Development of user-friendly, high throughput screening for ligands and inhibitors of carbohydrate modifying enzymes

This paper describes the impact of cloud computing and the use of GPUs on the performance of Autodock and Gromacs respectively. Cloud computing was applicable to reducing the ‘‘tail’’ seen in running Autodock on desktop grids and the GPU version of Gromacs showed significant improvement over the CPU version. A large (200,000 compounds) library of small molecules, seven sialic acid analogues of the putative substrate and 8000 sugar molecules were converted into pdbqt format and used to interrogate the Trichomonas vaginalis neuraminidase using Autodock Vina. Good binding energy was noted for some of the small molecules (~-9 kcal/mol), but the sugars bound with affinity of less than -7.6 kcal/mol. The screening of the sugar library resulted in a ‘‘top hit’’ with a-2,3-sialyllacto-N-fucopentaose III, a derivative of the sialyl Lewisx structure and a known substrate of the enzyme. Indeed in the top 100 hits 8 were related to this structure. A comparison of Autodock Vina and Autodock 4.2 was made for the high affinity small molecules and in some cases the results were superimposable whereas in others, the match was less good. The validation of this work will require extensive ‘‘wet lab’’ work to determine the utility of the workflow in the prediction of potential enzyme inhibitors.

Laccase-assisted approach to graft multifunctional materials of interest: keratin-EC based novel composites and their characterisation

This study focuses on the evaluation of raw keratin as a potential material to develop composites with novel characteristics. Herein, we report a mild and eco-friendly fabrication of in-house extracted feather keratin-based novel enzyme assisted composites consisting of ethyl cellulose (EC) as a backbone material. A range of composites between keratin and EC using different keratin: EC ratios were prepared and characterised. Comparing keratin to the composites, the FT-IR peak at 1,630 cm-1 shifted to a lower wavenumber of 1,610 cm-1 in keratin-EC which typically indicates the involvement of β-sheet structures of the keratin during the graft formation process. SEM analysis revealed that the uniform dispersion of the keratin increases the area of keratin-EC contact which further contributes to the efficient functionality of the resulting composites. In comparison to the pristine keratin and EC, a clear shift in the XRD peaks was also observed at the specific region of 2-Theta values of keratin-g-EC. The thermo- mechanical properties of the composites reached their highest levels in comparison to the keratin which was too fragile to be measured for its mechanical properties. Considerable improvement in the water contact angle and surface tension properties was also recorded.

Enzymatic grafting: A novel approach to develop multifunctional materials of interest

Today more than 99% of plastics are petroleum-based because of the availability and cost of the raw material. The durability of disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this issue. Enzymatic grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions, and increasing availability of scientific knowledge. Over the last several years, research covering various applications of robust enzymes like laccases and lipases has been increased rapidly, particularly in the field of polymer science, to graft multi-functional materials of interest. In principle, enzyme-assisted grafting may modify/impart a variety of functionalities to the grafted composites which individual materials fail to demonstrate on their own. The modified polymers through grafting have a bright future and their development is practically boundless. In the present study series of graft composites with poly(3-hydroxybutyrate) (P(3HB) as side chain and cellulose as a backbone polymer were successfully synthesised by introducing enzymatic grafting technique where laccase and lipase were used as model catalysts [1-3]. Subsequently, the resulting composites were removed from the casting surface under ambient environment and characterised by Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and X-ray diffraction (XRD) in detail. Moreover, the thermo-mechanical behaviours of the grafted composites were investigated by differential scanning calorimetry (DSC) and dynamic mechanical analyser (DMA) measurements, respectively. In addition, hydrophobic and hydrophilic characteristics of the grafted polymers were studied through drop contour analysis using water contact angle (WCA). In comparison to the individual counterparts improvement was observed in the thermo- mechanical properties of the composites to varied extent. The tensile strength, elongation at break, and Young’s modulus values of the composites reached their highest levels in comparison to the films prepared with pure P(3HB) only which was too fragile to measure any of the above said characteristics. Interestingly, untreated P(3HB) was hydrophobic in nature and after lipase treatment P(3HB) and P(3HB)-EC-based graft composite attained higher level of hydrophilicity. This is a desired characteristic that enhances the biocompatibility of the materials for proper cell adhesion and proliferation therefore suggesting potential candidates for tissue engineering/bio-medical type applications [3]. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the laccase/lipase assisted grafting of P(3HB) [1-3]. The newly grafted composites exhibit unique functionalities with wider range of potential applications in bio-plastics, pharmaceutical, and cosmetics industries, tissue engineering, and biosensors. [1] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Cellulose 21, 3613-3621 (2014). [2] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Carbohydrate Polymers 113, 131-137 (2014). [3] H.M.N. Iqbal, G. Kyazze, T. Tron and T. Keshavarz, Polymer Chemistry In-Press, DOI: 10.1039/C4PY0 0857J (2014).

A thin-layer chromatography-bioautographic method for detecting dipeptidyl peptidase IV inhibitors in plants

A thin-layer chromatography (TLC)-bioautographic method was developed with the aim to detect dipeptidyl peptidase IV (DPP IV) inhibitors from plant extracts. The basic principle of the method is that the enzyme (DPP IV) hydrolyzes substrate (Gly-Pro-p-nitroaniline) into p-nitroaniline (pNA), which diazotizes with sodium nitrite, and then reacts with N-(1-naphthyl) ethylenediamine dihydrochloride in turn to form a rose-red azo dye which provides a rose-red background on the TLC plates. The DPP IV inhibitors showed white spots on the background as they blocked enzymolysis of the substrate to produce pNA. The method was validated with respect to selectivity, sensitivity, linearity, precision, recovery, and stability after optimizing key parameters including plate type, time and temperature of incubation, concentration of substrate, enzyme and derivatization reagents, and absorption wavelength. The results showed good lineary within amounts over 0.01–0.1 μg range for the positive control, diprotin A, with the coefficient of determination (r2) = 0.9668. The limits of detection (LOD) and quantification (LOQ) were 5 and 10 ng, respectively. The recoveries ranged from 98.9% to 107.5%. The averages of the intra- and inter-plate reproducibility were in the range of 4.1–9.7% and 7.6–14.7%, respectively. Among the nine methanolic extracts of medicinal herbs screened for DPP IV inhibitors by the newly developed method, Peganum nigellastrum Bunge was found to have one white active spot, which was then isolated and identified as harmine. By spectrophotometric method, harmine hydrochloride was found to have DPP-IV inhibitory activity of 32.4% at 10 mM comparing to that of 54.8% at 50 μM for diprotin A.

Laccase from Aspergillus niger: A novel tool to graft multifunctional materials of interests and their characterization

In the present study, we propose a green route to prepare poly(3-hydroxybutyrate) [(P(3HB)] grafted ethyl cellulose (EC) based green composites with novel characteristics through laccase-assisted grafting. P(3HB) was used as a side chain whereas, EC as a backbone material under an ambient processing conditions. A novel laccase obtained from Aspergillus niger through its heterologous expression in Saccharomyces cerevisiae was used as a green catalyst for grafting purposes without the use of additional initiator and/or cross-linking agents. Subsequently, the resulting P(3HB)-g-EC composites were characterized using a range of analytical and imagining techniques. Fourier transform infrared spectroscopy (FT-IR) spectra showed an increase in the hydrogen-bonding type interactions between the side chains of P(3HB) and backbone material of EC. Evidently, X-ray diffraction (XRD) analysis revealed a decrease in the crystallinity of the P(3HB)-g-EC composites as compared to the pristine individual polymers. A homogeneous P(3HB) distribution was also achieved in case of the graft composite prepared in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a mediator along with laccase as compared to the composite prepared using pure laccase alone. A substantial improvement in the thermal and mechanical characteristics was observed for grafted composites up to the different extent as compared to the pristine counterparts. The hydrophobic/hydrophilic properties of the grafted composites were better than those of the pristine counterparts.

Synergy between histone deacetylase inhibitors and DNA- damaging agents is mediated by histone deacetylase 2 in colorectal cancer

Previous studies have associated the overexpression of histone deacetylase 2 (HDAC2) and the presence of TP53 mutations with the progression to advanced stage drug resistant colorectal cancer (CRC). However, the mechanistic link between HDAC2 expression and the TP53 mutational status has remained unexplored. Here, we investigated the function of HDAC2 in drug resistance by assessing the synergistic effects of DNA-targeted chemotherapeutic agents and HDAC inhibitors (HDACis) on two TP53-mutated colorectal adenocarcinoma CRC cell lines (SW480 and HT-29) and on the TP53-wild type carcinoma cell line (HCT116 p53+/+) and its TP53 deficient sub-line (HCT116 p53-/-). We showed that in the untreated SW480 and HT-29 cells the steady-state level of HDAC2 was low compared to a TP53-wild type carcinoma cell line (HCT116 p53+/+). Increased expression of HDAC2 correlated with drug resistance, and depletion by shRNA sensitised the multi-drug resistance cell line HT-29 to CRC chemotherapeutic drugs such as 5-fluorouracil (5-FU) and oxaliplatin (Oxa). Combined treatment with the HDACi suberoylanilide hydroxamic acid plus 5-FU or Oxa reduced the level of HDAC2 expression, modified chromatin structure and induced mitotic cell death in HT-29 cells. Non-invasive bioluminescence imaging revealed significant reductions in xenograft tumour growth with HDAC2 expression level reduced to <50% in treated animals. Elevated levels of histone acetylation on residues H3K9, H4K12 and H4K16 were also found to be associated with resistance to VPA/Dox or SAHA/Dox treatment. Our results suggest that HDAC2 expression rather than the p53 mutation status influences the outcome of combined treatment with a HDACi and DNA-damaging agents in CRC.

Modulation of CYP1A1 by PKC Inhibitors and TPA Pre-Treatments in MH1C1 Rat Hepatoma Cells Exposed to 3 -Methylcholanthrene

Cytochrome P4501A1 (CYP1A1), an enzyme known to metabolize polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR). The involvement of protein kinase C (PKC) in the regulation of AhR signal transduction pathway, has been widely studied but the role of specific PKC isoform(s) involved in this process it is not well clarified. To study which PKC isoform(s) is implicated in the regulation of CYP1A1, in the poorly tumorigenic MH1C1 rat hepatoma cells, we examined the effects of some PKC pharmacological inhibitors, Calphostin C (CAL), Staurosporine (STA) and H7, and of 12-0-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, on basal and 3- methylcholanthrene (MC)-induced CYP1A1 protein expression and mediated ethoxyresorufin O-deethylation (EROD) activity. In parallel, the activities of PKC-α, -βI, -δ and -ε isoforms, the most expressed in MH1C1 cells, were monitored. After pre-treatment with CAL, STA and H7, the MC-induced CYP1A1 protein and EROD activity were rapidly reduced with temporal profile similar to the profile of the activity of α and β1 PKC isoforms. Moreover, TPA pre-treatment induced a biphasic effect on EROD activity, and a decline of PKC -βI and -α, in first instance, and -δ and -ε activities later on. These findings clearly show that, in MH1C1 cells, PKC is involved in CYP1A1 regulation and that α and βI classic PKC isoforms play an active role in modulating this process.