Rescue of dendritic cells from glycolysis inhibition improves cancer immunotherapy in mice.

Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging. Herein, a strategy is presented where glycolysis is inhibited in cancer cells using PFK15 (inhibitor of PFKFB3, rate-limiting step in glycolysis), while simultaneously glycolysis and function is rescued in DCs by delivery of fructose-1,6-biphosphate (F16BP, one-step downstream of PFKFB3). To demonstrate the feasibility of this strategy, vaccine formulations are generated using calcium-phosphate chemistry, that incorporate F16BP, poly(IC) as adjuvant, and phosphorylated-TRP2 peptide antigen and tested in challenging and established YUMM1.1 tumours in immunocompetent female mice. Furthermore, to test the versatility of this strategy, adoptive DC therapy is developed with formulations that incorporate F16BP, poly(IC) as adjuvant and mRNA derived from B16F10 cells as antigens in established B16F10 tumours in immunocompetent female mice. F16BP vaccine formulations rescue DCs in vitro and in vivo, significantly improve the survival of mice, and generate cytotoxic T cell (Tc) responses by elevating Tc1 and Tc17 cells within the tumour. Overall, these results demonstrate that rescuing glycolysis of DCs using metabolite-based formulations can be utilized to generate immunotherapy even in the presence of glycolytic inhibitor.

Therapeutic Implications of Some Natural Products for Neuroimmune Diseases: A Narrative of Clinical Studies Review.

Neuroimmune diseases are a group of disorders that occur due to the dysregulation of both the nervous and immune systems, and these illnesses impact tens of millions of people worldwide. However, patients who suffer from these debilitating conditions have very few FDA-approved treatment options. Neuroimmune crosstalk is important for controlling the immune system both centrally and peripherally to maintain tissue homeostasis. This review aims to provide readers with information on how natural products modulate neuroimmune crosstalk and the therapeutic implications of natural products, including curcumin, epigallocatechin-3-gallate (EGCG), ginkgo special extract, ashwagandha, Centella asiatica, Bacopa monnieri, ginseng, and cannabis to mitigate the progression of neuroimmune diseases, such as Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, depression, and anxiety disorders. The majority of the natural products based clinical studies mentioned in this study have yielded positive results. To achieve the expected results from natural products based clinical studies, researchers should focus on enhancing bioavailability and determining the synergistic mechanisms of herbal compounds and extracts, which will lead to the discovery of more effective phytomedicines while averting the probable negative effects of natural product extracts. Therefore, future studies developing nutraceuticals to mitigate neuroimmune diseases that incorporate phytochemicals to produce synergistic effects must analyse efficacy, bioavailability, gut-brain axis function safety, chemical modifications, and encapsulation with nanoparticles.

Short term, low dose alpha-ketoglutarate based polymeric nanoparticles with methotrexate reverse rheumatoid arthritis symptoms in mice and modulate T helper cell responses.

Activated effector T cells induce pro-inflammatory responses in rheumatoid arthritis (RA) which then lead to inflammation of the joints. In this report, we demonstrate that polymeric nanoparticles with alpha keto-glutarate (aKG) in their polymer backbone (termed as paKG NPs) modulate T cell responses in vitro and in vivo. Impressively, a low dose of only three administrations of methotrexate, a clinically and chronically administered drug for RA, in conjunction with two doses of paKG NPs, reversed arthritis symptoms in collagen-induced arthritis (CIA) mice. This was further followed by significant decreases in pro-inflammatory antigen-specific T helper type 17 (TH17) responses and a significant increase in anti-inflammatory regulatory T cell (TREG) responses when CIA treated splenic cells were isolated and re-exposed to the CIA self-antigen. Overall, this study supports the concurrent and short term, low dose of paKG NPs and methotrexate for the reversal of RA symptoms.

Green synthesized Se nanoparticle-mediated alleviation of salt stress in field mustard, TS-36 variety.

During this decade, selenium nanoparticles have been found to play a crucial role in helping plants endure several stress conditions, which thereby helps enhance the production of crops in such harsh environments. Globally, high salinity is considered a long-term stress in the crop fields which affects the growth and production of many crops, including mustard-one of the most important oil crops. Here, the activities of spherical-shaped selenium nanoparticles with an average particle size of 55.81 nm, synthesized and functionalized by phytochemicals of fresh grape aqueous extract, were evaluated in the salinity stress (200 mM NaCl) tolerance of mustard plants grown hydroponically in modified Hoagland's solution. These bioactive nanoparticles (30 mg L-1) have exhibited significant activity in alleviating the salt stress complications in mustard, enhancing the activities of antioxidant enzymes (SOD 41.20 %, CAT 64.10 %, APX 63.06 %, and POX 70.43 %), phenolic content (98.88 %), flavonoid content (86.90 %), and free radical scavenging activity (61.89 %). The seed germination percentage, root and shoot length, fresh and dry weight per plant, water content percentage, chlorophyll content, carbohydrate content, and protein content were significantly improved by 39.66 %, 75 %, 60.64 %, 41.2 %, 22.11 %, 1.02 %, 81.92 %, 24.65 % and 79.14 % respectively by the nano selenium application during NaCl stress compared to the control group growing under salt stress without nanoparticles. Gas chromatography-mass spectrometry chromatogram analysis inferred the interaction between the nano-selenium and mustard plants under salt stress. Besides, the in-silico analysis revealed the active molecular interactions between selenium and 20 different proteins of mustard, including glutathione peroxidase, an important antioxidant enzyme.

Targeting Multiple End Organs in Lupus and Other Systemic Rheumatic Diseases by Inhibiting Bruton's Tyrosine Kinase.

Bruton tyrosine kinase (Btk) plays a vital role in activating and differentiating B-cells and regulating signaling in myeloid cells. Indeed, the potential use of Btk inhibitors in preventing lupus has been reported. Here, we extend these observations to 4 additional models of end-organ inflammation: (a) BWF1 lupus nephritis mice, (b) anti-GBM nephritis, (c) bleomycin-induced systemic sclerosis like skin disease, and (d) bleomycin-induced lung disease. In agreement with the previous studies, BTK inhibitor (BTKB66) treatment was effective in treating lupus nephritis in terms of reducing renal damage both functionally and histologically, accompanied by significant decrease in proteinuria. Both low-dose and high-dose BTKB66 profoundly blocked renal disease in the anti-GBM nephritis model, with efficacy that was comparable to that seen with dexamethasone. This study provides the first evidence that BTK inhibition has both therapeutic and preventative effects in bleomycin-induced SSc-like disease, in terms of reducing skin thickness, fibrosis, collagen deposition, and inflammation. Likewise, significantly lower lung inflammatory cell infiltration was observed after treatment with BTKB66. Therapeutic benefit was associated with lower numbers of macrophages, proliferating macrophages and activated T-cells in the respective injured organs. The observation that these immune cells play key roles in driving end organ inflammation in multiple systemic rheumatic diseases have broad implications for the use of BTKB66 in managing patients with systemic rheumatic diseases where multiple end organs are afflicted, including lupus and systemic sclerosis.

Epigallocatechin-3-Gallate Dampens Non-Alcoholic Fatty Liver by Modulating Liver Function, Lipid Profile and Macrophage Polarization.

Epigallocatechin-3-gallate (EGCG) has been shown to attenuate obesity, fatty liver disease, hepatic inflammation and lipid profiles. Here, we validate the efficacy of EGCG in a murine model of non-alcoholic fatty liver disease (NAFLD) and extend the mechanistic insights. NAFLD was induced in mice by a high-fat diet (HFD) with 30% fructose. EGCG was administered at a low dose (25 mg/kg/day, EGCG-25) or high dose (50 mg/kg/day, EGCG-50) for 8 weeks. In HFD-fed mice, EGCG attenuated body and liver weight by ~22% and 47%, respectively, accompanied by ~47% reduction in hepatic triglyceride (TG) accumulation and ~38% reduction in serum cholesterol, resonating well with previous reports in the literature. In EGCG-treated mice, the hepatic steatosis score and the non-alcoholic steatohepatitis activity score were both reduced by ~50% and ~57%, respectively, accompanied by improvements in hepatic inflammation grade. Liver enzymes were improved ~2-3-fold following EGCG treatment, recapitulating previous reports. Hepatic flow cytometry demonstrated that EGCG-fed mice had lower Ly6C+, MHCII+ and higher CD206+, CD23+ hepatic macrophage infiltration, indicating that EGCG impactedM1/M2 macrophage polarization. Our study further validates the salubrious effects of EGCG on NAFLD and sheds light on a novel mechanistic contribution of EGCG, namely hepatic M1-to-M2 macrophage polarization. These findings offer further support for the use of EGCG in human NAFLD.

Low dose Epigallocatechin Gallate Alleviates Experimental Colitis by Subduing Inflammatory Cells and Cytokines, and Improving Intestinal Permeability.

BACKGROUND: In this study, we investigate the impact of epigallocatechin gallate (EGCG), the most abundant and potent catechin in green tea, on a mouse model of inflammatory bowel disease (IBD) and the underlying mechanisms of action. METHODS: C57BL/6J mice were subjected to dextran sulfate sodium (DSS)-induced IBD-like disease and then randomly divided into three groups: Model group (MD), low-dose EGCG group (LE, 20 mg/kg/d), and high-dose EGCG group (HE, 50 mg/kg/d). DSS-induced clinical and macroscopic changes were monitored daily. Intestinal permeability was assessed by FITC-Dextran assay. RESULTS: Both high- and low-dose EGCG treatment alleviated clinical manifestations including body weight loss and disease activity index (DAI) of DSS-induced colitis. The DAI score was significantly improved after two days of EGCG treatment. At the end of the study, the macroscopic severity score (MSS) of HE and LE treatment groups were 2.4 ± 1.2, and 2.2 ± 1.0, respectively, significantly lower than that of the controls (5.0 ± 2.1). EGCG treatment also prevented colon shortening, and improved intestinal permeability and histopathological changes. In addition, EGCG treatment attenuated colon inflammation by suppressing colonic levels of pro-inflammatory cytokines IL-6, MCP-1, and TNF-alpha, and inhibited CD3+ T cell and CD68+ macrophage infiltration. CONCLUSION: EGCG is effective in inflammatory colitis because it reduces cellular and molecular inflammation, and reduces intestinal permeability.

PEGylated (NH4)xWO3 nanorod mediated rapid photonecrosis of breast cancer cells.

Photothermal therapy has emerged as a potential minimally invasive technique to destroy malignant cells with high selectivity. It utilizes low band gap nanoscale materials as photothermal agents dispersed at the affected area to increase the temperature locally by absorbing radiation in the near infrared (NIR) region and destroys the cells. In an effort to develop a photothermal agent with high efficacy for photothermal therapy, we found that (NH4)xWO3 nanorods of length 0.5-1.0 µm and diameter ∼100 nm could destroy breast cancer cells rapidly when irradiated with a wavelength in the therapeutic window. The material was prepared using a solvothermal route followed by PEGylation for improving the biocompatibility. X-ray diffraction and transmission electron microscopy studies revealed the hexagonal crystal lattice of the material. The uniform wrapping of polyethylene glycol (PEG) around the nanorods was confirmed using energy dispersive spectroscopy elemental mapping. An 808 nm laser was used to investigate the photothermal responses of the material on SUM-159 and MCF-7 breast cancer cells in vitro. The PEGylated-(NH4)xWO3 nanorods exhibited rapid temperature elevation from 20 °C to 60 °C within 3 min upon irradiation. A significant growth inhibition of SUM-159 and MCF-7 breast cancer cells with photonecrosis was observed. PEGylated (NH4)xWO3 nanorods could potentially be used in cancer therapy due to their strong photonecrotic properties at specific NIR wavelengths that suffer from minimal attenuation while passing through biological tissues.

Curcumin in Autoimmune and Rheumatic Diseases.

Over recent decades, many clinical trials on curcumin supplementation have been conducted on various autoimmune diseases including osteoarthritis, type 2 diabetes, and ulcerative colitis patients. This review attempts to summarize the highlights from these clinical trials. The efficacy of curcumin either alone or in conjunction with existing treatment was evaluated. Sixteen clinical trials have been conducted in osteoarthritis, 14 of which yielded significant improvements in multiple disease parameters. Eight trials have been conducted in type 2 diabetes, all yielding significant improvement in clinical or laboratory outcomes. Three trials were in ulcerative colitis, two of which yielded significant improvement in at least one clinical outcome. Additionally, two clinical trials on rheumatoid arthritis, one clinical trial on lupus nephritis, and two clinical trials on multiple sclerosis resulted in inconclusive results. Longer duration, larger cohort size, and multiple dosage arm trials are warranted to establish the long term benefits of curcumin supplementation. Multiple mechanisms of action of curcumin on these diseases have been researched, including the modulation of the eicosanoid pathway towards a more anti-inflammatory pathway, and the modulation of serum lipid levels towards a favorable profile. Overall, curcumin supplementation emerges as an effective therapeutic agent with minimal-to-no side effects, which can be added in conjunction to current standard of care.

Sunlight induced biosynthesis of silver nanoparticle from the bark extract of Amentotaxus assamica D.K. Ferguson and its antibacterial activity against Escherichia coli and Staphylococcus aureus.

Green synthesis of nanoparticles has gained importance due to its eco-friendly, low toxicity and cost effective nature. This study deals with the biosynthesis of silver nanoparticles (AgNPs) from the bark extract of Amentotaxus assamica. The AgNPs have been synthesised by reducing the silver ions into stable AgNPs using the bark extract of Amentotaxus assamica under the influence of sunlight irradiation. The characterisation of the biosynthesised AgNPs was carried out by UV-vis spectroscopy, X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray analysis. The UV-vis spectrum showed a broad peak at 472 nm. Also, the XRD confirmed the crystalline structure of the AgNPs. Moreover, the SEM analysis revealed that the biosynthesised AgNPs were spherical in shape. Also, dynamic light scattering techniques were used to evaluate the size distribution profile of the biosynthesised AgNPs. Furthermore, the biosynthesised AgNPs showed a prominent inhibitory effect against both Escherichia coli (MTCC 111) and Staphylococcus aureus (MTCC 97). Thus the biosynthesis of AgNPs from the bark extract of Amentotaxus assamica is found to eco-friendly way of producing AgNPs compared to chemical method.

Omega-3 Fatty Acids in Rheumatic Diseases: A Critical Review.

Many clinical trials of omega-3 fatty acids, supplied as fish oil supplements, have been carried out in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), lupus nephritis, and osteoarthritis (OA) over the past 3 decades. This review attempts to summarize the highlights of these studies to evaluate the clinical efficacy for omega-3 fatty acids to be added alongside existing treatment regimens. A total of 20 clinical trials have been carried out in RA, of which 16 exhibited significant improvements in multiple disease clinical outcomes. Nine clinical trials have been completed in SLE and lupus nephritis, of which 6 exhibited significant improvements in 1 or more clinical outcomes. A total of 4 clinical trials have been conducted in OA, of which 3 exhibited significant improvements in at least 1 clinical parameter. Multiple mechanisms for the clinical effects of omega-3 fatty acids have been implicated, including the modulation of eicosanoid synthesis toward a more anti-inflammatory profile and suppressed production of proinflammatory cytokines. Overall, fish oil supplements appear to be a safe and effective agent that could be added to the current treatment regimens in RA. Longer-term trials with larger patient cohort sizes are warranted to establish any long-term benefits of fish oil supplements in SLE, lupus nephritis, and OA.

Uptake of Total Petroleum Hydrocarbon (TPH) and Polycyclic Aromatic Hydrocarbons (PAHs) by Oryza sativa L. Grown in Soil Contaminated with Crude Oil.

The purpose of this study was to determine whether total petroleum hydrocarbon (TPH) and polycyclic aromatic hydrocarbons (PAHs) present in crude oil contaminated sites are transferred to roots, shoots and finally the grains of rice crops (Oryza sativa L.) grown in those sites. Soil was artificially contaminated with crude oil at concentrations of 0, 1000, 5000, 10,000, and 15,000 mg/kg, followed by planting of rice seedlings. After harvest, TPH in plant samples were measured, and it was determined that the uptake of TPH by the plants gradually increased as the concentration of oil in soil increased. Further, from GC-MS analysis, it was observed that PAHs including naphthalene and phenanthrene bioaccumulated in rice plant parts. Vital physico-chemical properties of soil were also altered due to crude oil contamination. Our study revealed that rice plants grown in crude oil polluted sites can uptake TPH including PAHs, thus emphasising the importance of prior investigation of soil condition before cultivation of crops.

Utilization of Paneer Whey Waste for Cost-Effective Production of Rhamnolipid Biosurfactant.

The present study aimed at isolating rhamnolipid biosurfactant-producing bacteria that could utilize paneer whey, an abundant waste source as sole medium for the production purpose. Pseudomonas aeruginosa strain, SR17, was isolated from hydrocarbon-contaminated soil that could efficiently utilize paneer whey for rhamnolipid production and reduce surface tension of the medium from 52 to 26.5 mN/m. The yield of biosurfactant obtained was 2.7 g/l, upgraded to 4.8 g/l when supplemented with 2 % glucose and mineral salts. Biochemical, FTIR, and LC-MS analysis revealed that extracted biosurfactant is a combination of both mono and di-rhamnolipid congeners. The critical micelle concentration (CMC) was measured to be 110 mg/l. Emulsification activity of the biosurfactant against n-hexadecane, olive oil, kerosene, diesel oil, engine oil, and crude oil were found to be 83, 88, 81, 92, 86, and 100 %, respectively. The rhamnolipid was detected to be non-toxic against mouse fibroblastic cell line L292.

Chrysin rich Scutellaria discolor Colebr. induces cervical cancer cell death via the induction of cell cycle arrest and caspase-dependent apoptosis.

AIMS: Scutellaria discolor Colebr. has been extensively used in traditional medicine against several diseases. The purpose of this study was to investigate the anticancer potential of S. discolor and to isolate the bioactive principle responsible for the anticancer activity. METHODS: Cytotoxicity experiments were performed on cancer and normal cells using MTT assay. The mechanism of cell death was evaluated using real time PCR array, fluorescence microscopy, flow cytometry and Western blotting. MTT assay guided isolation (partition and column chromatography) was performed to identify the antiproliferative principle. Quantification of the active principle was done using HPLC. KEY FINDINGS: Acetone extract of S. discolor (SDE) inhibited the growth and survival of cancer cells to varying degree, but the inhibition was found to be maximum in cervical cancer cell lines. There was no significant toxicity induced to normal cells. The cell death was mediated through apoptosis. There was increased mitochondrial membrane depolarization, expression of Bax, caspase-9, caspase-3 and cleaved-PARP indicating that SDE-induced caspase dependent apoptosis in HeLa cells. Moreover, SDE caused cell cycle arrest in G2 phase in HeLa cells. Cytotoxicity guided fractionation of SDE led to the isolation of chrysin as the active principle responsible for the antiproliferative activity for cervical cancer cells. Interestingly, chrysin was the major phytochemical constituent present in S. discolor. SIGNIFICANCE: S. discolor is an important anticancer plant and a new source of chrysin.

Placebo-controlled randomized clinical trial of fish oil's impact on fatigue, quality of life, and disease activity in Systemic Lupus Erythematosus.

INTRODUCTION: A recent metabolomic screen of sera from patients with Systemic Lupus Erythematosus (SLE) found reduction of antioxidants and substrates for energy generation. These metabolic alterations may underlie one of the most common features of SLE--fatigue. The metabolomic studies also noted reduced omega-3 fatty acids, which are powerful anti- oxidants. This deficiency may be causally related to oxidative stress, inflammation, disease activity, and fatigue in SLE. Supplementation of omega-3 fatty acids using fish oil in SLE has been shown to reduce oxidative stress in other studies. The objective of this study is to evaluate the effect of fish oil supplementation on clinical measures of fatigue, quality of life, and disease activity as part of a randomized clinical trial. METHODS: Fifty SLE patients recruited in outpatient clinics were randomized 1:1 to fish oil supplementation or olive oil placebo, and blinded to their treatment group. At baseline and after 6 months of treatment, RAND Short Form-36 (RAND SF-36), Fatigue Severity Scale (FSS), SLE Disease Activity Index (SLEDAI), and Physician Global Assessment (PGA) were completed; serum was also collected for soluble mediator analysis. RESULTS: Thirty-two patients completed the study. PGA improved significantly in the fish oil group compared with the placebo group (p = 0.015). The RAND SF-36 Energy/fatigue and Emotional well-being scores demonstrated improvement trends (p = 0.092 and 0.070). No clear difference was seen in FSS and SLEDAI (p = 0.350 and p = 0.417). Erythrocyte sedimentation rate and serum IL-12 were reduced (p = 0.008 and p = 0.058); while serum IL-13 was increased by fish oil supplementation (p = 0.033). CONCLUSIONS: In this randomized, placebo-controlled 6-month trial, SLE patients randomized to fish oil supplementation demonstrated improvement in their PGA, RAND SF-36, and some circulating inflammatory markers. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02021513 (registered 13 December 2013).

Green tea polyphenol (-)-epigallocatechin-3-gallate restores Nrf2 activity and ameliorates crescentic glomerulonephritis.

Crescentic glomerulonephritis (GN) is the most severe form of GN and is associated with significant morbidity and mortality despite aggressive immunotherapy with steroids, cytotoxic drugs, and plasmapheresis. We examined the therapeutic efficacy of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG, 50 mg/kg BW/day x3 weeks), a potent anti-inflammatory and anti-oxidant agent, on experimental crescentic GN induced in 129/svJ mice by administration of rabbit anti-mouse glomerular basement membrane sera. Routine histology and key molecules involved in inflammatory and redox signaling were studied. EGCG treatment significantly reduced mortality, decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. The improvements in renal function and histology were accompanied by the restoration of Nrf2 signaling (which was impaired in vehicle-treated mice) as shown by increased nuclear translocation of Nrf2 and cytoplasmic glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione peroxidase. EGCG-treated mice also showed reduction in p-Akt, p-JNK, p-ERK1/2 and p-P38 as well as restoration of PPARγ and SIRT1 levels. Lower dose of EGCG (25 mg/kg BW/day x2 weeks) treatment also significantly decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. Thus, our data illustrate the efficacy of EGCG in reversing the progression of crescentic GN in mice by targeting multiple signaling and inflammatory pathways as well as countering oxidative stress.

Effects of amino acid derivatives on physical, mental, and physiological activities.

Nutritional ergogenic aids have been in use for a long time to enhance exercise and sports performance. Dietary components that exhibit ergogenic activity are numerous and their consumption is common and popular among athletes. They often come under scrutiny by legal authorities for their claimed benefits and safety concerns. Amino acid derivatives are propagated as being effective aids to enhance physical and mental performance in many ways, even though studies have pointed out that individuals who are deficient are more likely to benefit from dietary supplementation of amino acid derivatives than normal humans. In this review, some of the most common and widely used amino acids derivatives in sports and athletics namely creatine, tyrosine, carnitine, HMB, and taurine have been discussed for their effects on exercise performance, mental activity as well as body strength and composition. Creatine, carnitine, HMB, and taurine are reported to delay the onset of fatigue, improve exercise performance, and body strength. HMB helps in increasing fat-free mass and reduce exercise induced muscle injury. Taurine has been found to reduce oxidative stress during exercise and also act as an antihypertensive agent. Although, studies have not been able to find any favorable effect of tyrosine administration on exercise performance, it has been proved to be very effective in fighting stress, improving mood and cognitive performance particularly in sleep-deprived subjects. While available data from published studies and findings are equivocal about the efficacy of creatine, tyrosine, and HMB, more comprehensive researches on carnitine and taurine are necessary to provide evidence for the theoretical basis of their ergogenic role in nutritional modification and supplementation.

Cephalotaxus griffithii Hook.f. needle extract induces cell cycle arrest, apoptosis and suppression of hTERT and hTR expression on human breast cancer cells.

BACKGROUND: Cephalotaxus spp. are known to possess anticancer potential. In this present work, for the first time the effects of C. griffithii needle (CGN) extracts on human cancer cells were examined. METHODS: The CGN was successively extracted with petroleum ether (PE), acetone and methanol. The extracts were tested for its effect on proliferation of cancer cells (MTT assay on HeLa, ZR751 and HepG2). Extract that showed the maximum growth inhibitory effect was subjected for mechanism of action study. These included apoptosis (morphological and DNA fragmentation assay), cell cycle (flow cytometry), caspase expression (Western blot) and activity (assay kit), p53 (western blot and TP53 siRNA interference) and telomerase expression (reverse transcriptase PCR) analysis. RESULTS: Among the extracts, PE extract induced maximum cytotoxicity, with highest death occurred in ZR751 cells. Since, PE extract induced cell death was highest among the CGN extracts, with maximum cancer cell death occurred in ZR751 cells; we carried out mechanism study of PE extract induced ZR751 cell death. It was observed that PE extract induced ZR751 cell death was associated with cell cycle arrest and apoptosis by activating both intrinsic and extrinsic apoptotic pathways. Knock down study revealed that p53 is essential for loss of ZR751 cell viability induced by PE extract. Further, PE extract down-regulated hTERT, hTR, and c-Myc expression. Thin layer chromatography analysis indicated the presence of unique phytochemicals in PE extract. CONCLUSION: Based on the observations, we concluded that PE extract of C. griffithii needle contains important phyto-components with multiple cellular targets for control of breast cancer and is worthy of future studies.

Metabolic disturbances associated with systemic lupus erythematosus.

The metabolic disturbances that underlie systemic lupus erythematosus are currently unknown. A metabolomic study was executed, comparing the sera of 20 SLE patients against that of healthy controls, using LC/MS and GC/MS platforms. Validation of key differences was performed using an independent cohort of 38 SLE patients and orthogonal assays. SLE sera showed evidence of profoundly dampened glycolysis, Krebs cycle, fatty acid ß oxidation and amino acid metabolism, alluding to reduced energy biogenesis from all sources. Whereas long-chain fatty acids, including the n3 and n6 essential fatty acids, were significantly reduced, medium chain fatty acids and serum free fatty acids were elevated. The SLE metabolome exhibited profound lipid peroxidation, reflective of oxidative damage. Deficiencies were noted in the cellular anti-oxidant, glutathione, and all methyl group donors, including cysteine, methionine, and choline, as well as phosphocholines. The best discriminators of SLE included elevated lipid peroxidation products, MDA, gamma-glutamyl peptides, GGT, leukotriene B4 and 5-HETE. Importantly, similar elevations were not observed in another chronic inflammatory autoimmune disease, rheumatoid arthritis. To sum, comprehensive profiling of the SLE metabolome reveals evidence of heightened oxidative stress, inflammation, reduced energy generation, altered lipid profiles and a pro-thrombotic state. Resetting the SLE metabolome, either by targeting selected molecules or by supplementing the diet with essential fatty acids, vitamins and methyl group donors offers novel opportunities for disease modulation in this disabling systemic autoimmune ailment.

Agrobacterium-mediated genetic transformation of Pogostemon cablin (Blanco) Benth. Using leaf explants: bactericidal effect of leaf extracts and counteracting strategies.

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 µM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.