Carboxyl-terminal truncated HBx regulates a distinct microRNA transcription program in Hepatocellular carcinoma development

Background: The biological pathways and functional properties by which misexpressed microRNAs (miRNAs) contribute to liver carcinogenesis have been intensively investigated. However, little is known about the upstream mechanisms that deregulate miRNA expressions in this process. In hepatocellular carcinoma (HCC), hepatitis B virus (HBV) X protein (HBx), a transcriptional trans-activator, is frequently expressed in truncated form without carboxyl-terminus but its role in miRNA expression and HCC development is unclear. Methods: Human non-tumorigenic hepatocytes were infected with lentivirus-expressing full-length and carboxyl-terminal truncated HBx (Ct-HBx) for cell growth assay and miRNA profiling. Chromatin immunoprecipitation microarray was performed to identify the miRNA promoters directly associated with HBx. Direct transcriptional control was verified by luciferase reporter assay. The differential miRNA expressions were further validated in a cohort of HBV-associated HCC tissues using real-time PCR. Results: Hepatocytes expressing Ct-HBx grew significantly faster than the full-length HBx counterparts. Ct-HBx decreased while full-length HBx increased the expression of a set of miRNAs with growth-suppressive functions. Interestingly, Ct-HBx bound to and inhibited the transcriptional activity of some of these miRNA promoters. Notably, some of the examined repressed-miRNAs (miR-26a, -29c, -146a and -190) were also significantly down-regulated in a subset of HCC tissues with carboxyl-terminal HBx truncation compared to their matching non-tumor tissues, highlighting the clinical relevance of our data. Conclusion: Our results suggest that Ct-HBx directly regulates miRNA transcription and in turn promotes hepatocellular proliferation, thus revealing a viral contribution of miRNA deregulation during hepatocarcinogenesis. © 2011 Yip et al.published_or_final_versio

Antifungal activity of compound

Background: Candidasis is one of the most commonly encountered human fungal infections and is caused by Candida species, most notably C. albicans. The opportunistic fungal pathogen exists as part of the normal microbiota, but can become invasive in immunocompromised patients with AIDs, cancers or organ transplantation. Superficial candidiasis is common but disseminated infections are usually fatal. Antifungal treatments include flucytasine, polyenes, azoles and echinocandines. Unfortunately, they may have toxic side effects, limited spectrum of activity and poor absorption. Alarmingly, extensive use of antifungal agents has resulted in the emergence of resistant Candida infections in clinical settings. There is thus an urgent need for new antifungal agents. Methods: Using broth dilution assay, we identified a compound (#205; 5,6,7-trihydroxy-2-phenylchromen-4-one) from a natural compund library (n = 400) with potent anti-Candidal activity against a clinical strain C. albicans CA12. Compound #205, also known as baicalein, is a major component of a Chinese herbal medicine Scutellaria baicalensis. In the present study, focused libraries of compound #205 were generated via acylation and the anti-Candidal activity was evaluated by broth dilution assay was used to evaluate the effects on membrane fluidity and efflux pumps. Results: Focused libraries (L1-L4) of compound #205 were generated. Besides C. albicans, broth dilution assay showed that both compound #205 and L1 exhibited similar antifungal activity against non-albicans Candida (NAC) species. Lower anti-Candidal activity was observed in L2-L4, in connection with the size of the carbon chain of the acyl donors. Compound #205 and L1 were fungistatic and inhibited efflux pump activity in C. albicans and NAC species. Conclusions: The findgins of the present study not only revealed the broad antifungal spectrum of compund #205, but also indicated the hydraxyl groups might play a pivotal role in anti-Candidal potency. Our data provided new information for stucture-activity relationship and antifungal spectrum of compound #205, which might help in further structural modifications to generate compounds with improved therapeutic potency

S11-1 [27] A Green Journey to the Making of Magic Bullets

Semi-synthetic cephalosporins, constituting the largest portion in the worldwide sales of beta-lactam antibiotics, have been used extensively for decades as “magic bullets” to combat microbial infections. The global cephalosporin market was valued at US$78,000 million in 2016 and it is estimated to increase by 14% by 2023. Most of the marketed cephalosporins are semi-synthetic. However, the methodologies involved are environmentally-damaging because toxic chemicals are used. The increasing annual demand of semi-synthetic cephalosporins and the concept of environmental sustainability pose a dire need to develop alternative approaches for “greener” production of antibiotics. Microorganisms have been heralded as a solution to many of the contemporary world’s most pressing issues. One of the frontiers in microbial biotechnology is to help solve environmental and sustainable resources problems, and scientists have been working vigorously to harness single-celled systems to produce high value fine chemicals such as commodity chemicals, therapeutic intermediates, and essential nutrients. Bioprocess technology has operational advantages: the procedures are low cost, high yield, and environmentally-sustainable. My research laboratory has been working on developing a sustainable and effective bioprocess using enzymes for the biosynthesis of 7-aminocephalosporanic acid (7-ACA), a cephem nucleus for the production of nearly two-thirds of the global commercial semi-synthetic cephalosporins. In particular, using bioinformatics and modeling, we have characterised a variant D-amino acid oxidase (DAAO) with enhanced catalytic properties suitable for industrial applications. By using variant DAAO and glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase), we have patented a two-enzyme biosystems for direct biosynthesis of 7-ACA with overall conversion rate approaching 100%. Immobilised enzymes are more robust and exhibit enhanced resistance to industrial and environmental challenges. However, immobilised enzymes are expensive and preparation involves tedious multi-step schemes. Yeast surface display technology represents a tangible and sustainable alternative approach to immobilised enzymes with industrial relevance. Enzyme biocatalysts are displayed on cell surface where a direct enzyme-substrate interaction is facilitated with higher stability and catalytic activity. The new paradigm in pharmaceutical industry is the development of multi-enzyme sequential reactions in one-pot to minimise enzyme inhibition and formation of by-products. We would like to address the problems and develop a streamlined bioprocess relevant to the concept of environmental sustainability by constructing a functional assembly of DAAO and GL-7-ACA acylase for one-pot production of 7-ACA. We reckon that yeast display technology is particularly suitable for biosynthesis of 7-ACA via an establishment of a functional assembly of enzymes with close proximity. Here, I would like to report the latest findings of my research on this area and the potential industrial applications

High level expression of secretory phytases in Pichia pastoris

Phytases catalyze the release of inorganic phosphate (Pi) from phytate, a major storage compound of phosphorus in plant seeds. However, non-ruminant animals, including humans, swine and poultry, could not digest phytate as they have little phytase activity in their guts. Discharge of undigested phytate into environment may cause phosphorus pollution. Phytate is also considered as an “anti-nutrient” for reducing dietary absorption of essential minerals as it can form insoluble and stable complexes with metal ions such as zinc, iron, magnesium and calcium. It is, therefore, necessary to develop sustainable approaches for environmentally-friendly utilization of this valuable and abundant natural resource. To this end, our team would like to employ baker’s yeast as a tiny factory to produce high level expression of secretory phytases for phytate hydrolysis

Differential phytate utilization in Candida species

The present study was undertaken to evaluate and characterize the phytase activity in different Candida species. A total of 113 Candida isolates representing eight species were examined for phytase activity by an agar plate assay using the calcium salt of phytic acid as the sole phosphorus source. A phytase-positive phenotype was identified by the formation of a clear halo around a fungal colony. Cell-bound differential phytase activity was observed in Candida isolates at inter- and intra-species levels. Although phytase activity was not affected by the supplementation of external phosphate in C. albicans, C. dubliniensis, C. glabrata, and C. kefyr, elevated phytase activity was evident in C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis in phosphate-free medium. Further characterization showed that, in general, relatively higher phytase activity was observed at more acidic pHs, and the phytase activity increased with incubation temperature, reaching a maximum at 55 or 65°C. Taken together, the findings demonstrated, for the first time, differential phytase activities in different Candida species. Phytase activity may be a contributing factor to fungal survival and proliferation within the human gastrointestinal tract, where nutrients are usually scarce

Antifungal activity of commercial mouthrinses against Candida albicans

Objective: To investigate the effectiveness of delmopinol (Decapinol®), sodium chlorite (TheraBreath®), povidone iodine (Betadine®), and lactoferrin-lysozyme-lactoperoxidase (OralSevenTM) mouthrinses against C. albicans planktonic cells and biofilms. Methods: Two-fold dilutions of the mouthrinse solutions were prepared (1:2, 1:4, 1:8, 1:16, 1:32, 1:64), and dispensed into 96 well microtitre plates. Standardized inocula of wild typ..

Baicalein inhibits energy-dependent efflux pump activity in Candida species

Objectives: To evaluate the in vitro antifungal activity of baicalein and its effect on energy-dependent efflux pumps of Candida species. Methods: Minimum inhibitory concentration (MIC) of baicalein was determined by broth microdilution assay (CLSI guidelines). MIC was defined as the lowest concentration of baicalein for 80% inhibition of visible growth when compared with control. Rhodamine 6G (R6G) efflux was evaluated to investigate the effect of