Breast Cancer Selective Disruption of Actin Cytoskeleton by Diallyl Trisulfide

Diallyl trisulfide (DATS) is an attractive anti-cancer phytochemical with in vitro and in vivo growth inhibitory effects against different solid tumors including breast cancer. We have shown previously that an immortalized mammary epithelial cell line (MCF-10A) is resistant to growth inhibition by DATS. In this study, we performed RNA-seq analysis using a breast cancer cell line (SK-BR-3) and MCF-10A cells to gain insights into cancer selective effects of DATS. The Gene Ontology analysis revealed upregulation of genes associated with actin cytoskeleton but downregulation of mitochondria-related genes in the SK-BR-3 human breast cancer cell line but not in the non-oncogenic MCF-10A cell line upon treatment with DATS. Quantitative real-time reverse transcription polymerase chain reaction confirmed DATS-mediated upregulation of several actin cytoskeleton-related genes in the SK-BR-3 cell line. The DATS treatment dose-dependently disrupted actin cytoskeleton in the SK-BR-3 cell line, whereas the MCF-10A cell line was more resistant to this effect. The DATS treatment caused a marked increase in phosphorylation of dynamin-1-like (DRP1) protein in the SK-BR-3 cell line. However, the DATS-mediated apoptosis was not affected by genetic deletion of DRP1 protein. The Reactome pathway analysis showed downregulation of genes associated with citric acid cycle in the SK-BR-3 cell line but not in the MCF-10A cells. However, expression of aconitase 2 or dihydrolipoamide S-succinyltransferase was not affected by DATS treatment. In conclusion, this study reveals that actin cytoskeleton is a novel target of DATS in the SK-BR-3 cell line, which may explain its inhibitory effect on breast cancer cell migration.

Mechanistic Targets of Diallyl Trisulfide in Human Breast Cancer Cells Identified by RNA-seq Analysis

Diallyl trisulfide (DATS), a metabolic by-product of processed garlic, is highly effective in inhibiting growth of human breast cancer cells in vitro and in vivo, but the underlying mechanisms are still not fully understood. In this study, we performed RNA-seq analyses using luminal-type (MCF-7) and basal-like (MDA-MB-231) human breast cancer cells to identify mechanistic targets of DATS. The Reactome Pathway Analysis revealed upregulation of genes associated with SLIT/ROBO tumor suppressor signaling following DATS treatment in both MCF-7 and MDA-MB-231 cells. However, the expression of SLIT2 and ROBO1 proteins or their downstream target C-X-C motif chemokine receptor 4 was not affected by DATS treatment in both cell lines. The Reactome as well as the Gene Ontology Pathways Analyses of the RNA-seq data from DATS-treated cells indicated downregulation of genes associated with G2 /M phase cell cycle arrest in comparison with vehicle-treated control cells. Consistent with the RNA-seq data, DATS treatment caused a significant increase in the fraction of the G2 /M population in both cell lines when compared to corresponding control cells. In addition, Ser10 phosphorylation of histone H3, a mitotic marker, was also increased significantly following DATS treatment in MCF-7 and MDA-MB-231 cells. These results indicate that while SLIT/ROBO signaling is not affected by DATS treatment, cell cycle arrest likely contributes to the antitumor effect of this phytochemical.

Mechanistic Targets of Diallyl Trisulfide in Human Breast Cancer Cells Identified by RNA-seq Analysis

Diallyl trisulfide (DATS), a metabolic by-product of processed garlic, is highly effective in inhibiting growth of human breast cancer cells in vitro and in vivo, but the underlying mechanisms are still not fully understood. In this study, we performed RNA-seq analyses using luminal-type (MCF-7) and basal-like (MDA-MB-231) human breast cancer cells to identify mechanistic targets of DATS. The Reactome Pathway Analysis revealed upregulation of genes associated with SLIT/ROBO tumor suppressor signaling following DATS treatment in both MCF-7 and MDA-MB-231 cells. However, the expression of SLIT2 and ROBO1 proteins or their downstream target C-X-C motif chemokine receptor 4 was not affected by DATS treatment in both cell lines. The Reactome as well as the Gene Ontology Pathways Analyses of the RNA-seq data from DATS-treated cells indicated downregulation of genes associated with G2 /M phase cell cycle arrest in comparison with vehicle-treated control cells. Consistent with the RNA-seq data, DATS treatment caused a significant increase in the fraction of the G2 /M population in both cell lines when compared to corresponding control cells. In addition, Ser10 phosphorylation of histone H3, a mitotic marker, was also increased significantly following DATS treatment in MCF-7 and MDA-MB-231 cells. These results indicate that while SLIT/ROBO signaling is not affected by DATS treatment, cell cycle arrest likely contributes to the antitumor effect of this phytochemical.

Increase in growth, productivity and nutritional status of wheat (Triticum aestivum L. cv. WH-711) and enrichment in soil fertility applied with organic matrix entrapped urea.

Field experiments were conducted during two consequent years in semi-arid, subtropical climate of Rohtak district situated in North-West Indian state Haryana to evaluate the effects of eco-friendly organic matrix entrapped urea (OMEU) on wheat (Triticum aestivum L. cv. WH-711). The OMEU prepared in granular form contained cow dung, rice bran (grain cover of Oryza sativa), neem (Azadirachta indica) leaves and clay soil (diameter of particles <0.002mm) in 1:1:1:1 ratios and saresh (plant gum of Acacia sp.) as binder entrapping half of the recommended dose of urea. A basal application of organic matrix entrapped urea showed increase in plant growth in terms of fresh and dry weights, root length, root number, leaf number, tillers, plant height, earlet number, earlet length and productivity in terms of grain yield and straw yield over free form of urea (FU) and no fertilizer (NF) application. The OMEU increased total soluble proteins, organic N and free ammonium content in the leaves at 45 and 60 days. The nutritional status of wheat grains in OMEU applied plants was almost similar to that observed for FU applied plants. An increase in organic carbon and available phosphorus (P) was observed in OMEU applied plots on harvest, whereas pH was slightly decreased over FU applied plots. The microbial population and activity in terms of fungal and bacterial colony count and activities soil dehydrogenase and alkaline phosphatase were significantly higher in OMEU applied plots as compared to the FU applied plots. Our data indicate that OMEU which are low cost, biodegradable and non-toxic can be used to replace the expensive chemical fertilizers for wheat cultivation in semi-arid, subtropical climate.

Growth, biomass production and remediation of copper contamination by Jatropha curcas plant in industrial wasteland soil.

The survival, biomass production and copper (Cu) remediation efficiency of Jatropha curcas L. was evaluated in Cu rich industrial wasteland soil (IWLS), collected from a local town, Sandila (Hardoi), Uttar Pradesh, India. The IWLS had high bulk density, water holding capacity (WHC), pH, electrical conductivity (EC), organic carbon and NPK. The Cu and Mn contents in IWLS were about 3 and 2 fold higher than that in the normal field soil (control). Stem cuttings of the J. curcas clones (BTP-A, BTP-N and BTP-K) were planted in IWLS as well as the same amended with cowdung or sand. The percent survival, net elongations and biomass accumulation of J. curcas were decreased slightly in IWLS, as compared to the control soil. The translocation of Cu from soil to the plants was higher in IWLS grown plants, which was more pronounced in IWLS amended with cowdung. J. curcas clones BTP-N, showed better survival and Cu removal efficiency from IWLS.

Remediation of nitrite contamination in ground and surface waters using aquatic macrophytes.

The study was carried out to determine the seasonal variation of nitrite levels in drinking and surface waters of urban, peri-urban and rural areas of Lucknow, during 2007-2008, and to evaluate the nitrite removal and accumulation potential of certain native aquatic macrophytes. Most of the drinking and surface water samples were collected from urbanized region of the city. All drinking water samples detected, showed higher nitrite level in winter, when compared with that in summer and rainy seasons. However, in drinking water samples nitrite level was below the permissible limit i.e. 3.29 mg l-1 NO2. The surface water showed more than 3 fold higher levels of nitrite over the permissible level i.e. 0.06 mg l-1, and the level was higher during rainy season than in summer and winter seasons. Eight macrophytes viz. Peltandra virginica, Utricularia vulgaris, Eichhornia crassipes, Trapa natans, Mimulus glabratus, Marsilea quadrifolia, Pistia stratiotes and Polygonum persicaria were studied for phytoremediation potential of nitrite from the water under simulated laboratory conditions. The gradual diminution in the level of nitrite in the water and simultaneously it’s increase in the plant tissues was recorded at 5th, 10th and 15th d after plant culture. All the plants selected, removed nitrite from water but Polygonum persicaria, Mimulus glabratus, Trapa natans and Pistia stratiotes were found more efficient and removed nitrite upto 60.91, 58.09, 60.97 and 72.28%, respectively. Observations revealed that Pistia stratiotes can be used for the effective removal of nitrite from the contaminated water.

Inhibition of angiogenic attributes by decursin in endothelial cells and ex vivo rat aortic ring angiogenesis model.

The present study was undertaken to observe the inhibition of angiogenesis by decursin. It was the first time to show that decursin offered strong anti-angiogenic activities under the biologically relevant growth (with serum) conditions. Decursin significantly inhibited human umbilical vein endothelial cell (HUVEC) proliferation concomitant with G1 phase cell cycle arrest. Decursin also inhibited HUVEC-capillary tube formation and invasion/migration in a dose-dependant manner which was associated with the suppression of matrix metalloproteinase (MMP) -2 and -9 activities. Decursin suppressed angiogenesis in ex vivo rat aortic ring angiogenesis model where it significantly inhibited blood capillary-network sprouting from rat aortic sections. Taken together, these findings suggested anti-angiogenic activity of decursin in biologically relevant condition, and warrants further pre-clinical studies for its potential clinical usefulness.

Organic matrix based slow release fertilizer enhances plant growth, nitrate assimilation and seed yield of Indian mustard (Brassica juncea L.).

Field experiments were conducted to study the effect of organic matrix based slow release fertilizers (SRFs) on plant growth, nitrate assimilation and seed yield of Brassica juncea L. cv. pusa bold. The agro-waste materials like cow dung, clay soil, neem leaves and rice bran were mixed together in 2:2:1:1 ratio and used as organic matrix for the immobilization of chemical fertilizer nutrients with commercial grade saresh (Acacia gum, 15% solution) as binder. Different fertilizer treatments were organic matrix based slow release fertilizers, SRF-I (542.0 kg ha-1); SRF-II (736.5 kg ha-1) and chemical fertilizer combinations, boron (3 kg ha-1)+sulphur (15 kg ha-1)+nitrogen (80 kg ha-1) and boron (3 kg ha-1) + sulphur (15 kg ha-1)+nitrogen (80 kg ha-1)+phosphorus (15 kg ha-1)+potassium (100 kg ha-1). Organic matrix based SRF-II released ammonium up to 50-d in wet soil under laboratory conditions which showed maximum retention of the nutrients. A very significant increase in plant growth, nitrate assimilation and seed yield was recorded in organic matrix based SRF-II applied plants. The maximum percent increase in biomass production was observed with organic matrix based SRF-II (increase of 65.8% in root fresh weight, 38.0% in root dry weight, 45.9% in leaf fresh weight plant-1 and 27.5 % in leaf dry weight plant-1 in 60-d old plants). It also increased the acquisition and assimilation of nitrate from the plant’s rhizosphere which was evident by 45.6% increase in nitrate, 27.5% in nitrite and 11.7% in nitrate reductase activity (NRA) in leaves of 45-d old plants over control. The organic matrix based SRF-II significantly increased the seed yield by 28% in Indian mustard. Cost analysis revealed that this formulation is cost effective as it is based on agro waste materials.

Conservation tillage, optimal water and organic nutrient supply enhance soil microbial activities during wheat (Triticum Aestivum L.) cultivation

The field experiments were conducted on sandy loam soil at New Delhi, during 2007 and 2008 to investigate the effect of conservation tillage, irrigation regimes (sub-optimal, optimal and supra-optimal water regimes), and integrated nutrient management (INM) practices on soil biological parameters in wheat cultivation. The conservation tillage soils has shown significant (p<0.05) increase in soil respiration (81.1 percent), soil microbial biomass carbon (SMBC) (104 percent) and soil dehydrogenase (DH) (59.2 percent) compared to the conventional tillage soil. Optimum water supply (3-irrigations) enhanced soil respiration over sub-optimum and supra-optimum irrigations by 13.32 percent and 79 percent respectively. Soil dehydrogenase (DH) activity in optimum water regime has also increased by 23.33 percent and 8.18 percent respectively over the other two irrigation regimes. Similarly, SMBC has also increased by 12.14 percent and 27.17 percent respectively in soil with optimum water supply compared to that of sub-optimum and supra-optimum water regime fields. The maximum increase in soil microbial activities is found when sole organic source (50 percent Farm Yard Manure+25 percent biofertilizer+25 percent Green Manure) has been used in combination with the conservation tillage and the optimum water supply. Study demonstrated that microbial activity could be regulated by tillage, water and nitrogen management in the soil in a sustainable manner.

Differential toxicity of cadmium to mustard (Brassica juncia L.) genotypes under higher metal levels.

Cadmium application inhibited various growth and biochemical parameters in seedlings of five cultivars of Brassica juncia L. with different magnitude at lower Cd supply, however, at higher metal applications the variation in Cd toxicity ranged with minor differences. The seedling vigour index (SVI) was inhibited more severely in Gangotri (62.25% over control) and least in Pusa Jai Kisan (8.95%) at 1.0 mM CdCl2. The SVI of all five mustard cultivars, however, severely inhibited (84.29-91.80%) at 5.0 mM Cd. The root and shoot elongation in 7 days old seedlings were inhibited by 32.39-40.38 and 11.83-56.40% respectively at 1.0 mM CdCl2, whereas the varietal differences in root and shoot elongation were 76.71-82.47 and 71.57-78.91 respectively at 5.0 mM CdCl2. The genotypic differences at lower Cd level were more pronounced in shoot elongation than that in the root elongation. The dry weight and moisture content of the seedlings, however, does not show much varietal differences even at lower Cd level, though the Cd toxicity increased at higher level of Cd application. The metal tolerance index (MTI) and % phytotoxicity of 3 days old seedlings ranged between 43.30-98.37 and 1.63-56.70% respectively at 1.0 mM CdCl2 for different mustard genotypes, whereas at 5.0 mM CdCl2 these factors ranged between 12.26- 20.92 and 80.08-87.74% respectively. The varietal differences of MTI and % phytotoxicity was, however, less pronounced at all the metal levels when the seedling attained an age of 7 days. A similar trend of genotypic variation was noticed in Cd accumulation in the seedlings at lower and higher levels of Cd supply to the seedlings. Amongst some biochemical parameters e.g. photosynthetic pigments, carbohydrates and proteins in the leaves, the photosynthetic pigments i.e. chlorophylls and carotenoids were decreased more drastically. The carbohydrate content of leaves, however, was the least affected component. Our data indicate that the differential toxicity of cadmium to Indian mustard genotypes was dependent on the level of contamination and growth phases.

Genotypic variation in Brassica juncea (L.) Czern. cultivars in growth, nitrate assimilation, antioxidant responses and phytoremediation potential during cadmium stress.

Four cultivars of Brassica juncea i.e. TM-4, TM-2, RH-30 and T-59 were screened for seed germination and seedling growth up to 15 days in the presence of 0.5-1.5 mM cadmium (Cd). The exposure to Cd reduced seed germination and seedling growth (root and shoot length and dry weight) in all four cultivars; the effect being more severe in TM-2 and RH-30 than in TM-4 and T-59 and at 3 d than at 7 d and 15 d. The cultivars TM-4 and T-59, with higher tolerance to Cd toxicity, were selected for further analysis including the estimation of nitrate reductase (NR) and peroxidase (POD) activities, total organic nitrogen (TON), total soluble proteins, proline levels and Cd accumulation. The NR activity and total soluble proteins decreased upon Cd exposure in a concentration dependent manner, whereas TON increased significantly in 3 d seedlings upon Cd exposure. The activity of POD and proline level increased significantly as compared to the respective controls. The level of Cd accumulation was higher in T-59 than in TM-4. Therefore, T-59 was found to be the most tolerant cultivar to Cd than other three cultivars possibly due to a better capacity to transport Cd in their vacuolar sink. The variety T-59, thus, appears to be suitable for Cd phytoremediation.

Early onset myasthenia gravis with atypical features.

A 14 year old boy with atypical myasthenia gravis is reported. The interesting features of the case were the onset in first decade with progressive weakness of limb muscles simulating limb girdle myopathy, presence of bilateral symmetrical non fluctuating external ophthalmoplegia with ptosis and the absence of diplopia. Differential response to choline esterase inhibitors was clinically apparent. In contrast to excellent improvement in limb weakness with choline esterase inhibitors, there was no improvement in external ophthalmoplegia or ptosis. The possibilities are discussed and literature is reviewed.

Trends in detection of new leprosy cases at two centres in Himachal Pradesh, India: a ten-year study.

An impressive decline in leprosy prevalence rate (PR) in all endemic districts of India is seen in the post-MDT era. However, the new case-detection rate, an important statistical indicator in leprosy control programmes, has not shown significant decline in spite of all efforts. In Himachal Pradesh, a decline in PR from 7.8 to 0.56/10000 between 1991 to 2000 is seen, and recently the State has won national acclaim for having achieved the goal of elimination of leprosy in all the districts. The vertical leprosy programme has been integrated into general health services of the state. An analysis of data from 1991 to 2000 of two leprosy control units of Himachal Pradesh, the Urban Leprosy Clinic in Shimla (ULC-S) and the District Leprosy Control Unit in Mandi (DLCU-M), showed no significant decline in the new cases detected. 277 and 271 new cases were detected at these centres respectively; these included 2.2% and 1.5% children of less than 14 years of age. Almost 75% of these cases were males and of MB type. A steadily increasing trend in the annual detection of new cases was seen at both the centres during the decade. The cases registered at DLCU-M were mainly indigenous to the district. At ULC-S, 45 migrant cases from other endemic areas-mainly from Nepal, Bihar, and Uttar Pradesh-had also contributed to the increased number of new cases. Other possible causes discussed for this higher new case detection, e.g. overdiagnosis, detection of backlog "hidden cases" and voluntary reporting of patients, do not differ from those seen in other parts of the country or the world.

Augmenting in vitro shoot multiplication by vipul (triacontanol) and adventitious rhizogenesis by rice bran extract in Dendrocalamus strictus.

Like other bamboo species, Dendrocalamus strictus flowers gregariously after a prolonged intermast period of 48 years and constitutes an ideal material for in vitro clonal propagation. In this study, MS liquid medium containing 0.5, 1.0 and 2.0 mL/L vipul (Godrej Agrovet, Ltd., Sachin, India), a commercial formulation of triacontanol, with or without BA (3.0 mg/L) was tested for in vitro shoot multiplication and 1.0, 2.5 and 5.0 mL/L of 20% (w/v) alcoholic/aqueous rice bran extract (alone or in combination) with NAA (3 mg/L) used for in vitro adventitious rhizogenesis in single node culture derived shoots of Dendrocalamus strictus.. After a multiplication cycle for 4-5 week, vipul (0.5 mL/L) with BA (3.0 mg/L) in the culture medium induced 4.59 fold shoot multiplication rate whereas application of BA and vipul alone had corresponding values of 3.29 and 0.53 fold respectively. Maximum vipul concentration (2 mL/L) with BA (3 mg/L) exhibited shoot multiplication higher than (or equal to) that of BA alone. Maximum in vitro rooting percentage (55.66%) was obtained on half MS medium enriched with alcoholic rice bran extract (2.5 mL/L) and NAA (3 mg/L). This is the first investigation reporting amelioration of in vitro shoot multiplication rate by triacontanol and rooting percentage by rice bran extract in explants from mature bamboo culms. The protocol is economical and rapid for in vitro clonal propagation of Dendrocalamus strictus.

Evaluation of lesions suitable for percutaneous transluminal coronary angioplasty after thrombolytic therapy of acute myocardial infarction.

Forty-two patients of acute myocardial infarction (AMI) and clinically successful thrombolysis underwent coronary angiography 7.6 +/- 3.6 days after the AMI. The infarct related artery was patent in 33 of 42 (78.5%) patients, and 27 of these 33 (82%) had residual diameter stenosis of 70 per cent or more. Arteries showing more than 70 per cent luminal diameter narrowing were considered suitable for percutaneous transluminal coronary angioplasty (PTCA) if the lesion was less than 1 cm in length and there was no significant left main or distal lesion. Based on the above criteria, 22 of the 33 patients (66%) with recanalised infarct-related artery were found to have lesions suitable for PTCA. Thus, after successful thrombolysis, significant proportion of patients of acute myocardial infarction have residual lesions that are suitable for PTCA.