Neoxanthin prevents H2O2-induced cytotoxicity in HepG2 cells by activating endogenous antioxidant signals and suppressing apoptosis signals.

BACKGROUND: The liver has a solid inbuilt antioxidant defense system to regulate oxidative stress. However, exposure to an excessive level of ROS causes liver injury. This study examined the cytoprotective effect of neoxanthin, a xanthophyll antioxidant molecule isolated from Solanum trilobatum in stress-induced HepG2 cells. METHODS AND RESULTS: The cytotoxic effect of H2O2 and cytoprotective potential of ß-carotene, lutein, and neoxanthin was analyzed by WST-1 assay. The intracellular ROS level and mitochondrial membrane potential (MMP) were measured using DCFH-DA (2', 7'-dichlorofluorescin diacetate) and JC-10 MMP assay. The expression of anti-oxidant and apoptotic markers was measured by western blot analysis. Neoxanthin pretreatment exhibited better protection than ß-carotene and lutein against cell death caused by H2O2. It significantly arrested H2O2-mediated elevation of intracellular ROS levels and protected MMP. The intracellular antioxidant enzymes HO-1 and SOD-2 were upregulated by neoxanthin pretreatment. Neoxanthin also activated the protein expression of redox-sensitive transactivation factors, Nrf2 and NF-kB. The cytoprotective effect of neoxanthin was associated with increased expression of the anti-apoptotic protein, Bcl-2 and decreased pro-apoptotic protein Bax. CONCLUSIONS: For the first time, our results demonstrate that neoxanthin offers adequate protection against stress-mediated cytotoxicity in hepatocytes by activating the intracellular antioxidant defense system and blocking apoptosis.

Extracellular urease from Arthrobacter creatinolyticus MTCC 5604: scale up, purification and its cytotoxic effect thereof.

Urease is a potent metalloenzyme with diverse applications. This paper describes the scale up and purification of an extracellular urease from Arthrobacter creatinolyticus MTCC 5604. The urease production was scaled-up in 3.7 L and 20 L fermentor. A maximum activity of 27 and 27.8 U/mL and a productivity of 0.90 and 0.99 U/mL/h were obtained at 30 h and 28 h in 3.7 and 20 L fermentor, respectively. Urease was purified to homogeneity with 49.85-fold purification by gel filtration and anion exchange chromatography with a yield of 36% and a specific activity of 1044.37 U/mg protein. The enzyme showed three protein bands with molecular mass of 72.6, 11.2 and 6.1 kDa on SDS-PAGE and ~ 270 kDa on native PAGE. The cytotoxic effect of urease was assessed in vitro using cancer cell lines (A549 and MG-63) and normal cell line (HEK 293). Urease showed its inhibitory effects on cancer cell lines through the generation of toxic ammonia, which in turn increased the pH of the surrounding medium. This increase in extracellular pH, enhanced the cytotoxic effect of weak base chemotherapeutic drugs, doxorubicin (50 µM) and vinblastine (100 µM) in the presence of urease (5 U/mL) and urea (0-4 mM) significantly.

Hierarchical Self-Assembly and Aggregation-Induced EmissionEnhancement in Tetrabenzofluorene-based Red Emitting Molecules.

The newly synthesized chiral active [5]helicene-like tetrabenzofluorene (TBF) based highly red-emitting molecules exhibit flower-like self-assembly. These molecules display photophysical and structural properties such as intramolecular charge transfer, dual state emission, large fluorescence  quantum yield, and solvatochromism. In TBFID, the indandione functional group attached on both sides as the terminal group offers an A-D-A push-pull effect and acts as a strong acceptor to cause more redshift in solution as well as in solid state as compared to TBFPA (TBF with benzaldehyde functional group in terminal position). The self-assembly studies of TBFID demonstrate the aggregation-induced emission enhancement (AIEE) attributed to the restriction of intramolecular rotation at the aggregated state. Furthermore, TBFID shows high quantum yield and intense red emission, making the molecule fit for organic light-emitting diodes (OLED) and bioimaging applications.

Sanggenol B, a plant bioactive, as a safer alternative to tackle cancer by antagonising human FGFR.

Fibroblast Growth Receptor Factor (FGFR) are a family of proteins which are, in addition to their biological role, are involved in various pathological functions, such as cancer cellular proliferation, and metastasis. Deregulation of FGFRs at various points could result in malignancy. A conformational transition of the DFG (Asp-Phe-Gly) motif can switch the enzyme from a catalytically active (DFG-in) to an inactive (DFG-out) state. There are a few FDFR inhibitors which have received approval from the FDA, but these have adverse side effects. Hence, there is a demand for safer alternatives. With this aim, Ligand and Structure based virtual screening was carried to identify suitable lead molecule. In this process, Four Featured atom-based 3D Pharmacophore with quantitative structure-activity relationship analysis (3D-QSAR) was developed. The External validation of the hypothesis was carried invoking criteria such as Area under the ROC curve. Natural plant compound databases such as the Traditional Chinese medicine, NPACT and the ZINC Natural databases were chosen for pharmacophore filtering, which was followed by virtual screening against FGFR isoforms. The compound Sanggenol B was identified as the most suitable lead molecule. Structural stability of the protein-ligand complex and interactions of the ligand (Sanggenol B & the reference compound Ponatinib) with FGFR were analysed for 1000 ns (triplicate) by means of molecular simulation and the binding free energy was calculated using MMGBSA. Sanggenol B (PubChem CID: 15233694) binds effectively at the active site with favourable energies and is proposed as a safe alternative from a natural source.Communicated by Ramaswamy H. Sarma.

Growth factor-mimicking 3,4-dihydroxyphenylalanine-encoded bioartificial extracellular matrix like protein promotes wound closure and angiogenesis.

The present work reports a new route to prepare a "smart biomaterial" by mimicking long-acting cellular growth factor showing enhanced cell-material interactions by promoting cell proliferation and angiogenesis. For that, reactive non-proteogenic amino acid 3,4-dihydroxyphenylalanine (DOPA) was genetically introduced into an intrinsic triple-helical hierarchical structure forming protein to initiate hierarchical self-assembly to form a macromolecular structure. The self-assembled scaffold displayed vascular endothelial growth factor mimicking the pro-angiogenic reactive group for repairing and remodeling of damaged tissue cells. We customized the recombinant collagen-like protein (CLP) with DOPA to promote rapid wound healing and cell migrations. Selective incorporation of catechol in variable and C-terminal region of CLP enhanced interaction between inter- and intra-triple-helical collagen molecules that resulted in a structure resembling higher-order native collagen fibril. Turbidity analysis indicated that the triple-helical CLP self-assembled at neutral pH via a catechol intra-crosslinking mechanism. After self-assembly, only DOPA-encoded CLP formed branched filamentous structures suggesting that catechol mediated network coordination. The catechol-encoded CLP also acted as a "smart material" by mimicking long-acting cellular growth factor showing enhanced cell-material interactions by promoting cell proliferation and angiogenesis. It eliminates release rate, stability, and shelf-life of hybrid growth factor conjugated biomaterials. The newly synthesized CLP has the potential to promote accelerated cell migration, pro-angiogenesis, and biocompatibility and could be used in the field of implantable medical devices and tissue engineering.

Non-enzymatic reduction of Cr (VI) and it's effective biosorption using heat-inactivated biomass: A fermentation waste material.

The effectiveness of heat-inactivated fungal biomass a fermentation waste of newly isolated laccase enzyme producer Leiotrametes flavida was studied for Cr (VI) removal in water and applied for Cr (VI) removal from tannery effluent. Adsorption parameters pH, biomass concentration and contact time were optimized using Box-Behnken design of response surface methodology. The adsorption process fits the Langmuir isotherm. Thermodynamic and kinetic studies showed that the process is spontaneous at ambient temperature and followed the second-order kinetics model, respectively. The values of the kinetic model indicated that the adsorption process is a combination of physisorption and chemisorption. Chromium adsorption onto the biomass was confirmed by SEM-EDAX, FTIR, XPS and XRD analysis. XPS analysis confirmed the reduction of Cr (VI) to Cr (III). The amount of chromium adsorbed was 72.38 % and 68.33 % for water and effluent, respectively. Chromium adsorbed onto biomass was desorbed at pH 9 with 1 M NaOH. Total chromium desorbed was 61.40 and 59.38 percent from water and effluent, respectively. The amount of Cr (III) in the desorbed sample was 71 and 68 percent, respectively. The heat-inactivated biomass of Leiotrametes flavida is a suitable material for efficient Cr (VI) removal and detoxification.

Side chain torsion dictates planarity and ionizability of green fluorescent protein's chromophore leading to spectral perturbations.

Spectral characteristics of fluorescent proteins (FPs) are well studied, and through protein engineering, several FP variants constituting entire visible spectrum have been created. One of the most common mechanisms attributed to spectral shifts in FP is excited state proton transfer (ESPT), hydroxyl moiety protonation and deprotonation, along with chromophore cis-trans isomerism. The most widely studied FPs are those derived from avGFP (Aequorea victoria GFP) and Dsred (Discosoma coral). Apart from the above mechanism, certain interacting residues are said to play a vital role in altering the proton transfer pathway leading to numerous spectral variants. Similarly, the hydrogen-bonded networks solely cannot dictate the energy landscape of FPs. Non-bonded interactions also can create secondary harmonic shifts by dipole-dipole inductions. Side chain contacts tend to alter the topological and torsional geometry, thereby disturbing the chromophore's planarity. Side chain torsional variations have almost been unaccounted for their distortions in FPs. We hypothesize the torsional landscape and altered residual interactions as prominent factors for the spectral shifts. Through our 200 ns molecular dynamics investigation, we prospect that van der Waals packing in Dsred is more compact than that of avGFP, thus creating a low solvent occupiable environment and reduced solvent interactions having higher red spectral shift. The torsional changes of wild avGFP, S65T avGFP and Dsred have been studied to comprehend the inter-residual contact distance and the geometrical descriptors. Communicated by Ramaswamy H. Sarma.

A self-assembly and higher order structure forming triple helical protein as a novel biomaterial for cell proliferation.

Collagen plays a critical role in the structural design of the extracellular matrix (ECM) and cell signaling in mammals, which makes it one of the most promising biomaterials with versatile applications. However, there is considerable concern regarding the purity and predictability of the product performance. At present, it is mainly derived as a mixture of collagen (different types) from animal tissues, where the selective enrichment of a particular type of collagen is generally difficult and expensive. Collagen derived from bovine sources poses the risk of transmitting diseases and can cause adverse immunologic and inflammatory responses. Hence, recombinant collagen can be a good alternative. Nevertheless, the necessity of post-translational hydroxyproline (Hyp) modification limits large-scale recombinant collagen production. Here, we recombinantly expressed the collagen-like protein (CLTP) and genetically introduced the Hyp in the CLTP to form a higher order self-assembled fibril structure, similar to human collagen. During the current study, it was observed that the Hyp incorporated CLTP protein (CLTHP) formed a stable triple helical polyproline-II like structure and self-assembled to form fibrils at neutral pH, which had an initial lag phase followed by a growth phase similar to animal collagen. In contrast, the higher order fibrillar assembly was missing in the nonhydroxylated CLTP. This study demonstrated that CLTHP self-association is based on the common underlying lateral interactions between triple helical structured proteins, where the hydroxyproline forms the significantly stable hydration network. Hence, this work will be the first fundamental empirical research for flexible modifications of recombinant collagen for structural analysis and biomedical applications.