(E)-N-Benzyl-2-cyano-3-phenyl­acryl­amide

In the title compound, C17H14N2O, the N-benzyl­formamide and phenyl groups are located on the opposite sides of the C=C bond, showing an E configuration; the terminal phenyl rings are twisted to each other at a dihedral angle of 63.61 (7)°. Inter­molecular classical N—H⋯N and weak C—H⋯O hydrogen bonds occur in the crystal structure

Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates

Transfection agents "from traditional to minaturised screening"

Three libraries of cationic lipids were synthesised and their transfection efficiency in vitro on the HEK293T cell line compared to available commercial compounds.  Two compounds were shown to be more effective and showed minimal cytotoxicity by both MTT and trypan blue assays showing the value of this novel class of compounds for gene delivery in vitro. Single bead screening was evaluated.  A small library of Arginine containing cationic lipids was synthesised on high-loading beads to assess the possibility of screening the material cleaved from single beads.  One compound was more efficient than EffecteneTM and seven was demonstrated transfection up to 80% efficiency.  These studies showed that single  bead screening was viable but further studies with accurate quantitation need to be undertaken to realise maximum activity. Finally, several compounds from the synthesised libraries were tested for their transfection activity using a microarray setup.  Poor transfection was observed, however this technique is very rapid, miniaturised and fully automated, and with further research and optimisation will offer an ideal screening technique.</p

DNA transfection screening from single beads

The solid-phase synthesis of a library of arginine-containing lipid transfection agents on high-loading beads is described. The transfection activity of the cationic lipids was determined using compound cleaved from single beads (single-bead screening) and showed, in some cases, comparable or higher DNA transfection activities as compared to commercially available reagents. Lipids with one arginine headgroup and a cholesterol tail were found to be the most active, even though their DNA binding strength (retardation assays) was relatively weak. Single-bead screening of transfection activity facilitates the rapid analysis of libraries of transfection reagents and will allow the rapid optimization of gene delivery into cells, both in culture and in vivo

Ethanolic extract of Ocimum sanctum leaf modulates oxidative stress, cell cycle and apoptosis in head and neck cancer cell lines

Ocimum sanctum Linn. is a medicinal herb that has cytotoxic effects by inducing oxidative stress in some carcinomas. This study aimed to examine the impact of O. sanctum leaf extract on oxidative stress, cell cycle progression, and apoptosis in cell lines of head and neck squamous cell carcinoma (HNSCC). Isogenic primary (HN18/HN30) and metastatic (HN17/HN31) HNSCC cell lines were used. Preparation of the ethanolic extract of O. sanctum leaf (EEOS) was carried out. HNSCC cell lines were exposed to varying concentrations (0.1–0.8 mg/ml) of EEOS for a duration of 72 h, and the MTT assay was utilized to determine the cytotoxic doses. To assess the impact of EEOS on HNSCC cells, the levels of reactive oxygen species (ROS) and malondialdehyde were measured using a fluorometric method. Flow cytometry was utilized to evaluate effects of EEOS on the cell cycle, DNA damage, and apoptosis in HNSCC cells. Caspase-3 and -9 levels in the EEOS-treated HNSCC cells were measured by ELISA. The chemical components in EEOS were detected using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. EEOS exhibited cytotoxicity against the HN18, HN17, HN30 and HN31 cells at minimum concentrations of 0.1, 0.3, 0.2 and 0.2 mg/ml, respectively. Treatment with EEOS resulted in a significant increase in ROS levels in HN18 and HN17 cells. Additionally, EEOS significantly induced the levels of malondialdehyde in HN18 and HN31 cells. Moreover, EEOS arrested the cell cycle in HN30 and HN31 cells, and significantly induced DNA damage and apoptosis in the HN18, HN30, and HN31 cells. EEOS selectively increased caspase-9 in the HN18 cells. However, caspase-3 was activated without apoptosis in the EEOS-treated HN17 cells. The constituents of EEOS were identified as rosmarinic acid, caffeic acid, and apigenin. In conclusion, EEOS exhibits various prooxidative and apoptotic effects between HNSCC cells

Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells

The ability of non-viral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common non-viral gene delivery system. Solid-phase synthesis was used to produce three libraries of polyamine-based cationic lipids with diverse hydrophobic tails. These were characterised, and structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. Two of the cationic lipids produced high-efficiency transfection of human cells. Surprisingly, these two compounds were from the library with two headgroups and one aliphatic tail, a compound class regarded as detergent-like and little investigated for transfection. These cationic lipids are promising reagents for gene delivery and illustrate the potential of solid-phase synthesis methods for lipoplex discovery

Anti-Acne Vulgaris Potential of the Ethanolic Extract of <i>Mesua ferrea</i> L. Flowers

Acne vulgaris is a common chronic inflammatory skin disease. In the present study, we reported the anti-acne vulgaris effect of the Mesua ferrea (M. ferrea) flower extract. The extract was evaluated for three anti-acne-causing bacteria properties including Cutibacterium acnes (C. acnes), Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus). The results indicated that the M. ferrea flower extract could be considered as the bactericidal agent against S. epidermidis and S. aureus with MIC values of 0.78 and 6.25 mg mL−1 and MBC values of 1.56 and 12.50 mg mL−1 and the bacteriostatic agent against C. acnes with MIC and MBC values of 3.12 and 25.00 mg mL−1, respectively. The extract at a concentration of 25 µg mL−1 also presented potent anti-inflammatory activity with a significant decrease of nitric oxide (NO) and tumor necrosis factor (TNF)-α productions in RAW 264.7 macrophage cells stimulated by LPS. In addition, the extract showed moderate to weak anti-oxidative capacities against DPPH, ABTS, FRAP and NO assays and also showed weak anti-tyrosinase activity. M. ferrea flower extract may serve as the alternative natural anti-acne formulations