Combined targeting of SET and tyrosine kinases provides an effective therapeutic approach in human T-cell acute lymphoblastic leukemia.

Recent evidence suggests that inhibition of protein phosphatase 2A (PP2A) tumor suppressor activity via the SET oncoprotein contributes to the pathogenesis of various cancers. Here we demonstrate that both SET and c-MYC expression are frequently elevated in T-ALL cell lines and primary samples compared to healthy T cells. Treatment of T-ALL cells with the SET antagonist OP449 restored the activity of PP2A and reduced SET interaction with the PP2A catalytic subunit, resulting in a decrease in cell viability and c-MYC expression in a dose-dependent manner. Since a tight balance between phosphatases and kinases is required for the growth of both normal and malignant cells, we sought to identify a kinase inhibitor that would synergize with SET antagonism. We tested various T-ALL cell lines against a small-molecule inhibitor screen of 66 compounds targeting two-thirds of the tyrosine kinome and found that combined treatment of T-ALL cells with dovitinib, an orally active multi-targeted small-molecule receptor tyrosine kinase inhibitor, and OP449 synergistically reduced the viability of all tested T-ALL cell lines. Mechanistically, combined treatment with OP449 and dovitinib decreased total and phospho c-MYC levels and reduced ERK1/2, AKT, and p70S6 kinase activity in both NOTCH-dependent and independent T-ALL cell lines. Overall, these results suggest that combined targeting of tyrosine kinases and activation of serine/threonine phosphatases may offer novel therapeutic strategies for the treatment of T-ALL.

Effect of sequential bio-processing conditions on the content and composition of vitamin K2 and isoflavones in fermented soy food.

In the present research, effect of sequential addition of Bifidobacterium bifidum, Bacillus subtilis and Rhizopus oligosporus on content and composition of vitamin K2 and isoflavones in fermented soy foods have been investigated. Initially, soybeans were fermented with B. bifidum; then this fermented mass was re-fermented with co-culture of B. subtilis and R. oligosporus. The evolved sequence of microbes inoculation tended towards significantly (p < 0.5) higher enzymes levels (126.16 ± 2.23 IU/mg lipase, 36.52 ± 1.25 IU/mg phytase and 8.52 ± 1.12 IU/mg ß-glucosidase); maximum menaquinone-7 production (9.3 ± 1.27 µg/g); and isoflavone content (84.64 ± 1.97 % daidzein, 99.29 ± 0.86 % genistein, 96.42 ± 1.32 % glycitein) after 72 h of solid-state fermentation. The study showed that co-fermentation of soybean with different microbes in a particular sequence can enhance nutritional value batter than the mono-culture fermentation due to the positive correlation between enzymes (lipase, phytase, ß-glucosidase) levels, menaquinone-7 and soy isoflavones content.

Development of menaquinone-7 enriched nutraceutical: inside into medium engineering and process modeling.

Menaquinone 7 (MK-7) is nutritionally important metabolite found by fermentation mainly using B. subtilis species. In this study, soybean medium was modified to improve the MK-7 production using Bacillus subtilis NCIM 2708 under solid state fermentation. The objective of this study was to produce large amount of MK-7 within a short period of time. Nine nutritional components viz. glycerol, mannitol, dextrose, sucrose, yeast extract, malt extract, K2HPO4, MgSO4.7H2O and CaCl2 were investigated to obtain the maximum MK-7 concentration. The highest MK-7 concentration 39.039 µg/g was obtained after 24 h of fermentation in the following optimised medium components: soybean 20 g, glycerol 40 ml/kg, mannitol 60 g/kg, yeast extract 4 g/kg, malt extract 8 g/kg and calcium chloride 4 g/kg. The maximum production of MK-7 56.757 µg/g was predicted by point prediction tool of Design Expert 7.1 software (Statease Inc. USA). This data shows 68.78 % validity of the predicted model.

Simultaneous estimation of glycosidic isoflavones in fermented and unfermented soybeans by TLC-densitometric method.

A simple, accurate and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of three glycosidic isoflavones (daidzin, genistin and glycitin) in soybean (Glycine max L.) has been established and validated. Chromatography was performed on aluminum foil-backed silica gel 60 F254 HPTLC plates and found compact spots for daidzin, genistin and glycitin (Rf value of 0.39, 0.51 and 0.32, respectively) with mobile phase toluene : ethyl acetate : formic acid : acetic acid in the ratio of 1 : 8 : 1 : 0.5, v/v/v/v. Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 260 nm. The method was validated for precision, recovery, robustness, specificity, limit of detection (LOD) and limit of quantification (LOQ), in accordance with the ICH guidelines. The LOD (2.9, 19.3 and 3.5 µg mL(-1)), LOQ (9.03, 58.6 and 10.7 µg mL(-1)), recovery (95.9-106.66, 86.97-106.56 and 98.54-105.65%) and precision (≤2.12, ≤0.722 and ≤0.066) were satisfactory for glycosidic form of isoflavones daidzin, genistin and glycitin, respectively. Soybean variety Kh-09 bragg was found to have relatively higher amount of glycosidic isoflavones, namely daidzin, genistin and glycitin 278, 597.5 and 109.4 µg g(-1), respectively, and after fermentation the glycosidic isoflavones concentration in soybean fermented with Bacillus subtilis strain were decreased significantly after 24 h of incubation; conversely, aglycone isoflavones were increased significantly. The method for quantification of isoflavones in unfermented and fermented soybeans, with good resolution has been developed.

Five new secondary metabolites from Monascus purpureus-fermented Hordeum vulgare and Sorghum bicolor.

Long grains of Hordeum vulgare and Sorghum bicolor were individually fermented with Monascus purpureus MTCC 369 under solid state fermentation. The aqueous extract of Monascus which fermented H. vulgare and S. bicolor was found to contain five different new metabolites. Silica gel column chromatography of the aqueous extract with a linear gradient of ethyl acetate, acetonitrile and carbon tetrachloride (v/v) yielded five new metabolites named benzopranyl capriate (9H-1-isoprenyl-benzopyran-5-isopropanoic acid-6-ol-6-n-decanoate), shorghumoic acid (n-octadec-8,11-dien-7α-ol-1-oic acid) and sorghumflavin A (2-n-butyloxo-6-ß-hydroxy-7-ß-isoprenyl ankaflavin) from Monascus-fermented S. bicolor, while hordeumflavin B (2-n-undecanyloxo-7-ß-isoprenyl ankaflavin) and vulgaredilone (2-dodecanyl-7-ß isopranyl monoscodilone) from Monascus-fermented H. vulgare.

Development of an HPTLC-based diagnostic method for invasive aspergillosis.

A rapid, sensitive and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for determination of gliotoxin in Aspergillus infected immunocompromised patients with invasive aspergillosis (IA). Densitometric analysis of gliotoxin was carried out in the absorbance mode at 254 nm after single-step extraction with chloroform. The method uses TLC aluminum plates pre-coated with silica gel 60F-254 as a stationary phase and toluene-isoamyl alcohol-methanol (10:0.5:0.5, v/v/v) as mobile phase, which gives compact spot of gliotoxin (R(f) = 0.51). The calibration curve was linear (r(2) > or = 0.994) between peak area and concentration in the tested range of 100-1000 ng spot(-1) with minimum detectable range 0.025 ng mu(-1) of serum sample. The mean +/- SD value of slope and intercept of the standard chromatogram of gliotoxin were found to be 523.2 +/- 1.555635 and 915.8 +/- 30.68843, respectively. The developed method is simple, rapid, precise and less costly than earlier diagnostic methods, and different serum samples can be run on a single TLC plate for comparative analysis. The proposed method can be used to analyze gliotoxin in patient serum for easy, rapid and cost-effective diagnosis of IA.