2.

Cytochrome P450 BM3 of Bacillus megaterium - a possible endosulfan biotransforming gene.

Computing chemistry was applied to understand biotransformation mechanism of an organochlorine pesticide, endosulfan. The stereo specific metabolic activity of human CYP-2B6 (cytochrome P450) on endosulfan has been well demonstrated. Sequence and structural similarity search revealed that the bacterium Bacillus megaterium encodes CYP-BM3, which is similar to CYP-2B6. The functional similarity was studied at organism level by batch-scale studies and it was proved that B. megaterium could metabolize endosulfan to endosulfan sulfate, as CYP-2B6 does in human system. The gene expression analyses also confirmed the possible role of CYP-BM3 in endosulfan metabolism. Thus, our results show that the protein structure based in-silico approach can help us to understand and identify microbes for remediation strategy development. To the best of our knowledge this is the first report which has extrapolated the bacterial gene for endosulfan biotransformation through in silico prediction approach for metabolic gene identification.

Correlative electron and fluorescence microscopy of magnetotactic bacteria in liquid: toward in vivo imaging.

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

Nucleation of iron oxide nanoparticles mediated by Mms6 protein in situ.

Biomineralization proteins are widely used as templating agents in biomimetic synthesis of a variety of organic-inorganic nanostructures. However, the role of the protein in controlling the nucleation and growth of biomimetic particles is not well understood, because the mechanism of the bioinspired reaction is often deduced from ex situ analysis of the resultant nanoscale mineral phase. Here we report the direct visualization of biomimetic iron oxide nanoparticle nucleation mediated by an acidic bacterial recombinant protein, Mms6, during an in situ reaction induced by the controlled addition of sodium hydroxide to solution-phase Mms6 protein micelles incubated with ferric chloride. Using in situ liquid cell scanning transmission electron microscopy we observe the liquid iron prenucleation phase and nascent amorphous nanoparticles forming preferentially on the surface of protein micelles. Our results provide insight into the early steps of protein-mediated biomimetic nucleation of iron oxide and point to the importance of an extended protein surface during nanoparticle formation.

Degradation of Di- Through Hepta-Chlorobiphenyls in Clophen Oil Using Microorganisms Isolated from Long Term PCBs Contaminated Soil.

Present work describes microbial degradation of selected polychlorinated biphenyls (PCBs) congeners in Clophen oil which is used as transformer oil and contains high concentration of PCBs. Indigenous PCBs degrading bacteria were isolated from Clophen oil contaminated soil using enrichment culture technique. A 15 days study was carried out to assess the biodegradation potential of two bacterial cultures and their consortium for Clophen oil with a final PCBs concentration of 100 mg kg(-1). The degradation capability of the individual bacterium and the consortium towards the varying range of PCBs congeners (di- through hepta-chlorobiphenyls) was determined using GCMS. Also, dehydrogenase enzyme was estimated to assess the microbial activity. Maximum degradation was observed in treatment containing consortium that resulted in up to 97 % degradation of PCB-44 which is a tetra chlorinated biphenyl whereas, hexa chlorinated biphenyl congener (PCB-153) was degraded up to 90 % by the consortium. This indicates that the degradation capability of microbial consortium was significantly higher than that of individual cultures. Furthermore, the results suggest that for degradation of lower as well as higher chlorinated PCB congeners; a microbial consortium is required rather than individual cultures.

Investigation of polybrominated diphenyl ethers in old consumer products in India.

Polybrominated diphenyl ethers (PBDEs) used extensively over the past 3 decades as flame retardants in most types of polymers, all over the world, have been identified as global pollutants. PBDEs pose various health problems such as thyroid hormone disruption, permanent learning and memory impairment, behavioral changes, hearing deficits, delayed puberty onset, fetal malformations, and possibly cancer. Many measurements of PBDEs in various matrices from Sweden, Holland, Japan, the USA, and elsewhere have been reported, but few measurements are available for India. In this study, a preliminary screening of different congeners of PBDEs has been performed in different old electronic and consumer products with an objective to build capacity in order to analyze PBDEs and BFRs. Six different samples, foam from upholstery, motherboard of a computer, children toy composite sample, old vanishing window blind sample, electrical wire sample, and PVC flooring sample, were collected and analyzed for the presence of the following PBDE congeners: BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183, and BDE-209. It was found that three out of six samples were positive for the presence of PBDEs. Three congeners were detected in the samples, i.e., BDE-47, BDE-153, and BDE-209, of which, highest concentration was of BDE-209. Among the samples, motherboard of computer showed the highest concentration of BDE-209 followed by window blind and foam from upholstery. The results of this preliminary investigation indicate that PBDEs are still present in the old consumer products which can be an important additional source of exposure to the population.

Treatment of refractory nano-filtration reject from a tannery using Pd-catalyzed wet air oxidation.

We attempted catalytic wet air oxidation (CWAO) of nanofiltration (NF)-reject using Pd based catalyst viz., Pd/activated charcoal (AC) and PdCl2 with the objective of degradation of refractory organic pollutants. Refractory organic pollutants in NF-reject before and after WAO and CWAO were confirmed by GC-MS analysis. Experiments were conducted to investigate the effects of temperature, catalyst dosage and air partial pressure on the rate of removal of total organic carbon (TOC). The reaction kinetics can be conveniently described by considering two-stage first order kinetics. The use of Pd/AC afforded 85% TOC removal, the corresponding rate constant (k) was 2.90 ± 0.075 × 10(-3)min(-1) (Pd/AC, 100mg/L; T, 473.15K; Pair, 0.69 MPa). On the other hand, 75% TOC was removed with k=2.31 ± 0.075 × 10(-3)min(-1) using Pd(2+) catalyst (Pd(2+), 16.66 mg/L; T, 473.15K; Pair, 0.69 MPa). The observed rate of mineralization under Pd-catalyzed conditions was significantly higher than that of the uncatalyzed oxidation (41%) under the similar experimental conditions. Catalyst stability experiments were performed and TEM, SEM, XRD, Raman and XPS characterization data collected. Despite some morphological transformation of support, Pd catalyst was stable under CWAO conditions.

Diversity of aromatic ring-hydroxylating dioxygenase gene in Citrobacter.

Genetic and functional diversity of Citrobacter spp. for their abilities to degrade aromatic compounds was evaluated to develop mixed cultures or a consortium for bioremediation technology. Thirty Citrobacter strains isolated from various effluent treatment plants were found to degrade a range of aromatic compounds: phenol, benzoate, hydroxy benzoic acid and biotransform mono-chlorophenols and di-chlorophenol within 24 to 48 h of incubation at 30 °C. Sequence similarity and phylogeny of the ARHD gene transcripts (730 nucleotides) depicted their diversity within 9 Citrobacter strains: HPC255, HPC369, HPC560, HPC570, HPC784, HPC1196, HPC1216, HPC1276 and HPC1299. Here, the degree of associations varied up to 84% with (i) ARHD α-sub unit (SU), (ii) LSU of Phenylpropionate dioxygenase (PDO), (iii) Phenol hydroxylase α-SU, (iv) Benzoate 1,2-dioxygenase, α-SU, (v) Naphthalene dioxygenase LSU, etc. This study has provided basic information, which can be used to develop a consortium of bacteria with mutually beneficial characteristics.

"Dioxin releases in waste incinerations and thermal processes".

The releases of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) from waste incinerators and thermal processes were investigated. The characteristics of mean PCDD/Fs I-TEQ concentrations and congener profiles were studied over the samples of water, soil, fly ash and bottom ash of individual source. The TEQ value for fly ash ranges from 0.013 to 17.01 pg-TEQ/g. Moreover, the TEQ value for bottom ash was 12.06 pg-TEQ/g and the TEQ values for the water samples were found to be in a consistent range from 0.41-0.56 ng-TEQ/L. In almost all the analyzed matrices the congener OCDD/OCDF was found in highest concentration raising the critical concerns over the overall PCDD/Fs emissions from incinerations and thermal processes.

Assaying baseline status of particulate laden polyaromatic hydrocarbon for a grass root level industrial project.

A study of particulate laden polyaromatic hydrocarbon was conducted at 13 selected locations in a 10 km radial distance of a proposed site for a grass root level industry. Suspended particulate matter samples were continuously monitored for 24 h over a period of 3 months. The Polyaromatic Hydrocarbons (PAHs) were extracted from the particulate samples and analysed using Gas Chromatograph-Mass Spectrometer. Limit of Quantification was also established for individual PAHs. Coal combustion and traffic emission were the major contributors for PAHs in the region. The relative contribution of 2, 3, 4, 5, and 6 ring PAHs in particulates of different sampling sites was also investigated and it is observed that 4 ring (29.76%) and 5 ring (29.06%) compounds are prominent in the particulates measured in the region.

Rapid analysis of 2,4-D in soil samples by modified Soxhlet apparatus using HPLC with UV detection.

The 2,4-dichlorophenoxy acetic acid (2,4-D) is used as a systemic herbicide to control broadleaf weeds in wheat, corn, range land/pasture land, sorghum, and barley. In this study, a fast and efficient method is developed by selection of modified extraction apparatus and high-performance liquid chromatography (HPLC)-UV conditions for the determination of 2,4-D in soil samples. The method is applied to the study of soil samples collected from the agricultural field. The herbicide is extracted from soil samples by acetonitrile in a modified Soxhlet apparatus. The advantages of the apparatus are that it uses small volume of organic solvent, reduced time of extraction, and better recovery of the analyte. The extract is filtered using a very fine microfiber paper. The total extract is concentrated in a rotatory evaporator, dried under ultrahigh pure N2, and finally reconstituted in 1 mL of acetonitrile. HPLC-UV at 228 nm is used for analysis. The herbicide is identified and quantitated using the HPLC system. The method is validated by the analysis of spiked soil samples. Recoveries obtained varied from 85% to 100% for spiked soil samples. The limit of quantitation (LOQ) and the limit of detection (LOD) are 0.010 and 0.005 parts per million (ppm), respectively, for spiked soil samples. The LOQ and LOD are 0.006 and 0.003 ppm for unspiked soil samples. The measured concentrations of 2,4-D in spiked soil samples are between 0.010 and 0.020 ppm with an average of 0.016 +/- 0.003 ppm. For unspiked soil samples it is between 0.006 ppm and 0.012 ppm with an average of 0.009 +/- 0.002 ppm. The measured concentrations of 2,4-D in soil samples are generally low and do not exceed the regulatory agencies guidelines.