datPAV--an online processing, analysis and visualization tool for exploratory investigation of experimental data

SUMMARY: Data processing, analysis and visualization (datPAV) is an exploratory tool that allows experimentalist to quickly assess the general characteristics of the data. This platform-independent software is designed as a generic tool to process and visualize data matrices. This tool explores organization of the data, detect errors and support basic statistical analyses. Processed data can be reused whereby different step-by-step data processing/analysis workflows can be created to carry out detailed investigation. The visualization option provides publication-ready graphics. Applications of this tool are demonstrated at the web site for three cases of metabolomics, environmental and hydrodynamic data analysis. AVAILABILITY: datPAV is available free for academic use at http://www.sdwa.nus.edu.sg/datPAV/.     

Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.     

Antitumor efficacy of a thrombospondin 1 mimetic CovX-body

CVX-045 is produced by covalently attaching a thrombospondin 1 (TSP-1) mimetic comprising a peptidic sequence and a linker to the Fab binding site of a proprietary scaffold antibody. CVX-045 possesses the potency of the TSP-1-derived peptide, along with the advantageous pharmacokinetics of an antibody. Antitumor activity of CVX-045 was evaluated in human xenograft models alone and in combination with standard chemotherapies and targeted molecules. In A549 and A431 xenograft models, CVX-045 demonstrated significant (P < .05) antiangiogenic activity, reducing tumor microvessel density and increasing the levels of necrosis within treated tumors. In an HT-29 xenograft model, CVX-045 in combination with 5-fluorouracil significantly (P < .01) decreased tumor growth rate compared with vehicle, CVX-045, or 5-fluorouracil alone. Cotreatment of CVX-045 plus CPT-11 delayed progression of tumor growth from day 28 to 60. In contrast CVX-045 alone treatment did not delay the progression of tumor growth, and CPT-11 alone delayed progression of tumor growth to day 39. Cotreatment of CVX-045 with sunitinib extended the time to reach tumor load from day 26 to 40. In summary, CVX-045 exhibits significant antiangiogenic activity in several tumor models and enhances antitumor activity in combination with chemotherapy or targeted therapies. These data suggest future avenues for effective combination therapy in treating solid tumors. CVX-045 has recently completed a phase 1 trial in solid tumors where it has been well tolerated.     

Rescue of photoreceptor degeneration by curcumin in transgenic rats with P23H rhodopsin mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects.     

Funding translational work in cell-based therapy

The cell therapy branch of the regenerative medicine field has been innovative in developing new models of delivery and development and identifying alternative sources of funding. We discuss the implications of these changes for pharmaceutical companies and the opportunities they offer to a new entrepreneur.     

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in?vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs?form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.     

Estimation of nuclear DNA content of various bamboo and rattan species

We determined the nuclear DNA content (genome size) of over 35 accessions each of bamboo and rattan species from Southeast Asia. The 2C DNA per nucleus was quantified by flow cytometry. The fluorescence of nuclei isolated from the leaves and stained with propidium iodide was measured. The genome size of the bamboo species examined was between 2.5 and 5.9 pg DNA per 2C nucleus. The genome size of the rattan species examined ranged from 1.8 to 10.5 pg DNA per 2C nucleus. This information will be useful for scientists working in diverse areas of plant biology such as biotechnology, biodiversity, genome analysis, plant breeding, physiology and molecular biology. Such data may be utilized to attempt to correlate the genome size with the ploidy status of bamboo species in cases where ploidy status has been reported.     

Recombinant dengue virus type 3 envelope domain III protein from Escherichia coli

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed-batch cultivation process were also developed in optimized medium. After fed-batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced ～649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS-PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.     

Minor diterpenoid glycosides from the leaves of Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, three new diterpenoid glycosides were isolated besides eight known steviol glycosides including stevioside, rebaudiosides A–F and dulcoside A. The structures of the three compounds were identified as 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-β-d-xylopyranosyl-β-d-glucopyranosyl) ester (1), 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (2), and 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-oxo-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (3) on the basis of extensive NMR and MS spectral studies. Another known diterpenoid glycoside, 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (4) was also isolated and its complete NMR spectral assignments were made on the basis of COSY, HSQC and HMBC spectral data.