A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-dependent RNA polymerase

The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3'UTR-U(30) RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT(PPi), which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3'dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC(50) values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3'UTR-C(30) RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.     

A two-step acid-catalyzed process for the production of biodiesel from rice bran oil

A study was undertaken to examine the effect of temperature, moisture and storage time on the accumulation of free fatty acid in the rice bran. Rice bran stored at room temperature showed that most triacylglyceride was hydrolyzed and free fatty acid (FFA) content was raised up to 76% in six months. A two-step acid-catalyzed methanolysis process was employed for the efficient conversion of rice bran oil into fatty acid methyl ester (FAME). The first step was carried out at 60 degrees C. Depending on the initial FFA content of oil, 55-90% FAME content in the reaction product was obtained. More than 98% FFA and less than 35% of TG were reacted in 2 h. The organic phase of the first step reaction product was used as the substrate for a second acid-catalyzed methanolysis at 100 degrees C. By this two-step methanolysis reaction, more than 98% FAME in the product can be obtained in less than 8 h. Distillation of reaction product gave 99.8% FAME (biodiesel) with recovery of more than 96%. The residue contains enriched nutraceuticals such as gamma-oryzanol (16-18%), mixture of phytosterol, tocol and steryl ester (19-21%).     

Incidence and remediation of fungi in a sick building in Malaysia: a case study

Problems caused by indoor microbial, especially fungal growth, have been further exaggerated by the increased incidence of water intrusions, condensation from air-conditioning system, and other factors. While fungal contamination in a building can be one of the indicators of indoor air quality (IAQ), air quality assessment and remediation should be considered as important and should be carried out systematically. This study reports the incidence and remediation of an excessive fungal growth in a building in Universiti Sains Malaysia (USM), Pulau Pinang, Malaysia. Acting on an official report by the building owner and occupants, an immediate walk-through investigation was carried out between June and December 2009. A thorough sampling comprising swab and spore impactor techniques showed that the colony-forming unit per cubic meter of air (CFU/m³) levels were far above those in the guidelines of most developed countries. Thus, the building was declared a ‘sick building’ and closed to the publics. However, through immediate action with recommended methods to overcome the problem, the premise has been pronounced safe. This is a good example of successful teamwork that involved a continuous investigation by a team of experts along with comprehensive action taken by the occupants and other authorities responsible for the building.     

Root colonisation of Pseudomonas aeruginosa strain UPMP3 and induction of defence‐related genes in oil palm (Elaeis guineensis)

Pseudomonas aeruginosa strain UPMP3 labelled with β‐glucuronidase (gusA) and green fluorescent protein (gfp) by electrotransformation yielded ca 1 × 10⁷ transformants µg^?1 DNA. The data obtained from the dilution plate count showed that over 28 days both epiphytic and endophytic populations of P. aeruginosa strain UPMP3 increased from 5.76 log₁₀ [colony forming unit (CFU) + 1] g^?1 fresh weight (FW) to 8.19 log₁₀ (CFU + 1) g^?1 FW and 4.10 log₁₀ (CFU + 1) g^?1 FW to 6.23 (CFU + 1) g^?1 FW, respectively. Confocal laser scanning microscopic analysis of oil palm roots treated with gusA:gfp‐tagged P. aeruginosa strain UPMP3 showed intense root colonisation over the sampling period. The root surface colonisation by P. aeruginosa strain UPMP3 was followed by a second stage, characterised by cortical infection, and a third stage, which involves xylem ingression. The colonisation of oil palm roots by the gusA:gfp‐tagged strain was concentrated on root areas potentially rich in nutrients such as the elongation zones, ridges between epidermal cells and points of secondary adventitious root emergence. Different expression levels of defence‐related genes, namely, chitinase and β‐1,3‐glucanase in the strain UPMP3–host interaction recorded over 28 days, suggested the potential role of P. aeruginosa strain UPMP3 in triggering the defence mechanism in oil palm. This is the first report on root colonisation and upregulation of defence‐related genes on oil palm roots by P. aeruginosa strain UPMP3 and shows the potential of this strain to be used as a biocontrol agent in oil palm.     

Identification of cancer cell-line origins using fluorescence image-based phenomic screening

Universal phenotyping techniques that can discriminate among various states of biological systems have great potential. We applied 557 fluorescent library compounds to NCI's 60 human cancer cell-lines (NCI-60) to generate a systematic fluorescence phenotypic profiling data. By the kinetic fluorescence intensity analysis, we successfully discriminated the organ origin of all the 60 cell-lines.     

DNA Barcoding Reveals Cryptic Diversity within Commercially Exploited Indo-Malay Carangidae (Teleosteii: Perciformes)

Background

DNA barcodes, typically focusing on the cytochrome oxidase I gene (COI) in many animals, have been used widely as a species-identification tool. The ability of DNA barcoding to distinguish species from a range of taxa and to reveal cryptic species has been well documented. Despite the wealth of DNA barcode data for fish from many temperate regions, there are relatively few available from the Southeast Asian region. Here, we target the marine fish Family Carangidae, one of the most commercially-important families from the Indo-Malay Archipelago (IMA), to produce an initial reference DNA barcode library.

Methodology/Principal Findings

Here, a 652 bp region of COI was sequenced for 723 individuals from 36 putative species of Family Carangidae distributed within IMA waters. Within the newly-generated dataset, three described species exhibited conspecific divergences up to ten times greater (4.32–4.82%) than mean estimates (0.24–0.39%), indicating a discrepancy with assigned morphological taxonomic identification, and the existence of cryptic species. Variability of the mitochondrial DNA COI region was compared within and among species to evaluate the COI region''s suitability for species identification. The trend in range of mean K2P distances observed was generally in accordance with expectations based on taxonomic hierarchy: 0% to 4.82% between individuals within species, 0% to 16.4% between species within genera, and 8.64% to 25.39% between genera within families. The average Kimura 2-parameter (K2P) distance between individuals, between species within genera, and between genera within family were 0.37%, 10.53% and 16.56%, respectively. All described species formed monophyletic clusters in the Neighbour-joining phylogenetic tree, although three species representing complexes of six potential cryptic species were detected in Indo-Malay Carangidae; Atule mate, Selar crumenophthalmus and Seriolina nigrofasciata.

Conclusion/Significance

This study confirms that COI is an effective tool for species identification of Carangidae from the IMA. There were moderate levels of cryptic diversity among putative species within the central IMA. However, to explain the hypothesis of species richness in the IMA, it is necessary to sample the whole family across their broad geographic range. Such insights are helpful not only to document mechanisms driving diversification and recruitment in Carangidae, but also to provide a scientific framework for management strategies and conservation of commercially-important fisheries resources.     

Honey--a novel antidiabetic agent

Diabetes mellitus remains a burden worldwide in spite of the availability of numerous antidiabetic drugs. Honey is a natural substance produced by bees from nectar. Several evidence-based health benefits have been ascribed to honey in the recent years. In this review article, we highlight findings which demonstrate the beneficial or potential effects of honey in the gastrointestinal tract (GIT), on the gut microbiota, in the liver, in the pancreas and how these effects could improve glycemic control and metabolic derangements. In healthy subjects or patients with impaired glucose tolerance or diabetes mellitus, various studies revealed that honey reduced blood glucose or was more tolerable than most common sugars or sweeteners. Pre-clinical studies provided more convincing evidence in support of honey as a potential antidiabetic agent than clinical studies did. The not-too-impressive clinical data could mainly be attributed to poor study designs or due to the fact that the clinical studies were preliminary. Based on the key constituents of honey, the possible mechanisms of action of antidiabetic effect of honey are proposed. The paper also highlights the potential impacts and future perspectives on the use of honey as an antidiabetic agent. It makes recommendations for further clinical studies on the potential antidiabetic effect of honey. This review provides insight on the potential use of honey, especially as a complementary agent, in the management of diabetes mellitus. Hence, it is very important to have well-designed, randomized controlled clinical trials that investigate the reproducibility (or otherwise) of these experimental data in diabetic human subjects.     

Recent advances in elucidating the biological properties of Withania somnifera and its potential role in health benefits

Withania somnifera (L.) Dunal (Solanaceae), also known as ashwagandha, is an important medicinal plant that is widely used as a home remedy for several diseases in the Indian subcontinent and other parts of the world. W. somnifera is a dietary supplement composed of various nutrients, polyphenols and alkaloids that have free radical scavenging capacity, as well as other chemical constituents that possess anti-inflammatory, antitumor, anti-stress, antioxidant, immunomodulatory, and rejuvenating properties. The mechanism of action for these properties are not fully understood. W. somnifera also appears to influence the endocrine, cardiopulmonary and central nervous systems. Toxicity studies reveal that W. somnifera can be used without side effects. The findings presented in this review are very encouraging and indicate that this herb should be studied more extensively to confirm these results and to reveal other potential therapeutic effects.