Identification and characterization of a hydrophobin Vmh3 from Pleurotus ostreatus.

Hydrophobins (HPs) are relatively small surface-active proteins of fungal origin. Being an industrially important protein, isolation of new molecules from GRAS (Generally Regarded as Safe) strains like mushrooms is the need of the time. In the present work, hydrophobin Vmh3-1 is isolated, purified, and identified from a culture broth and vegetative mycelia of Pleurotus ostreatus grown in a Potato dextrose broth (PDB) in static culture conditions. Purified proteins from the broth and the cell wall showed bands of 11 kDa and 17 kDa when analyzed on SDS-PAGE. Hydrophobin Vmh3-1 was identified in purified protein samples by the Orbitrap-HR-LC-MS/MS analysis with a maximum of 66% sequence coverage. The amphipathic nature of the protein was revealed by an increase in the water contact angle (WCA) of the hydrophilic surface of glass by 87% as well as a decrease in the WCA of the hydrophobic surface of Teflon by 19%. The emulsification property was tested with food-grade oils and Hexane. A maximum activity (EI 24) of 87.64% was recorded for Sunflower oil. In CD (Circular dichroism) spectra, Vmh3-1 showed the typical spectra of hydrophobin with a dominance of ß-sheets (51%) in the secondary structure and a minimum percentage of the α-helix (2%). The protein did not show a self-aggregating property on vigorous shaking making it suitable for numerous industrial applications. The identification of Vmh3-1 with detailed amino acid sequencing and the characterization of the protein to evaluate its potential in surface modifications for various industrial applications is demonstrated herein for the first time.

Positive Selection on a Regulatory Insertion-Deletion Polymorphism in FADS2 Influences Apparent Endogenous Synthesis of Arachidonic Acid.

Long chain polyunsaturated fatty acids (LCPUFA) are bioactive components of membrane phospholipids and serve as substrates for signaling molecules. LCPUFA can be obtained directly from animal foods or synthesized endogenously from 18 carbon precursors via the FADS2 coded enzyme. Vegans rely almost exclusively on endogenous synthesis to generate LCPUFA and we hypothesized that an adaptive genetic polymorphism would confer advantage. The rs66698963 polymorphism, a 22-bp insertion-deletion within FADS2, is associated with basal FADS1 expression, and coordinated induction of FADS1 and FADS2 in vitro. Here, we determined rs66698963 genotype frequencies from 234 individuals of a primarily vegetarian Indian population and 311 individuals from the US. A much higher I/I genotype frequency was found in Indians (68%) than in the US (18%). Analysis using 1000 Genomes Project data confirmed our observation, revealing a global I/I genotype of 70% in South Asians, 53% in Africans, 29% in East Asians, and 17% in Europeans. Tests based on population divergence, site frequency spectrum, and long-range haplotype consistently point to positive selection encompassing rs66698963 in South Asian, African, and some East Asian populations. Basal plasma phospholipid arachidonic acid (ARA) status was 8% greater in I/I compared with D/D individuals. The biochemical pathway product-precursor difference, ARA minus linoleic acid, was 31% and 13% greater for I/I and I/D compared with D/D, respectively. This study is consistent with previous in vitro data suggesting that the insertion allele enhances n-6 LCPUFA synthesis and may confer an adaptive advantage in South Asians because of the traditional plant-based diet practice.

DNA methylation analysis of phenotype specific stratified Indian population.

BACKGROUND: DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes. METHODS: Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing. RESULTS: Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5'-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5'-UTR CpG methylation was also found to be associated with higher body mass index (BMI). CONCLUSION: Differential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

Molecular mechanisms of anti-tumor properties of P276-00 in head and neck squamous cell carcinoma.

BACKGROUND: Tumors of the head and neck present aggressive pathological behavior in patients due to high expression of CDK/CCND1 proteins. P276-00, a novel CDK inhibitor currently being tested in clinic, inhibits growth of several cancers in vitro and in vivo. The pre clinical activity of P276-00 in head and neck cancer and its potential mechanisms of action at molecular level are the focus of the current studies. METHOD: We have investigated the anti-cancer activity of P276-00 in head and neck tumors in vitro and in vivo. Candidate gene expression profiling and cell based proteomic approaches were taken to understand the pathways affected by P276-00 treatment. RESULTS: It was observed that P276-00 is cytotoxic across various HNSCC cell lines with an IC50 ranging from 1.0-1.5 µmoles/L and culminated in significant cell-cycle arrest in G1/S phase followed by apoptosis. P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets. Further, we observed that apoptosis was mediated through P53 activation leading to higher BAX/BCL-2 ratio and cleaved caspase-3 levels. It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8. Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis. CONCLUSION: In summary, we have observed that P276-00 inhibits cyclin-D/CDK4/P16/pRB/E2F axis and induces apoptosis by increased P53 phosphorylation in HNSCC cells. These results suggest a novel indication for P276-00 in head and neck cancer with a potential role for IL-6 and HSPA8 as candidate serum biomarkers.

Potentiation of in vitro and in vivo antitumor efficacy of doxorubicin by cyclin-dependent kinase inhibitor P276-00 in human non-small cell lung cancer cells.

BACKGROUND: In the present study, we show that the combination of doxorubicin with the cyclin-dependent kinase inhibitor P276-00 was synergistic at suboptimal doses in the non-small cell lung carcinoma (NSCLC) cell lines and induces extensive apoptosis than either drug alone in H-460 human NSCLC cells. METHODS: Synergistic effects of P276-00 and doxorubicin on growth inhibition was studied using the Propidium Iodide (PI) assay. The doses showing the best synergistic effect was determined and these doses were used for further mechanistic studies such as western blotting, cell cycle analysis and RT-PCR. The in vivo efficacy of the combination was evaluated using the H-460 xenograft model. RESULTS: The combination of 100 nM doxorubicin followed by 1200 nM P276-00 showed synergistic effect in the p53-positive and p53-mutated cell lines H-460 and H23 respectively as compared to the p53-null cell line H1299. Abrogation of doxorubicin-induced G2/M arrest and induction of apoptosis was observed in the combination treatment. This was associated with induction of tumor suppressor protein p53 and reduction of anti-apoptotic protein Bcl-2. Furthermore, doxorubicin alone greatly induced COX-2, a NF-κB target and Cdk-1, a target of P276-00, which was downregulated by P276-00 in the combination. Doxorubicin when combined with P276-00 in a sequence-specific manner significantly inhibited tumor growth, compared with either doxorubicin or P276-00 alone in H-460 xenograft model. CONCLUSION: These findings suggest that this combination may increase the therapeutic index over doxorubicin alone and reduce systemic toxicity of doxorubicin most likely via an inhibition of doxorubicin-induced chemoresistance involving NF-κB signaling and inhibition of Cdk-1 which is involved in cell cycle progression.

Molecular pharmacology of multitarget cyclin-dependent kinase inhibitors in human colorectal carcinoma cells.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer death. Certain signaling pathways are implicated in colorectal carcinogenesis. Cyclin-dependent kinases (CDKs) are commonly hyperactivated in CRC and hence multitarget CDK inhibitors serve as promising therapeutic drugs against CRC. OBJECTIVE: Off-target effects of multitarget CDK inhibitors with differential CDK inhibitory spectrum viz. P276-00 (also known as riviciclib), roscovitine and UCN-01 on CRC cell lines of varied genetic background were delineated. METHOD: Protein expression was analyzed for key signaling proteins by western blotting. ß-catenin localization was assessed using immunofluorescence. HIF-1 transcriptional activity and target gene expression were studied by reporter gene assay and RT-PCR respectively. Anti-migratory and anti-angiogenic potential was evaluated by wound healing assay and endothelial tube formation assay. RESULTS: CDK inhibitors modulated various signaling pathways in CRC and for certain proteins showed a highly cell line-dependent response. Riviciclib and roscovitine inhibited HIF-1 transcriptional activity and HIF-1α accumulation in hypoxic HCT116 cells. Both of these drugs also abrogated migration of HCT116 and in vitro angiogenesis in HUVECs. CONCLUSION: Anticancer activity of multitarget CDK inhibitors can be certainly attributed to their off-target effects and should be analyzed while assessing their therapeutic utility against CRC.

Genome-wide DNA methylation profiling after Ayurveda intervention to bronchial asthmatics identifies differential methylation in several transcription factors with immune process related function.

BACKGROUND: The Indian traditional medicinal system, Ayurveda, describes several lifestyle practices, processes and medicines as an intervention to treat asthma. Rasayana therapy is one of them and although these treatment modules show improvement in bronchial asthma, their mechanism of action, particularly the effect on DNA methylation, is largely understudied. OBJECTIVES: Our study aimed at identifying the contribution of DNA methylation changes in modulating bronchial asthma phenotype upon Ayurveda intervention. MATERIALS AND METHODS: In this study, genome-wide methylation profiling in peripheral blood DNA of healthy controls and bronchial asthmatics before (BT) and after (AT) Ayurveda treatment was performed using array-based profiling of reference-independent methylation status (aPRIMES) coupled to microarray technique. RESULTS: We identified 4820 treatment-associated DNA methylation signatures (TADS) and 11,643 asthma-associated DNA methylation signatures (AADS), differentially methylated [FDR (≤0.1) adjusted p-values] in AT and HC groups respectively, compared to BT group. Neurotrophin TRK receptor signaling pathway was significantly enriched for differentially methylated genes in bronchial asthmatics, compared to AT and HC subjects. Additionally, we identified over 100 differentially methylated immune-related genes located in the promoter/5'-UTR regions of TADS and AADS. Various immediate-early response and immune regulatory genes with functions such as transcription factor activity (FOXD1, FOXD2, GATA6, HOXA3, HOXA5, MZF1, NFATC1, NKX2-2, NKX2-3, RUNX1, KLF11), G-protein coupled receptor activity (CXCR4, PTGER4), G-protein coupled receptor binding (UCN), DNA binding (JARID2, EBF2, SOX9), SNARE binding (CAPN10), transmembrane signaling receptor activity (GP1BB), integrin binding (ITGA6), calcium ion binding (PCDHGA12), actin binding (TRPM7, PANX1, TPM1), receptor tyrosine kinase binding (PIK3R2), receptor activity (GDNF), histone methyltransferase activity (MLL5), and catalytic activity (TSTA3) were found to show consistent methylation status between AT and HC group in microarray data. CONCLUSIONS: Our study reports the DNA methylation-regulated genes in bronchial asthmatics showing improvement in symptoms after Ayurveda intervention. DNA methylation regulation in the identified genes and pathways represents the Ayurveda intervention responsive genes and may be further explored as diagnostic, prognostic, and therapeutic biomarkers for bronchial asthma in peripheral blood.

P276-00, a cyclin-dependent kinase inhibitor, modulates cell cycle and induces apoptosis in vitro and in vivo in mantle cell lymphoma cell lines.

BACKGROUND: Mantle cell lymphoma (MCL) is a well-defined aggressive lymphoid neoplasm characterized by proliferation of mature B-lymphocytes that have a remarkable tendency to disseminate. This tumor is considered as one of the most aggressive lymphoid neoplasms with poor responses to conventional chemotherapy and relatively short survival. Since cyclin D1 and cell cycle control appears as a natural target, small-molecule inhibitors of cyclin-dependent kinases (Cdks) and cyclins may play important role in the therapy of this disorder. We explored P276-00, a novel selective potent Cdk4-D1, Cdk1-B and Cdk9-T1 inhibitor discovered by us against MCL and elucidated its potential mechanism of action. METHODS: The cytotoxic effect of P276-00 in three human MCL cell lines was evaluated in vitro. The effect of P276-00 on the regulation of cell cycle, apoptosis and transcription was assessed, which are implied in the pathogenesis of MCL. Flow cytometry, western blot, immunoflourescence and siRNA studies were performed. The in vivo efficacy and effect on survival of P276-00 was evaluated in a Jeko-1 xenograft model developed in SCID mice. PK/PD analysis of tumors were performed using LC-MS and western blot analysis. RESULTS: P276-00 showed a potent cytotoxic effect against MCL cell lines. Mechanistic studies confirmed down regulation of cell cycle regulatory proteins with apoptosis. P276-00 causes time and dose dependent increase in the sub G1 population as early as from 24 h. Reverse transcription PCR studies provide evidence that P276-00 treatment down regulated transcription of antiapoptotic protein Mcl-1 which is a potential pathogenic protein for MCL. Most importantly, in vivo studies have revealed significant efficacy as a single agent with increased survival period compared to vehicle treated. Further, preliminary combination studies of P276-00 with doxorubicin and bortezomib showed in vitro synergism. CONCLUSION: Our studies thus provide evidence and rational that P276-00 alone or in combination is a potential therapeutic molecule to improve patients' outcome in mantle cell lymphoma.

Molecular evidence for increased antitumor activity of gemcitabine in combination with a cyclin-dependent kinase inhibitor, P276-00 in pancreatic cancers.

BACKGROUND: P276-00 is a novel cyclin-dependent kinase inhibitor currently in Phase II clinical trials. Gemcitabine is a standard of care for the treatment of pancreatic cancer. The present study investigated the effect of the combination of P276-00 and gemcitabine in five pancreatic cancer cell lines. METHODS: Cytotoxic activity was evaluated by Propidium Iodide assay. Cell cycle and apoptosis was analyzed by flow cytometry. Genes and proteins known to inhibit apoptosis and contribute to chemoresistance were analysed using western blot analysis and RT-PCR. In vivo efficacy was studied in PANC-1 xenograft model. RESULTS: The combination of gemcitabine followed by P276-00 was found to be highly to weakly synergistic in various pancreatic cancer cell lines as assessed by the combination index. Enhancement of apoptosis in PANC-1 cells and decrease in the antiapoptotic protein Bcl-2 and survivin was seen. P276-00 potentiated the gemcitabine-induced cytotoxicity by modulation of proteins involved in chemoresistance to gemcitabine and cell cycle viz. antiapoptotic proteins p8 and cox-2, proapoptotic protein BNIP3 and cell cycle related proteins Cdk4 and cyclin D1. The above results could explain the novel mechanisms of action of the combination therapy. We also show here that gemcitabine in combination with P276-00 is much more effective as an antitumor agent compared with either agent alone in the PANC-1 xenograft tumor model in SCID mice. CONCLUSIONS: The chemosensitzation of pancreatic tumors to gemcitabine would likely be an important and novel strategy for treatment of pancreatic cancer and enable the use of lower and safer concentrations, to pave the way for a more effective treatment in this devastating disease. Phase IIb clinical trials of P276-00 in combination with gemcitabine in pancreatic cancer patients are ongoing.

Promising Anticancer Activity of Multitarget Cyclin Dependent Kinase Inhibitors against Human Colorectal Carcinoma Cells.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer death worldwide, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Yet, efficacy of multitarget CDK inhibitors as therapeutic agents has not been much explored against CRC. OBJECTIVE: The anticancer potential of multitarget CDK inhibitor riviciclib (also known as P276-00), was investigated against CRC cell lines of varied genetic background. METHODS: Cytotoxicity of riviciclib - potent CDK1, CDK4 and CDK9-specific inhibitor was evaluated in vitro. Further, its effect on clonogenic potential, cell cycle, apoptosis and transcription was tested using colony forming assay, flow cytometry and western blot analysis, respectively. Also, efficacy of riviciclib in combination with standard chemotherapeutic agents was assessed. Dependency of CRC cells on specific CDKs for their survival was confirmed using siRNA studies. RESULTS: Riviciclib exerted significant cytotoxicity against CRC cells and inhibited their colony forming potential. It induced apoptosis along with inhibition of cell cycle CDKs and cyclins as well as transcriptional CDKs and cyclins. Moreover, dual combination of riviciclib with standard chemotherapeutic drugs exhibited synergism in CRC cells. siRNA studies indicated that CRC cells are dependent on specific CDKs for their survival which are targets of riviciclib. CONCLUSION: This study provides evidence that multitarget CDK inhibitors can serve as promising therapeutic agents against CRC alone or in combination.

A novel inhibitor of hypoxia-inducible factor-1α P3155 also modulates PI3K pathway and inhibits growth of prostate cancer cells.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. It is essential for angiogenesis and is associated with tumor progression and overexpression of HIF-1α has been demonstrated in many common human cancers. Therefore, HIF-1α is one of the most compelling anticancer targets. METHODS: To identify HIF-1α inhibitors, luciferase reporter gene assay under hypoxia and normoxia was used. Detailed studies such as western blotting, RT-PCR, immunofluorescence were carried out to elucidate its mechanism of action. Antiangiogenic activity of P3155 was demonstrated by migration assay and tube formation assay. Efficacy study of P3155 was performed on PC-3 xenograft model. RESULTS: P3155 showed specific HIF-1α inhibition with IC50 of 1.4 µM under hypoxia. It suppressed HIF-1α expression as well as PI3K/Akt pathway and abrogated expression of HIF-1-inducible gene viz. vascular endothelial growth factor (VEGF). P3155 in combination with HIF-1α siRNA showed significant synergistic effect. In addition, it demonstrated significant in vivo efficacy and antiangiogenic potential in prostate cancer cell lines. CONCLUSION: We have identified a novel HIF-1α inhibitor P3155 that also modulates PI3K/Akt pathway, which may contribute to its significant in vitro and in vivo antitumor activity.

Two new polyoxygenated flavonoids from Blumea eriantha DC, methanol extract and their anti-proliferative activity.

Two new flavonoids, 3, 3', 4' -Trihydroxy- 6, 7, 8 -trimethoxy flavone (1) and 3-Hydroxy-6, 7, 8, 3', 4'-pentamethoxy flavone (2) have been isolated from the methanol extract of Blumea eriantha DC. The structures of these compounds were determined on the basis of spectroscopic techniques such as IR, MS, 1 D, and 2 D NMR spectroscopy. The compounds (1) and (2) were tested for their anti-proliferative activity on NCI-H23 cell line. A standard drug Paclitaxel showed potent anti-proliferative effect (56%) at 100 nM concentration after 24 h treatment whereas compound (2) did not show any anti-proliferative effect. Only compound (1) showed significant reduction in cell viability at concentration 25773 nM (89%) and 257731 nM (68%) after 24 h when compared with 0.1% DMSO. The compound (1) showed significant but much lower reduction in cell viability as compared to the standard drug Paclitaxel.

Development of novel inhibitors targeting HIF-1α towards anticancer drug discovery.

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulatory protein of cellular response to hypoxia, and regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, vascularization, and invasion/metastasis. HIF-1α has been implicated in the regulation of genes involved in angiogenesis, for example, VEGF and is associated with tumor progression. In the last decade, over expression of HIF-1α has been demonstrated in many common human cancers and emerging as a validated target for anticancer drug discovery. Here we report the discovery of newly designed and synthesized pyridylpyrimidine based potent and selective inhibitors of HIF-1α. P2630 has been found as potent antiproliferative, antiangiogenic and orally efficacious compound in PC-3 xenograft mice model.

In vitro antitumor properties of a novel cyclin-dependent kinase inhibitor, P276-00.

Cyclin-dependent kinases (Cdk) and their associated pathways represent some of the most attractive targets for the development of anticancer therapeutics. Based on antitumor activity in animal models, a variety of Cdk inhibitors are undergoing clinical evaluation either as a single agent or in combination with other approved drugs. In our anticancer drug discovery program, a novel series of flavones have been synthesized for evaluation against the activity of Cdk4-D1. This enzyme catalyzes the phosphorylation of retinoblastoma protein, thus inhibiting its function. We have identified a series of potent Cdk4-D1 inhibitors with IC(50) below 250 nmol/L. In this report, we have described the properties of one of the best compound, P276-00 of the flavone's series. P276-00 shows 40-fold selectivity toward Cdk4-D1, compared with Cdk2-E. The specificity toward 14 other related and unrelated kinases was also determined. P276-00 was found to be more selective with IC(50)s <100 nmol/L for Cdk4-D1, Cdk1-B, and Cdk9-T1, as compared with other Cdks, and less selective for non-Cdk kinases. It showed potent antiproliferative effects against various human cancer cell lines, with an IC(50) ranging from 300 to 800 nmol/L and was further compared for its antiproliferative activity against cancer and normal fibroblast cell lines. P276-00 was found to be highly selective for cancer cells as compared with normal fibroblast cells. To delineate its mechanism of action, the effect of P276-00 on cell cycle proteins was studied in human breast cancer cell line (MCF-7) and human non-small cell lung carcinoma (H-460). A significant down-regulation of cyclin D1 and Cdk4 and a decrease in Cdk4-specific pRb Ser(780) phosphorylation was observed. P276-00 produced potent inhibition of Cdk4-D1 activity that was found to be competitive with ATP and not with retinoblastoma protein. The compound also induced apoptosis in human promyelocytic leukemia (HL-60) cells, as evidenced by the induction of caspase-3 and DNA ladder studies. These data suggest that P276-00 has the potential to be developed as an anti-Cdk chemotherapeutic agent.

P276-00, a novel cyclin-dependent inhibitor induces G1-G2 arrest, shows antitumor activity on cisplatin-resistant cells and significant in vivo efficacy in tumor models.

P276-00, a flavone that inhibits cyclin-dependent kinases, has been identified by us recently as a novel antineoplastic agent. In this study, we have selected a panel of human tumor cell lines and xenografts to allow determination of selectivity and efficacy of P276-00. When tested against a panel of 16 cisplatin-sensitive and cisplatin-resistant cell lines, the antiproliferative potential of P276-00 was found to be approximately 30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was approximately 26-fold more potent than cisplatin, and further, it was also found to be active against cisplatin-resistant tumors of central nervous system, melanoma, prostate, and renal cancers. Further, we studied the effects of P276-00 on cell cycle progression by flow cytometry using asynchronous and synchronous population of tumor and normal cells. Asynchronous population of human prostate carcinoma (PC-3) and human promyelocytic leukemia (HL-60) cells when exposed to P276-00 showed arrest of slow-growing PC-3 cells in G(2)-M with no significant apoptosis observed up to 72 h. Unlike PC-3, significant apoptosis was seen in fast-growing HL-60 cells at 6 h. However, synchronized human non-small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells showed arrest of cells in G(1). H-460 cells undergo apoptosis, which increases with longer exposure to the compound and also after exposure to P276-00 for 48 h followed by recovery. In contrast, the normal cells (WI-38) remain arrested in G(1) with no significant apoptosis up to 72 h of exposure and also after 48 h of P276-00 treatment followed by recovery, confirming our previous results that P276-00 was less effective against normal cells compared with cancer cells. After promising in vitro results, P276-00 was checked for in vivo efficacy in murine tumor and human xenograft models. Growth inhibition of murine colon cancer (CA-51) was significant when P276-00 was administered i.p. at 50 mg/kg daily for 20 treatments. However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for seven treatments showed significant inhibition in the growth. Further studies were undertaken to establish the efficacy profile of P276-00 in human tumor xenograft models. In the two xenograft models studied, P276-00 showed potent in vivo antitumor potential. Compound P276-00 at a dose of 35 mg/kg administered daily via the i.p. route for 10 days showed significant (P < 0.05) inhibition in the growth of human colon carcinoma HCT-116 xenograft. Furthermore, P276-00 at a dose of 50 mg/kg once daily and 30 mg/kg twice daily administered via i.p. route for 20 treatments significantly (P < 0.05) inhibited growth of human non-small cell lung carcinoma H-460 xenograft. Thus, the in vitro cellular potency, together with in vivo antitumor activity, confirms the potential of P276-00, a cyclin-dependent kinase inhibitor as an anticancer molecule.

Dosha phenotype specific Ayurveda intervention ameliorates asthma symptoms through cytokine modulations: Results of whole system clinical trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Over the past few decades, there have been significant scientific advances leading to improved understanding of asthma as a disease and treatment providing immediate relief. However, prevention of recurrent attacks, exacerbations and disease cure remains a challenge. Ayurveda refers to bronchial asthma as Tamaka Swasa and it is well explained in Charaka Samhita. Management of asthma in Ayurveda includes removal of vitiated Kapha through Shodhana, Shamana procedures, herbal and herbomineral formulations in addition to advising a healthy lifestyle and diet. Several clinical trials on Ayurvedic formulations for treatment of asthma are reported, however, whole system management of asthma has rarely been studied in the manner in which it is actually being practiced. Ayurveda therapeutics provides Dosha specific approaches, which needs biological investigation. AIM OF THE STUDY: The objective of our study was to investigate lung functions and cytokine changes in Asthmatic individuals in response to Ayurvedic intervention. METHODS: The study design was approved by the Institutional Ethics Committee of Tilak Ayurveda Mahavidyalaya (TAMV) & Sheth Tarachand Ramnath Charitable Ayurveda Hospital and followed guidelines of the Declaration of Helsinki and Tokyo for humans. It was conducted as a whole system individualized pragmatic clinical trial and written consent of patients was collected before enrollment. One hundred and fifteen patients with mild-to-moderate asthma were divided into 2 sub-groups depending on their disease subsets and administered phenotype specific ayurvedic interventions. Seventy six asthma patients completed the treatment. Serum IgE levels, blood eosinophil counts, spirometry and blood cytokine levels were measured before the start of treatment and six months at the end of treatment. Age and sex matched healthy participants (n=69) were recruited in the study for comparison of cytokines levels. RESULTS: Significant improvements in FEV1(% predicted) (p<0.0001) and FVC (% predicted) (p=0.0001) was observed in asthmatic patients who underwent Ayurvedic treatment. Circulating levels of IgE (p<0.03) and eosinophil numbers (p=0.001) reduced significantly in the asthmatics after Ayurvedic treatment. This was associated with significant reduction in levels of circulating cytokines. Levels of Th2, Th1 and inflammatory cytokines in the peripheral blood were higher than healthy control participants at baseline (p values <0.0001) and reduced significantly after ayurvedic intervention. CONCLUSION: This proof of concept study highlights the potential benefits and possible mechanism of Ayurvedic interventions in patients with mild-to-moderate asthma. The interventions significantly reduced IgE and eosinophil count, also improved lung function and reduced levels of circulating Th2 cytokines.

DNA methylation detection at single base resolution using targeted next generation bisulfite sequencing and cross validation using capillary sequencing.

With a purpose of accurate and simultaneous determination of DNA methylation from multiple loci in multiple samples, here, we are demonstrating a method to aid rapid DNA methylation detection of genomic sequences. Using genomic DNA of peripheral blood from 14 healthy individuals, DNA methylation in 465 CpG sites from 12 loci of genes (ADAM22, ATF2, BCR, CD83, CREBBP, IL12B, IL17RA, MAP2K2, RBM38, TGFBR2, TGFBR3, and WNT5A) was analysed by targeted next generation bisulfite sequencing. Analysed region for three genes, BCR, IL17RA and RBM38 showed an absolute mean DNA methylation of 25.6%, 89.2% and 38.9% respectively. Other nine gene loci were unmethylated and exhibited <10% absolute mean DNA methylation. Two genes, IL17RA and RBM38 were technically validated using direct capillary sequencing and results were comparable with positive correlation (P=0.0088 & P<0.0001 respectively) in the CpG sites for DNA methylation. All CpG sites analysed from RBM38 genes locus displayed 95% limits of agreement for DNA methylation measurements from the two methods. The present approach provides a fast and reliable DNA methylation quantitative data at single base resolution with good coverage of the CpG sites under analysis in multiple loci and samples simultaneously. Use of targeted next generation bisulfite sequencing may provide an opportunity to explore genes in the discovery panel for biomarker identification and facilitate functional validation.

Genome-wide analysis correlates Ayurveda Prakriti.

The practice of Ayurveda, the traditional medicine of India, is based on the concept of three major constitutional types (Vata, Pitta and Kapha) defined as "Prakriti". To the best of our knowledge, no study has convincingly correlated genomic variations with the classification of Prakriti. In the present study, we performed genome-wide SNP (single nucleotide polymorphism) analysis (Affymetrix, 6.0) of 262 well-classified male individuals (after screening 3416 subjects) belonging to three Prakritis. We found 52 SNPs (p ≤ 1 × 10(-5)) were significantly different between Prakritis, without any confounding effect of stratification, after 10(6) permutations. Principal component analysis (PCA) of these SNPs classified 262 individuals into their respective groups (Vata, Pitta and Kapha) irrespective of their ancestry, which represent its power in categorization. We further validated our finding with 297 Indian population samples with known ancestry. Subsequently, we found that PGM1 correlates with phenotype of Pitta as described in the ancient text of Caraka Samhita, suggesting that the phenotypic classification of India's traditional medicine has a genetic basis; and its Prakriti-based practice in vogue for many centuries resonates with personalized medicine.

Docosahexaenoic acid content is significantly higher in ghrita prepared by traditional Ayurvedic method.

BACKGROUND: Ghee (clarified butter) also known as ghrita, has been utilized for thousands of years in Ayurveda. Ghee is mostly prepared by traditional method in Indian households or by direct cream method at industry level. Ayurvedic classics mention that ghrita made from cow milk is superior. However, there is no scientific comparison available on preparation methods and essential fatty acids content of ghrita. OBJECTIVE: To investigate fatty acid composition of ghrita prepared by traditional/Ayurvedic method and commercial method (direct cream method). MATERIALS AND METHODS: Fatty Acid Methyl Esters (FAME) extracted from ghrita samples were analysed on Gas Chromatography (GC) Shimadzu B using capillary column BPX70 (0.32 mm*60 m, ID of 0.25 mm). The fatty acids in the samples were identified by comparing peaks with the external standard 68A (Nu-Chek-Prep, Inc.USA). Significant differences between the experimental groups were assessed by analysis of variance. RESULTS: Distribution of fatty acids was compared in ghrita samples prepared by traditional method and direct cream method which is commercially used. Saturated fatty acids were predominant in both the groups. Mono unsaturated fatty acids and poly unsaturated fatty acids were in the range of 17-18% and 3-6% respectively. DHA content was significantly higher in ghee prepared by traditional method using curd starter fermentation. CONCLUSION: The findings suggested that ghrita prepared by traditional ayurvedic methods contains higher amount of DHA; Omega-3 long-chain polyunsaturated fatty acids, which is a major component of retinal and brain tissues and remains important in prevention of various diseases.

Insights from Ayurveda for translational stem cell research.

Ayurveda, the traditional Indian system of medicine has given great emphasis to the promotion of health. Ayurveda therapies are based on restoration of body balance and nourishment of dhatus or tissues. Rasayana concept of Ayurveda explains tissue regeneration and cell renewal. The drugs and therapies explained as rasayana provide research opportunities for biology of regeneration. Specific rasayana stimulate and nourish respective dhatus. Interpretation of this description offers clues for specific differentiation of stem cells with appropriate extract. The preliminary experiments on Medhya drugs suggest neuronal stem cells differentiation. Authors highlight the potential of Ayurveda and its possible contributions in regenerative medicine. Authors propose a protocol based on integrative approach derived from Ayurveda concepts and current understanding of regenerative medicine. The advanced understanding about adult and embryonic stem cells along with concepts of regeneration in Ayurveda has immense potential in the development of regenerative medicine.