Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris

Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS–PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72 h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection

Identification of protein biomarkers for candida parapsilosis and candida tropicalis

Candida species are the major human fungal pathogens and incidence of systemic candidiasis has been rising over the years with Candida albicans as the main species isolated. However, Candida parapsilosis and Candida tropicalis have emerged recently as increasingly prevalent pathogens, but only few studies have focused on them thus far. In the first part of this study, systemic infection of C. parapsilosis and C. tropicalis were generated in mice via intravenous challenge and their pathogenicity was studied. It was demonstrated that mice challenged with C. parapsilosis and C. tropicalis exhibited different survival rate, with death only observed for C. tropicalis-infected mice. Besides, C. tropicalis-infected mice displayed higher fungal tissue burden and more severe kidney damage. Overall, the results indicate that C. tropicalis was more virulent than C. parapsilosis and suggests that specific virulence factors such as morphogenesis may account for variation in pathogenesis. In another context, difficulty in establishing definitive diagnosis for candidasis has prompted the search of biomarkers for the disease. Squalene synthase is a novel antigenic protein of C. tropicalis that was discovered from a previous study. To investigate its potential as a biomarker candidate, this protein was expressed in Pichia pastoris and the fusion protein was purified by affinity chromatography. The results showed that the purified recombinant protein was specifically recognized by polyclonal antibodies from C. tropicalis-infected mice on Western blot, suggesting that the protein could be a potential biomarker for C.tropicalis. However, further testing is needed to confirm its utility. To further discover protein biomarkers for C. parapsilosis and C. tropicalis and to understand their host-pathogen interactions, an immunoproteomic analysis was performed. For this purpose, cell wall proteins-enriched fractions of C. parapsilosis and C. tropicalis were systemically screened for antigens using antisera obtained from experimentally infected mice. This analysis led to the identification of 12 immunogenic proteins each for C. parapsilosis and C. tropicalis, of which 8 were common antigens for both species. Among these antigens, 14 have been previously reported as antigens of C.albicans, whereas isocitrate dehydrogenase (Idh2p) and dihydrolipoyllysine-residue succinyltransferase (Kgd2p) were novel immunogenic proteins that were reported for the first time for Candida species. The present work showed that these antigens were expressed in vivo during infection and are likely to play important roles in pathogenesis. Next, the newly reported antigens, Idh2p and Kgd2p were overexpressed as recombinant proteins in Escherichia coli and subsequently purified by affinity chromatography. The antigenicity of the recombinant proteins was verified by immunoblotting using antisera from infected mice. This preliminary work suggests that the two proteins may find potential application as biomarker for C.parapsilosis and C. tropicalis. However, additional work is required to evaluate the usefulness of these proteins. Collectively, findings from the mouse model of infection and antigen profiling by immunoproteomics help to improve understanding on host response to C. parapsilosis and C. tropicalis infection, as well as discovering new protein antigens to be employed as disease biomarker candidates. This work also described the production of several antigenic recombinant proteins that lays the foundation for further research

Domed survival : a multimedia project.

This report documents our development of a multimedia educational game based on an original concept and script.Bachelor of Communication Studie

Regulation of signal transduction pathways in colorectal cancer: implications for therapeutic resistance

Resistance to anti-cancer treatments is a critical and widespread health issue that has brought serious impacts on lives, the economy and public policies. Mounting research has suggested that a selected spectrum of patients with advanced colorectal cancer (CRC) tend to respond poorly to both chemotherapeutic and targeted therapeutic regimens. Drug resistance in tumours can occur in an intrinsic or acquired manner, rendering cancer cells insensitive to the treatment of anti-cancer therapies. Multiple factors have been associated with drug resistance. The most well-established factors are the emergence of cancer stem cell-like properties and overexpression of ABC transporters that mediate drug efflux. Besides, there is emerging evidence that signalling pathways that modulate cell survival and drug metabolism play major roles in the maintenance of multidrug resistance in CRC. This article reviews drug resistance in CRC as a result of alterations in the MAPK, PI3K/PKB, Wnt/β-catenin and Notch pathways

The position of home insurance in Singapore

Home ownership in Singapore had increased significantly from the 1960's to 1990's. This was the result of increasing affluence, relaxation of rules governing the use of Central Provident Funds (CPF) for purchasing private residential properties and the accelerated building programmes ofHousing Development Board (HDB) flats in the 1980's. Despite a marked growth in home ownership for the past decades, the increase in demand for home insurance has been very gradual and very slow. There are two main objectives in this study. The first objective is to present the current situation of home insurance in Singapore in terms of the existing home insurance products, premium rating, marketing distribution, claims experience and the perceptions of the insurers. The second objective is to investigate and analyze the current demand for home insurance among the homeowners in Singapore. The demographic characteristics as well as the reasons for purchasing or not purchasing home insurance will be examined. To achieve the stated objectives, a total of twelve interviews were carried out with the executives of insurance companies. To assess the consumer demand, questionnaires were administered to a sample of 292 homeowners or adults of marriageable age. The data gathered were analyzed and collated. Views differ among the insurers regarding the outlook of home insurance market. For the consumer survey, it was found that some co-relation exists between the level of income, the type of housing property owned and the purchase of home insurance. Some limitations of this study were highlighted and recommendations were made on the marketing efforts to better penetrate the consumer market for home insurance in Singapore.BUSINES

Metaproteomic analysis of human gut microbiota: where are we heading?

Abstract The human gut is home to complex microbial populations that change dynamically in response to various internal and external stimuli. The gut microbiota provides numerous functional benefits that are crucial for human health but in the setting of a disturbed equilibrium, the microbial community can cause deleterious outcomes such as diseases and cancers. Characterization of the functional activities of human gut microbiota is fundamental to understand their roles in human health and disease. Metaproteomics, which refers to the study of the entire protein collection of the microbial community in a given sample is an emerging area of research that provides informative details concerning functional aspects of the microbiota. In this mini review, we present a summary of the progress of metaproteomic analysis for studying the functional role of gut microbiota. This is followed by an overview of the experimental approaches focusing on fecal specimen for metaproteomics and is concluded by a discussion on the challenges and future directions of metaproteomic research

Proteogenomics:From next-generation sequencing (NGS) and mass spectrometry-based proteomics to precision medicine

One of the best-established area within multi-omics is proteogenomics, whereby the underpinning technologies are next-generation sequencing (NGS) and mass spectrometry (MS). Proteogenomics has contributed significantly to genome (re)-annotation, whereby novel coding sequences (CDS) are identified and confirmed. By incorporating in-silico translated genome variants in protein database, single amino acid variants (SAAV) and splice proteoforms can be identified and quantified at peptide level. The application of proteogenomics in cancer research potentially enables the identification of patient-specific proteoforms, as well as the association of the efficacy or resistance of cancer therapy to different mutations. Here, we discuss how NGS/TGS data are analyzed and incorporated into the proteogenomic framework. These sequence data mainly originate from whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. We explain two major strategies for sequence analysis i.e., de novo assembly and reads mapping, followed by construction of customized protein databases using such data. Besides, we also elaborate on the procedures of spectrum to peptide sequence matching in proteogenomics, and the relationship between database size on the false discovery rate (FDR). Finally, we discuss the latest development in proteogenomics-assisted precision oncology and also challenges and opportunities in proteogenomics research