Isolation, Screening for Bioactivities and Identification of Selected Endophyte Fungi by Sequencing of 18S RRNA/Its Genes

Endophytic fungi occur within plant tissues such as leaves and stems of healthy plants without producing any apparent infections and symptoms. Experiments were conducted to isolate endophytic fungi from healthy medicinal plants as well as detecting any reoccurrence of any particular endophytic fungi predominantly in selected local medicinal plants. Endophytic fungi have also been recognised as repository of novel secondary metabolites, which have beneficial biological activities, biocontrol of insects and oligosaccharides degrading enzymes. Thus, isolated endophytic fungi were screened for their bioactive properties by using Thin Layer Chromatography and Agar Diffusion Assay. Specially engineered yeast strains i.e. UCS and UCK from Kyowa Hakko Company, Japan were used to screened for anti-tumour activity. 18S ribosomal Ribonucleic Acid (18S rRNA) I Internal Transcribed Spacer (ITS) gene sequencing were conducted to identified certain isolates. Isolation of the endophytic fungi from healthy local medicinal plants showed that 61 out of 72 (84.7%) yielded endophytic fungi. Most of the endophytes were obtained from the leaves and very few from the stems. The reoccurrence rate of the endophytic fungi was 1.39% (1 in 72). Nevertheless, no predominant endophytic fungi association with types of medicinal plant was observed. All the isolated endophytic fungi were able to degrade starch, xylan, mannan and inulin. 98.33% of the endophytic tested were able to degrade sago starch and rice starch. About 96.67% of the isolates were detected producing potato starch and starch wheat unmodified degrading enzyme. However, 95% of the endophytes produced tapioca starch and com starch degrading enzyme. Among the isolated endophytic fungi, 16.9% of them were considered important with regard to the bioactive screening results. Thus, 22 isolates from 130 isolated fungi gave satisfactory results in bioactive screening. There were 16 isolates that gave positive results in bio-activity test against UCS/UCK yeast strain. This showed that 16 out of 130 isolates produced potential bioactive compound for anti-tumour. Four out of the twenty two important isolates were identified through microscopic examination and 18S IITS gene sequencing. Isolate 12L was identified as Penicillium spp. through microscopic morphology observation. Those isolates identified by 18S IITS gene sequencing included 1B, Endothia gyrosa; 19L, Colfetotrichum gloeosporioides and 22L, Botryosphaeria ribis . BLAST programme was used as a tool to determine the homology of the sequence obtained with the database sequence

Surface plasmon resonance biosensor for real-time detection of genetically modified organisms

Application of surface plasmon resonance (SPR) biosensor in detection of genetically modifed organism (GMO) is demonstrated. A total of four biotinylated probes namely Tnosb, P35Sb, LECb and TSQb were successfully immobilized onto the SA chip. Results analysis indicated that the SPR system with the sensor chip immobilized with the Tnosb, P35Sb, LECb and TSQb biotinylated probes potentially detect complementary standard fragments as low as 1 nM. Biospecifc interaction analysis (BIA), employing surface plasmon resonance (SPR) and biosensor technologies provide easy, rapid and automatable approach in detection of GMOs. Short assay times, label free DNA hybridization reaction and no toxic compounds are required, i.e. ethidium bromide, and the reusability of the sensor surface are some of the factors that contribute to the general advantages of the surface plasmon resonance (SPR) biosensor system in detection of GMOs

Molecular quantitation and characterization of Vibrio cholerae from different seafood obtained from wetmarket and supermarket

Vibrio cholerae still represents a significant threat to human health worldwide despite the advances in hygiene, consumer knowledge, food treatment and food processing. In Malaysia, statistics in year 2009 have shown that among the food and water borne diseases, food poisoning has the highest incidence rate of 36.17 per 100,000 populations and with a mortality rate of 0.01 per 100,000 populations. In this study, 22 seafood samples comprising of fish, squid, crustacean and mollusks purchased from wet market and supermarket were analyzed. The Most Probable Number (MPN) and real time PCR was used to enumerate the Vibrio cholerae in seafood sample. The results showed that MPN-real time PCR of the samples from wet market had a maximum of > 1100 MPN/g compare to 93 MPN/g enumerated from the MPN plate. The MPN-real time PCR in the samples from supermarket indicated 290 MPN/g as compared to 240 MPN/g enumerated from the MPN plate. The standard curves showed that there was a good linear correlation between the Ct values. The minimum level of detection of Vibrio cholerae standard DNA at targeted gene was 3 × 10 -5 ng/μl

The used of recombinant plasmid DNA in GMO quantitative analysis of insect resistance maize targeted unapproved StarLink corn and approved Bt176 corn in food and feed sold commercially sold Malaysia

Genetically modified organisms (GMO) are increased remarkably from year to year and the estimated global area cultivated with genetically modified (GM) crops reached 125 million hectares in year 2008. However, insect resistance maize based on Bacillus thuringienses (Bt) is of the most cultivated GM crop in worldwide. Bacillus thuringiensis (Bt) is an aerobic, gram-positive bacterium that synthesize one or more Cry protein that are toxic to various types crop and forestry insects pests. To date, several cry genes have been introduced into GM plant to combat with various type of insect. Worldwide commercialization of GM crops has raised the customers’ concern about the Biosafety issues, and thus, many countries have implemented the labeling legislations for GM food and their derivatives. In this study, we introduced the quantitative analysis method based on the recombinant plasmid DNA as calibrators that can be used to determine the percentage of GMO content in various types of food and feed samples. Therefore, we have reported 7.5% (6/80) of the samples were contained StarLink maize and 1.25% (1/80) samples were contained Bt176 maize. Additionally, the percentage of GM content in each positive sample were further determined with the developed quantitative method. The percentage of the StarLink corns that present in the positive samples were varies from 0.09% to 2.53% and Bt176 corn that present in the positive sample was 16.90%. The present study demonstrated that the recombinant plasmid DNA that used in quantitative real-time method as good alternative quantitative analysis of GM content

Evaluation of injury to Saccharomyces rouxii YSa40 cells in low water activity/pH glycerol/CPB stress system

Mid-exponential phase Saccharomyces rouxii YSa40 cells subsequently stressed at low a w/pH in the 0.64 a w/pH 3.5 glycerol/CPB system became injured. Such injury was detected by the loss of ability of the stressed population to form colonies on secondary-stress plating medium (glycerol/BM agar at 0.94 a w/pH 3.5 (lactic acid)) while colony forming ability on secondary non-stress plating medium (sugars/BM agar at 0.94 a w/pH 3.5 (lactic) was unaffected. The injury was shown to be due to sensitivity to glycerol/lactic acid. Results of the present study will be useful for achieving complete decontamination of 'Intermediate Moisture Foods' against xerotolerant molds and yeast

Quantitative analysis of Roundup Ready soybean content in soy-derived food and animal feed by using Real-time PCR incorporated with cloned DNA fragments

Malaysia, Biosafety Bill 2006 was approved by Parliament in July 2007, and labeling legislation will be implemented soon. In this study, duplex polymerase chain reaction (PCR) was carried out to detect endogenous soybean lectin gene and exogenous cp4-epsps (5’-enolpyruvylshikimate-3-phospate synthase) gene simultaneously. Additionally, real-time PCR utilizing SYBR Green fluorescence dye were established for the quantitative analysis of Roundup Ready soybean (RRS), which is based on the two established calibration curve from cloned fragment of cp4-epsps gene and lectin gene respectively. Approximately, 39.5% (45/114) of the samples examined in this study contain RRS, animal feeds (31), processed food (13) and raw soybean (1). Additionally, 75.6% (34/45) of the positive samples were found contained RRS above 0.9%. The sensitive GMO quantitative approach described in this study enable the analysis of various samples and this will facilitate the labeling process

The interplay between microRNAs and cellular components of tumour microenvironment (TME) on non-small-cell lung cancer (NSCLC) progression

Cellular components of the tumour microenvironment (TME) are recognized to regulate the hallmarks of cancers including tumour proliferation, angiogenesis, invasion, and metastasis, as well as chemotherapeutic resistance. The linkage between miRNA, TME, and the development of the hallmarks of cancer makes miRNA-mediated regulation of TME a potential therapeutic strategy to complement current cancer therapies. Despite significant advances in cancer therapy, lung cancer remains the deadliest form of cancer among males in the world and has overtaken breast cancer as the most fatal cancer among females in more developed countries. Therefore, there is an urgent need to develop more effective treatments for NSCLC, which is the most common type of lung cancer. Hence, this review will focus on current literature pertaining to antitumour or protumourigenic effects elicited by nonmalignant stromal cells of TME in NSCLC through miRNA regulation as well as current status and future prospects of miRNAs as therapeutic agents or targets to regulate TME in NSCLC

Metagenomic studies of the gut microbiota: the snake gut microbiota as a model organism

Microbes play a very important role in each individual. The microbial communities and its genetic blueprint greatly influence in many human diseases. Most of the microbe populations are grow in an individual’s gut. Therefore, metagenomics studies on gut microbes are essential to understand the microbial diversity in gut and the knowledge on microbial composition associates with terrestrial animals will be very important for further understand nutrition, diseases and physiological state. Besides, the availability of next generation sequencing technologies gives a better understanding on gut microbiotas communities compare to the first generation sequencing. This paper, we suggested snakes as a model to study microbial metagenomics due to its various compounds can help to cure various illnesses, even kill off unwanted germs from body. Therefore, this paper mainly review on snake gut microbes, secondary metabolites produce by microbes and the benefits of molecular technologies used in metagenomics which can be useful in medical industries and treatment of infectious diseases

Detection and cloning of Phospholipase C‐zeta (PLCζ) gene fragments from testes of Rattus tiomanicus (Malayan wood rat).

PLC‐zeta is a novel, sperm‐specific phospholipase‐C that is highly effective in causing Ca2+ oscillation and activation in mouse eggs during fertilization. Upon sperm‐oocyte fusion, PLC‐zeta diffuse into the oocyte cytosol and stimulate the inositol1, 4, 5‐trisphosphate (IP3) pathway thus increasing IP3 levels and activating IP3 receptor‐mediated Ca2+ release from intracellular stores. This event will trigger oocyte activation, essential for embryo development. The importance of PLC‐zeta was demonstrated when PLC‐zeta removal from sperm extracts stops Ca2+ release in eggs. In addition, sperm from transgenic mice expressing short hairpin RNAs targeting PLC‐zeta mRNA has been shown to reduce PLC‐zeta protein and significantly disturbs the calcium oscillatory behavior of eggs inseminated with these sperm. Rattus tiomanicus (Malayan wood rat) is the predominant rodent pest targeting oil palm estates in Malaysia. Various methods have been used to control their population. In the current study, PLC‐zeta expression was detected using one‐step RT‐PCR from the testes. The PLC‐zeta sequence of R. tiomanicus showed strong similarity on alignment with PLC‐zeta sequence of R. norvegicus. Discovery of a highly conserved PLC‐zeta among rodents and understanding of the molecular properties of PLC‐zeta can lead to further development of alternative approaches of controlling them in a more environmental friendly way. Disruption of the expression of PLC‐zeta in R. tomianicus could lead to poor fertility in the male thus lowering the population of this pest and its deleterious effects to the agriculture sector especially oil palm, one of the most broadly grown crops in South East Asia