C2GAP2 is a common regulator of Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

Phagocytosis, macropinocytosis, and G protein coupled receptor-mediated chemotaxis are Ras-regulated and actin-driven processes. The common regulator for Ras activity in these three processes remains unknown. Here, we show that C2GAP2, a Ras GTPase activating protein, highly expressed in the vegetative growth state in model organism Dictyostelium. C2GAP2 localizes at the leading edge of chemotaxing cells, phagosomes during phagocytosis, and macropinosomes during micropinocytosis. c2gapB- cells lacking C2GAP2 displayed increased Ras activation upon folic acid stimulation and subsequent impaired chemotaxis in the folic acid gradient. In addition, c2gaB- cells have elevated phagocytosis and macropinocytosis, which subsequently results in faster cell growth. C2GAP2 binds multiple phospholipids on the plasma membrane and the membrane recruitment of C2GAP2 requires calcium. Taken together, we show a shared negative regulator of Ras signaling that mediates Ras signaling for chemotaxis, phagocytosis, and macropinocytosis.

A CRISPR-based method for testing the essentiality of a gene.

The CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene essentiality (CRISPR-E test) that allows the identification of essential genes. Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading frame of a gene in the genome and analyze the in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations in the targeted region of the gene in surviving cells. If the gene is non-essential, the cells would carry both in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations. In contrast, the cells would carry only in-frame (3n) mutations if the targeted gene is essential, and this selective elimination of frameshift (3n + 1 and 3n + 2) mutations of the gene indicate its essentiality. As a proof of concept, we have used this CRISPR-E test in the model organism Dictyostelium discoideum to demonstrate that Dync1li1 is an essential gene while KIF1A and fAR1 are not. We further propose a simple method for quantifying the essentiality of a gene using the CRISPR-E test.

Xanthan gum production using jackfruit-seed-powder-based medium: optimization and characterization.

Xanthan gum (XG) production by Xanthomonas campestris NCIM 2961 using jackfruit seed powder (JSP) as a novel substrate was reported. Central composite design (CCD) of response surface method (RSM) was used to evaluate the linear and interaction effects of five medium variables (JSP, peptone, citric acid, K2HPO4 and KH2PO4) for XG production. Maximum XG production (51.62 g/L) was observed at the optimum level of JSP (4 g/L), peptone (0.93 g/L), citric acid (0.26 g/L), K2HPO4 (1.29 g/L) and KH2PO4 (0.5 g/L). K2HPO4 and KH2PO4 were found as significant medium components, which served as buffering agents as well as nutrients for X. campestris growth. The obtained biopolymer was characterized as XG by XRD and FTIR analysis. Results of this study revealed that JSP was found to be a suitable low cost substrate for XG production.

Protective effect of bacillopeptidase CFR5 from Bacillus subtilis CFR5 on cerulein-induced pancreatitis.

Bacillopeptidase is a serine peptidase, known for its fibrinolytic activity. However, a very little information is known about its in vivo inflammatory and/or anti-inflammatory properties. Thus, to understand whether bacillopeptidase incorporation can regulate pancreatitis or not, the cerulein-induced pancreatitis model was used, and the role of bacillopeptidase on pancreatitis was studied. In this study, 46 kDa protein was purified from Bacillus subtilis and identified as bacillopeptidase CFR5 (BPC) through MS/MS analysis. The nutritional prophylactic group was orally fed with two doses of BPC (100 µg/Kg/BW of rat) 6 h before cerulein administration and analyzed for its effect on intestine and pancreas inflammation, cytokines, and pancreatitis marker gene expression. BPC administration significantly reduced the severity of pancreatitis by decreasing serum amylase, lipase, pancreatic edema and myeloperoxidase activity. The pretreatment with BPC suppressed the pancreatic pro-inflammatory and inflammatory cytokines production including IL-6, IL-1ß, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-13 in both pancreas and serum samples. Moreover, BPC supplementation restored pancreatitis mediated disruption of intestinal barrier integrity by upregulating tight junction proteins (ZO-1, occludin), antimicrobial peptides (DEFB1, CRAMP), MUC-2, TFF3 expression and by enhancing SCFA's production. Pretreatment with BPC suppressed the intestinal inflammation with reduced cytokines production in the colon and ileal region of cerulein-induced pancreatitis. Thus, BPC based pretreatment protocol is a novel intervention to prevent acute pancreatitis.

Nimbolide inhibits androgen independent prostate cancer cells survival and proliferation by modulating multiple pro-survival signaling pathways.

BACKGROUND: Prostate cancer is the most prominent cancer in men, experiencing a relapse in disease often express high serum TNF-α levels. It has been correlated with increased cell survival and proliferation of prostate cancer cells. Previous studies reported that nimbolide, a terpenoid derived from the leaves and flowers of neem tree inhibits cancer growth through selective modulation of cell signaling pathways linked to inflammation, survival, proliferation, angiogenesis and metastasis. METHODS: The present study aimed to examine the effect of nimbolide at 1 and 2µM concentrations on TNF-α/TNFR1 mediated signaling molecules involved in cell survival and proliferation in PC-3 cell line via NF-κB and MAPK pathways by real time PCR and western blot. Protein and compound interaction were performed by Molecular docking analysis. RESULTS: Our results indicate that nimbolide treatment suppressed expression of TNF-α, SODD, Grb2, SOS mRNA and modulated TNF-α/TNFR1 regulated NF-κB and MAPK signaling molecules in PC-3 cells. Additional molecular dynamics simulation studies confirmed the stability of nimbolide and signaling molecules binding interactions. Binding pose analysis revealed the significance of hydrogen bond interactions for effective stabilization of virtual ligand protein complexes. CONCLUSION: Nimbolide inhibited prostate cancer cell survival and proliferation via NF-κB and MAPK pathways.

Phytochemical composition, GC-MS analysis, in vitro antioxidant and antibacterial potential of clove flower bud (Eugenia caryophyllus) methanolic extract.

Plant derived pharmacologically active compounds have gained importance in food and pharmaceutical industries. The aim of the present study is to identify and study the antioxidant, antimicrobial properties of the phytochemicals present in the crude extract of Eugenia caryophyllus flower buds. The antioxidant activity of the methanol, acetone and chloroform extract was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The methanol extract showed better radical scavenging activity than other selected solvents. Preliminary screening of phytochemicals was carried out in methanol extract and total phenol content was found high. Antibacterial activity was determined by well diffusion assay and methanol extract was found effective against Klebsiella pneumonia. FTIR and GC-MS results indicate the presence of aromatic compounds and major constituents were found to be eugenol and eugenyl acetate. Results of this study implied that Eugenia caryophyllus flower bud extract could be considered as health nutriments in food and pharmaceutical industries.

Association between physiological falls risk and physical performance tests among community-dwelling older adults.

BACKGROUND: Physical performance and balance declines with aging and may lead to increased risk of falls. Physical performance tests may be useful for initial fall-risk screening test among community-dwelling older adults. Physiological profile assessment (PPA), a composite falls risk assessment tool is reported to have 75% accuracy to screen for physiological falls risk. PPA correlates with Timed Up and Go (TUG) test. However, the association between many other commonly used physical performance tests and PPA is not known. The aim of the present study was to examine the association between physiological falls risk measured using PPA and a battery of physical performance tests. METHODS: One hundred and forty older adults from a senior citizens club in Kuala Lumpur, Malaysia (94 females, 46 males), aged 60 years and above (65.77±4.61), participated in this cross-sectional study. Participants were screened for falls risk using PPA. A battery of physical performance tests that include ten-step test (TST), short physical performance battery (SPPB), functional reach test (FRT), static balance test (SBT), TUG, dominant hand-grip strength (DHGS), and gait speed test (GST) were also performed. Spearman's rank correlation and binomial logistic regression were performed to examine the significantly associated independent variables (physical performance tests) with falls risk (dependent variable). RESULTS: Approximately 13% older adults were at high risk of falls categorized using PPA. Significant differences (P<0.05) were demonstrated for age, TST, SPPB, FRT, SBT, TUG between high and low falls risk group. A significant (P<0.01) weak correlation was found between PPA and TST (r=0.25), TUG (r=0.27), SBT (r=0.23), SPPB (r=-0.33), and FRT (r=-0.23). Binary logistic regression results demonstrated that SBT measuring postural sways objectively using a balance board was the only significant predictor of physiological falls risk (P<0.05, odds ratio of 2.12). CONCLUSION: The reference values of physical performance tests in our study may be used as a guide for initial falls screening to categorize high and low physiological falls risk among community-dwelling older adults. A more comprehensive assessment of falls risk can be performed thereafter for more specific intervention of underlying impairments.

Nimbolide inhibits invasion and migration, and down-regulates uPAR chemokine gene expression, in two breast cancer cell lines.

OBJECTIVES: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women, worldwide. Urokinase type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis of breast cancer. Nimbolide is a potent cytotoxic limnoid isolated from Azadirachta indica. Our previous studies have shown that nimbolide elicits pleiotropic effects on breast cancer cells; however, its roles in invasion and migration have not previously been fully elucidated. MATERIALS AND METHODS: Protein expression of pEGFR, VEGFR, NFκB, IKKα, IKKß, MMP-2, MMP-9 and TIMP-2 were analysed by western blotting. We also analysed expressions of uPA, uPAR genes and chemokines by real-time PCR. Breast cancer cell invasion was assessed by transwell invasion assay and cell migration analysed by scratch wound healing assay. RESULTS: Our results showed that reduced protein expression of pEGFR, VEGFR, NFκB, IKKα, ß, MMP-2, MMP-9 and TIMP-2 was higher in nimbolide-treated breast cancer cells. mRNA expression of uPA, uPAR, chemokines and their receptors were also significantly reduced in response to nimbolide treatment. Nimbolide inhibited breast cancer cell migration and invasion as shown in transwell invasion and wound healing assays. CONCLUSION: These results clearly proved inhibitory effects of nimbolide on tumour cell invasion and migration by down-regulating proteins critically involved in regulation of cell invasion and metastasis, suggesting a possible therapeutic role of nimbolide for breast cancer.

Quercetin reverses EGF-induced epithelial to mesenchymal transition and invasiveness in prostate cancer (PC-3) cell line via EGFR/PI3K/Akt pathway.

Epidermal growth factor (EGF) plays an important role in metastasis and tumorigenesis of prostate cancer. Epithelial-mesenchymal transition (EMT) is a process in tumor progression during which cancer cells undergo dramatic changes acquiring highly invasive properties. The purpose of this study was to determine the effect of quercetin on EGF-induced EMT in prostate cancer (PC-3) cell line. Quercetin, a plant flavonoid, prevented EGF-induced invasion and migration of PC-3 cells. The protein and mRNA expressions of E-cadherin and N-cadherin were studied by immunocytochemistry, Western blotting and real-time polymerase chain reaction. Quercetin prevented EGF-induced expression of N-cadherin and vimentin and increased the expression of E-cadherin in PC-3 cells, therefore preventing EGF-induced EMT. EGF-induced cell adhesion proteins, intercellular adhesion molecule and vascular cell adhesion molecule were significantly decreased by quercetin treatment. Furthermore, mRNA and protein expressions of Snail, Slug and Twist showed that quercetin significantly decreased EGF-induced expressions of Snail, Slug and Twist. The protein expressions of epidermal growth factor receptor (EGFR)/phosphatidylinositide 3-kinases (PI3K)/Akt/extracellular signal-regulated kinase (ERK)1/2 pathway showed that quercetin prevents EGF-induced EMT via EGFR/PI3k/Akt/ERK1/2 pathway and by suppressing transcriptional repressors Snail, Slug and Twist in PC-3 cells. Thus, it is concluded from the present study that quercetin may prevent cancer metastasis by targeting EMT.

Quercetin, a natural dietary flavonoid, acts as a chemopreventive agent against prostate cancer in an in vivo model by inhibiting the EGFR signaling pathway.

Prostate cancer incidence and mortality rates have increased over the past years. The purpose of the present study was to examine the molecular mechanism underlying the chemopreventive effects of quercetin on prostate cancer in an in vivo model. Sprague-Dawley male rats were divided into four groups, Group I: vehicle control (propylene glycol), Group II: chemically induced cancer model (MNU + T); Group III: chemically induced cancer model + quercetin (200 mg per kg b.w.); Group IV: quercetin (200 mg per kg b.w.). Serum levels of quercetin were assessed by high performance liquid chromatography (HPLC). EGFR, PI3K/Akt protein levels were significantly increased in chemically induced cancer rats, which were brought back to normalcy in both DLP & VP (dorsolateral prostate & ventral prostate) by quercetin supplementation. Also, the protein expression levels of proliferating cell nuclear antigen (PCNA), N-cadherin, vimentin, and cyclin D1 exhibited a significant increase in both DLP & VP of chemically induced cancer rats. However, simultaneous quercetin supplementation significantly decreased PCNA, N-cadherin, vimentin, and cyclin D1 protein levels compared to chemically induced cancer rats. The E-cadherin expression was decreased in chemically induced cancer animals. Simultaneous quercetin supplementation prevented it. Real time PCR was used to study the mRNA expression of snail, slug and twist. Quercetin significantly decreased snail, slug, and twist mRNA levels in chemically induced cancer rats. To conclude from the present study, quercetin was effective in preventing prostate cancer progression by inhibiting the EGFR signaling pathway and by regulating cell adhesion molecules in Sprague Dawley rats.

Chemopreventive effect of quercetin, a natural dietary flavonoid on prostate cancer in in vivo model.

BACKGROUND & AIM: Prostate cancer is one of the frequently diagnosed cancers in men. Increased Growth factor IGF-1/IGF-1R axis activation mediated by both PI3K/Akt or RAF/MEK/ERK system and AR expression remains important in the development and progression of prostate cancer. Targeting such system by dietary agents quercetin in vivo model could aid its application in both treatment as well as prevention of prostate cancer. METHODS: In our study the rats were divided into four groups; Group I: control (propylene glycol-vehicle), Group II: cancer-induced (MNU and Testosterone treated) rats, Group III: cancer-induced + Quercetin (200 mg/kg body wt/orally) and Group IV: Quercetin (200 mg/kg body wt) thrice a week. After the treatment period rats were sacrificed and the ventral and dorsolateral prostate lobes were dissected. RESULTS: Antioxidant enzymes and apoptotic proteins were significantly decreased in cancer-induced animal and upon quercetin supplement its level was increased. The IGFIR, AKT, AR, cell proliferative and anti-apoptotic proteins were increased in cancer-induced group whereas supplement of quercetin decreased its expression. CONCLUSIONS: Quercetin down regulates the cell survival, proliferative and anti-apoptotic proteins thereby prevents prostate cancer, by acting as a chemopreventive agent in preclinical model.

Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway.

We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy.

Effect of diallyl disulfide on insulin-like growth factor signaling molecules involved in cell survival and proliferation of human prostate cancer cells in vitro and in silico approach through docking analysis.

PURPOSE: Diallyl Disulfide (DADS) is one of the major components of garlic, which inhibits the proliferation of various cancer cells. Our previous studies showed that DADS inhibits cell growth and induces apoptosis on prostate cancer cells. Insulin like growth factor signaling pathway plays a significant role on prostate cancer cell growth and survival and it's over expression also identified in human prostate cancers. The molecular mechanism of IGF mediated PI3K/Akt signaling remains to be elucidated. The present study was designed to evaluate the effects of diallyl disulfide on IGF signaling in androgen independent prostate cancer cells (PC-3). METHODS: DADS (10-50 µM) caused dose-dependent inhibition of PC-3 cells, were analyzed by MTT, IC50 value of PC-3 cells was 40 µM for 24h. Interestingly, DADS also altered the mRNA and protein expressions of IGF signaling and apoptotic molecules which were confirmed by semi quantitative PCR and western blot method. Further the docking study of DADS with IGF receptor was carried out by Ligand Fit of Discovery studio. Accord Excel Package was used for the prediction of ADME properties of the compound. RESULTS: The results suggests that DADS decreases the survival rate of androgen independent prostate cancer cells by modulating the expression of IGF system, which leads to inhibition of phosphorylation of Akt, thereby inhibits cell cycle progression and survival by lowering the expression of cyclin D1, NFkB and anti-apoptotic Bcl-2 molecule and increasing the level of pro-apoptotic (Bad and Bax) signaling molecules which leads to apoptosis. CONCLUSION: The present investigation showed downregulation of Akt and a concomitant increase in apoptosis in DADS treated prostate cancer cells. Since inhibition of this Akt pathway by DADS leads to inhibition in cancer cell progression, it is highly suggested that DADS has the potential use as a therapy for prostate cancer.

Chemopreventive effects of zinc on prostate carcinogenesis induced by N-methyl-N-nitrosourea and testosterone in adult male Sprague-Dawley rats.

PURPOSE: Zinc is an important micronutrient involved in structural and regulatory functions in mammalian cells. It inhibits proliferation of both androgen-dependent and -independent prostate cancer in vitro. However, no report is available on the chemopreventive role of zinc on prostate cancer initiation in in vivo model. The main purpose of this study was to assess the chemopreventive effects of zinc on prostate carcinogenesis induced by a single dose of N-methyl-N-nitrosourea (MNU) and continuous testosterone administration in Sprague-Dawley rats. METHODS: In this study, prostate cancer was induced in Sprague-Dawley rats using MNU+ testosterone (MNU + T). Rats were simultaneously treated with zinc (100 ppm) thrice a week. Serum and tissue activity of prostatic acid phosphatase (PAcP) was measured using biochemical analysis. Serum and tissue zinc levels were assessed by atomic absorption spectrophotometry. The ventral prostatic citrate level, phase I drug-metabolizing enzymes such as cytochrome P450, cytochrome b(5), cytochrome b(5) reductase, cytochrome C reductase, phase II enzyme like glutathione-S-transferase, lipid peroxidation, hydrogen peroxide (H(2)O(2)), and reduced glutathione were also analyzed by biochemical assays. Protein expressions of p53, proliferating cell nuclear antigen (PCNA), caspase-3, and B-cell lymphoma protein-X(L) (Bcl-X(L)) were detected by Western blot analysis. Histopathological evaluation of ventral prostate was studied using hematoxylin and eosin staining method. RESULTS: MNU + T-treated rats showed 60, 50, and 30% of hyperplastic, dysplastic, and prostatic intraepithelial neoplastic changes, respectively, in ventral prostate, whereas MNU + T along with zinc-treated rats showed an incidence of each 10% of hyperplasia, dysplasia, and prostatic intraepithelial neoplasia in the ventral prostate. Serum zinc level and PAcP activity were significantly increased in MNU + T-treated rats, whereas these were decreased in zinc-treated rats. The ventral prostatic PAcP and glutathione-S-transferase activities, zinc, citrate, reduced glutathione levels, and protein levels of p53, caspase-3 were significantly decreased in MNU + T-treated rats, whereas increased in zinc-treated rats. Phase I drug-metabolizing enzyme activities, lipid peroxidation, H(2)O(2) levels, PCNA, and Bcl-X(L) levels were increased in MNU + T-treated rats, whereas these levels were restored to within normal limits in zinc-treated rats. CONCLUSION: This study suggests that zinc may have a beneficial effect against MNU and testosterone-induced prostate carcinogenesis. Thus, it may act as a potential chemopreventive agent in targeting the prostate cancer.

Prevalence of sleep-disordered breathing and sleep apnea in middle-aged urban Indian men.

No data are available on the prevalence of sleep-disordered breathing (SDB) and obstructive sleep apnea-hypopnea syndrome (OSAHS) in Indians. We conducted a two-phase cross-sectional prevalence study for the same in healthy urban Indian males (35-65 years) coming to our hospital in Bombay for a routine health check. We also investigated its risk factors and evaluated the significance of the most commonly asked questions that best correlated with the presence of OSAHS. In the first phase, 658 subjects (94%) returned completed questionnaires regarding their sleep habits and associated medical conditions. In the second phase, 250 of these underwent an overnight home sleep study. The estimated prevalence of SDB (apnea-hypopnea index of 5 or more) was 19.5%, and that of OSAHS (SDB with daytime hypersomnolence) was 7.5%. Multiple stepwise logistic regression determined body mass index, neck girth, and history of diabetes mellitus as the principal covariates of SDB. The presence of snoring, nocturnal choking, unrefreshing sleep, recurrent awakening from sleep, daytime hypersomnolence, and daytime fatigue was each statistically significant for identifying patients with OSAHS. The higher prevalence of OSAHS in urban Indian men is striking and may have major public health implications in a developing country.