Wei Ning Chen

High Potency and Broad-Spectrum Antimicrobial Peptides Synthesized via Ring-Opening Polymerization of α-Aminoacid-N-carboxyanhydrides

Antimicrobial peptides (AMPs), particularly those effective against methicillin-resistant Staphylococcus aureus ( S. aureus ) and antibiotic-resistant Pseudomonas aeruginosa ( P. aeruginosa ), are important alternatives to antibiotics. Typical peptide synthesis methods involving solid-phase sequential synthesis are slow and costly, which are obstacles to their more widespread application. In this paper, we synthesize peptides via ring-opening polymerization of alpha-amino acid N-carboxyanhydrides (NCA) using a transition metal initiator. This method offers high potential for inexpensive synthesis of substantial quantities of AMPs. Lysine (K) was chosen as the hydrophilic amino acid and alanine (A), phenylalanine (F), and leucine (L) as the hydrophobic amino acids. We synthesized five series of AMPs (i.e., P(KA), P(KL), P(KF), P(KAL), and P(KFL)), varied the hydrophobic amino acid content from 0 to 100%, and determined minimal inhibitory concentrations (MICs) against clinically important Gram-negative and Gram-positive bacteria and fungi (i.e., Escherichia coli ( E. coli ), P. aeruginosa , Serratia marcescens ( S. marcescens ), and Candida albicans ( C. albicans ). We found that P(K(10)F(7.5)L(7.5)) and P(K(10)F(15)) show the broadest activity against all five pathogens and have the lowest MICs against these pathogens. For P(K(10)F(7.5)L(7.5)), the MICs against E. coli , P. aeruginosa , S. marcescens , S. aureus , and C. albicans are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 62.5 microg/mL, while for P(K(10)F(15)) the respective MICs are 31 microg/mL, 31 microg/mL, 250 microg/mL, 31 microg/mL, and 125 microg/mL. These are lower than the MICs of many naturally occurring AMPs. The membrane depolarization and SEM assays confirm that the mechanism of microbe killing by P(K(10)F(7.5)L(7.5)) copeptide includes membrane disruption, which is likely to inhibit rapid induction of AMP-resistance in pathogens.

Pharmacogenetics of Target Genes Across Doxorubicin Disposition Pathway: A Review

Increased understanding of the molecular mechanisms of tumor heterogeneity combined with rapid advances in the field of pharmacogenetics and pharmacogenomics have fuelled studies on individualizing anticancer therapy. Doxorubicin (Adriamycin), is an anthracycline glycoside antibiotic originally produced by Streptomyces peucetius var. caesius, and is widely used either as a single agent or in combination with other chemotherapeutic regimens for curative, adjuvant, and palliative treatment in cancer patients. The pharmacogenetics of doxorubicin has not been well characterized. The polygenic influence of functional candidate gene variants across doxorubicin biochemical pathway is hypothesized to contribute to its heterogeneity in disposition, influencing the efficacy of treatment and occurrence of adverse effects like cardiomyopathy in patients undergoing doxorubicin based adjuvant and neo-adjuvant chemotherapy. The pharmacogenetics of Asian population differs from that of other ethnic groups, particularly from Caucasian and African populations, and indicates an important role of ethnicity in determining predictive end points during chemotherapy and in individualizing treatment. This review comprehensively examines the pharmacogenetics of the regulatory nuclear receptor Pregnane-X Receptor (PXR), influx (SLC22A16) and efflux drug transporters (ABCB1, ABCG2, ABCC5, ABCB5 and RLIP76) and drug metabolizing enzymes (CBR1, CBR3) across the biochemical pathway of doxorubicin in Asian breast cancer patients receiving doxorubicin based adjuvant chemotherapy. The influence of functional genetic variants on the inter-individual variability in pharmacokinetics of doxorubicin and its major metabolite are also discussed. The incorporation of non-genetic factors and subsequent validation of these findings in different patient and population groups will be valuable in tailoring doxorubicin dosage regimens to an individual to maximize therapeutic efficacy and minimize adverse reactions, leading to improved clinical outcomes.

Metabolic engineering for enhanced fatty acids synthesis in Saccharomyces cerevisiae

Microbial production of biofuel has attracted significant attention in recent years. The fatty acids are important precursors for the production of fuels and chemicals, and its biosynthesis is initiated by the conversion of acetyl-CoA to malonyl-CoA which requires acetyl-CoA as key substrate. Herein, the yeast Saccharomyces cerevisiae was proposed to be metabolically engineered for cytosol acetyl-CoA enhancement for fatty acid synthesis. By gene disruption strategy, idh1 and idh2 genes involved in citrate turnover in tricarboxylic acid cycle (TCA cycle) were disrupted and the citrate production level was increased to 4- and 5-times in mutant yeast strains. In order to convert accumulated citrate to cytosol acetyl-CoA, a heterologous ATP-citrate lyase (ACL) was overexpressed in yeast wild type and idh1,2 disrupted strains. The wild type strain expressing acl mainly accumulated saturated fatty acids: C14:0, C16:0 and C18:0 at levels about 20%, 14% and 27%, respectively. Additionally, the idh1,2 disrupted strains expressing acl mainly accumulated unsaturated fatty acids. Specifically in Δidh1 strain expressing acl, 80% increase in C16:1 and 60% increase in C18:1 was detected. In Δidh2 strain expressing acl, 60% increase in C16:1 and 45% increase in C18:1 was detected. In Δidh1/2 strain expressing acl, there was 92% increase in C16:1 and 77% increase in C18:1, respectively. The increased fatty acids from our study may well be potential substrates for the production of hydrocarbon molecules as potential biofuels.

Human hepatitis B virus mutants: significance of molecular changes

Human hepatitis B virus, the leading pathogen for hepatitis B, is a compact DNA virus with viral genes that largely overlap. An increasing number of mutations have emerged following human interventions such as vaccination and anti‐viral therapy. While vaccine escape mutants are characterized by mutations on the antigenic hepatitis B surface antigen, those carrying mutations in other viral proteins are either resistant to anti‐viral therapy or implicated in acute liver diseases. Molecular identification of these various mutants should shed new lights on the underlying mechanism of hepatitis B virus viral escape and resistance and provide helpful information on their effective eradication.

Comparative Proteomics Analysis of Vascular Smooth Muscle Cells Incubated with S- and R-Enantiomers of Atenolol Using iTRAQ-coupled Two-dimensional LC-MS/MS

Atenolol is a β1-selective drug, which exerts greater blocking activity on β1-adrenoreceptors than on β2-adrenoreceptors, with the S-enantiomer being more active than R-enantiomer. The aim of this study was to investigate the proteins with differential protein expression levels in the proteome of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of atenolol using an iTRAQ-coupled two-dimensional LC-MS/MS approach. Our results indicated that some calcium-binding proteins such as calmodulin, protein S100-A11, protein S100-A4, and annexin A6 were down-regulated and showed relatively lower protein levels in cells incubated with the S-enantiomer of atenolol than those incubated with the R-enantiomer, whereas metabolic enzymes such as aspartate aminotransferase, glutathione S-transferase P, NADH-cytochrome b5 reductase, and α-N-acetylgalactosaminidase precursor were up-regulated and displayed higher protein levels in cells incubated with the S-enantiomer relative to those incubated with the R-enantiomer. The involvement of NADH-cytochrome b5 reductase in the intracellular anabolic activity was validated by NAD+/NADH assay with a higher ratio of NAD+/NADH correlating with a higher proportion of NAD+. The down-regulation of the calcium-binding proteins was possibly involved in the lower intracellular Ca2+ concentration in A7r5 cells incubated with the S-enantiomer of atenolol. Ca2+ signals transduced by calcium-binding proteins acted on cytoskeletal proteins such as nestin and β-tropomyosin, which can play a complex role in phenotypic modulation and regulation of the cytoskeletal modeling. Our preliminary results thus provide molecular evidence on the metabolic effect and possible link of calcium-binding proteins with treatment of hypertension associated with atenolol.

HBx genotype D represses GSTP1 expression and increases the oxidative level and apoptosis in HepG2 cells

Epigenetics has been implicated in human cancer development. Epigenetic factors include HBx protein, which is able to induce hypermethylation and suppresses tumor suppressor genes. One of such tumor suppressor genes, GSTP1, shows reduced expression in many human cancers. Hypermethylation of GSTP1 is the most studied mechanism of its silence. In the present study, we reported that GSTP1 expression was completely depleted in HBV integrated HepG2.2.15 cells due to the hypermethylation in its promoter region. And it was HBx, especially HBx genotype D, that played the key role in repressing GSTP1 expression. Further functional studies like ROS assay and apoptosis detection were also used to confirm this repression. Our findings should facilitate the understanding of HBV and their influences on the epigenetic modulations for epigenetic tumorigenesis during HBV‐mediated hepatocellular carcinogenesis.

Novel short antibacterial and antifungal peptides with low cytotoxicity: Efficacy and action mechanisms.

Short antimicrobial peptides with nine and eleven residues were developed against several clinically important bacterial and fungal pathogens (specifically Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Fusarium solani). Twelve analogues of previously reported peptides BP76 (KKLFKKILKFL) and Pac-525 (KWRRWVRWI) were designed, synthesized, and tested for their antimicrobial activities. Two of our eleven amino acid peptides, P11-5 (GKLFKKILKIL) and P11-6 (KKLIKKILKIL), have very low MICs of 3.1-12.5microg ml(-1) against all five pathogens. The MICs of these two peptides against S. aureus, C. albicans and F. solani are four to ten times lower than the corresponding MICs of the reference peptide BP76. P9-4 (KWRRWIRWL), our newly designed nine-amino acid analogue, also has particularly low MICs of 3.1-6.2microg ml(-1) against four of the tested pathogens; these MICs are two to eight times lower than those reported for Pac-525 (6.2-50microg ml(-1)).These new peptides (P11-5, P11-6 and P9-4) also exhibit improved stability in the presence of salts, and have low cytotoxicity as shown by the hemolysis and MTT assays. From the results of field-emission scanning electron microscopy, membrane depolarization and dye-leakage assays, we propose that these peptides exert their action by disrupting membrane lipids. Molecular dynamics simulation studies confirm that P11-6 peptide maintains relatively stable helical structure and exerts more perturbation action on the order of acyl tail of lipid bilayer.

Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ-coupled 2-D LC-MS/MS analysis: Insights on liver angiogenesis

Hepatitis B virus (HBV) infection and in particular Hepatitis B Virus X Protein have been shown to modulate angiogenesis. However, a comprehensive and coordinated mechanism in the HBV‐induced angiogenesis remains to be established. In this study, transient transfection of replicative HBV genome was carried out in rat primary hepatocytes (RPHs) as well as HepG2 cells. Angiogenesis was assessed by tube formation assay. 2‐D LC‐MS/MS analysis was used to detect differentially expressed proteins in cells, supporting HBV replication compared with those transfected with the empty vector. A cell‐based HBV replication was established in both RPHs and HepG2 cells. HBV replication‐induced angiogenesis was indicated by tube formation of endothelial cells cultured in condition medium from RPHs or HepG2 cells supporting HBV replication. Enzymes associated with angiogenesis, namely fumarate hydratase and tryptophanyl‐tRNA synthetase, were identified by 2‐D LC‐MS/MS analysis in HBV replicating RPHs and HepG2 cells. Our results indicated that the application of quantitative proteomics based on iTRAQ can be an effective approach to evaluate the effects of HBV replication on liver angiogenesis. The angiogenesis‐associated proteins identified in our study may eventually lead to novel anti‐angiogenic hepatocellular carcinoma cancer therapy based on tumor vascular targeting or be the markers for hepatocellular carcinoma diagnosis.

Altered antigenicities of hepatitis B virus surface antigen carrying mutations outside the common "a" determinant.

Altered antigenicities of hepatitis B virus surface antigen carrying mutations outside the common “a” determinant

iTRAQ-coupled 2-D LC-MS/MS analysis of protein profile associated with HBV-modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.