A preliminary study on the occurrence of Aspergillus spp. and aflatoxin B1 in imported wheat and barley in Penang, Malaysia

Thirty samples consisting of wheat (15) and barley (15) were collected from different markets in Penang, Malaysia, originating from India and Thailand, respectively. All samples were analyzed for occurrence of Aspergillus spp. and aflatoxin B₁ (AFB1). Aspergillus flavus was dominant in all samples followed by A. niger. AFB1 could be detected in three wheat samples ranging from 0.42 to 1.89 μg/kg and one barley sample had 0.58 μg/kg of AFB1. The AFB1 levels in all the samples were below the Malaysian regulatory limits (<35 μg/kg). The frequency and quantity of AFB1 levels in this study were very low in wheat and barley samples compared to other agricultural commodities reported in India and Thailand. This is the first report on determination of Aspergillus spp. and AFB1 in imported wheat and barley grains in Penang, Malaysia.     

Cloning and characterization of Thermotoga maritima beta-glucuronidase

The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.     

Screening and docking chemical ligands onto pocket cavities of a protease for designing a biocatalyst

A detailed study of the trypsin surface has been carried out to gain insight into its biological functions and interactions which helped to determine the binding specificity. Twenty-four cavity pockets were automatically identified on trypsin from PDB file entry 1AUJ using CASTp (Computed Atlas of Surface Topography of proteins). Molecular docking was exploited as an efficient in silico screening tool for studying protein-ligand interactions. A systematic docking study using Autodock 3.05 has been performed on the five largest binding pockets in trypsin. A set of ten putative chemical ligands was used to dock into selected binding pockets. Docking of ligands into the five largest pockets in trypsin showed that 1,10-phenanthroline and ethanolamine preferentially bound at pocket 24 and benzamidine at pocket 22. Thermodynamically, we also found that ethanol, propanol, propandiol and phosphoethanolamine preferentially bound at pocket 21 whereas p-aminobenzamidine, phenylacetic acid and phenylalanine interacted mainly at pocket 20 based on their lowest interaction free energy.     

Monoclonal antibody production: viability improvement of RC1 hybridoma cell in different types of bioreactor

The study was done to improve the viability of the RC1 hybridoma cell in order to produce more amount of monoclonal antibody (mAb). By using the optimized media, the cell had been cultured in two bioreactor systems which were the MiniPerm and Stirred Tank bioreactor (ST bioreactor), and the results were compared to the one obtained by using the T-Flask bioreactor which was used as a standard. The results showed that the ST bioreactor was able to improve the viability of the cell to the value of 91.8% which was a little bit better than the one obtained by the MiniPerm bioreactor (88.6%) and far better than that of achieved by the T-Flask bioreactor (76.4%). This was well correlated with the good growth performance of the cell in the ST bioreactor with the specific growth rate (μ) value of 0.0289 h⁻¹ followed by MiniPerm bioreactor with the value of 0.0243 h⁻¹ and then the T-Flask with the value of 0.0151 h⁻¹. The low value of doubling time (t _d ) obtained in the ST bioreactor (24 h) compared to the one obtained in the MiniPerm (29 h) and T-Flask bioreactor (46 h) had also contributed to the higher value of cell viability. As a result a higher concentration of mAb was able to be produced by the ST bioreactor (0.42 g l⁻¹) compared to that of the MiniPerm (0.37 g l⁻¹) and T-Flask bioreactor (0.23 g l⁻¹).     

Accelerated rates of floral evolution at the upper size limit for flowers

Evolutionary theory explains phenotypic change as the result of natural selection, with constraint limiting the direction, magnitude, and rate of response [1]. Constraint is particularly likely to govern evolutionary change when a trait is at perceived upper or lower limits. Macroevolutionary rates of floral-size change are unknown for any angiosperm family, but it is predicted that rates should be diminished near the upper size limit of flowers, as has been shown for mammal body mass [2]. Our molecular results show that rates of floral-size evolution have been extremely rapid in the endoholoparasite Rafflesia, which contains the world's largest flowers [3]. These data provide the first estimates of macroevolutionary rates of floral-size change and indicate that in this lineage, floral diameter increased by an average of 20 cm (and up to 90 cm)/million years. In contrast to our expectations, it appears that the magnitude and rate of floral-size increase is greater for lineages with larger flowered ancestors. This study suggests that constraints on rates of floral-size evolution may not be limiting in Rafflesia, reinforcing results of artificial- and natural-selection studies in other plants that demonstrated the potential for rapid size changes [4-6].     

MATIN: A Random Network Coding Based Framework for High Quality Peer-to-Peer Live Video Streaming

In recent years, Random Network Coding (RNC) has emerged as a promising solution for efficient Peer-to-Peer (P2P) video multicasting over the Internet. This probably refers to this fact that RNC noticeably increases the error resiliency and throughput of the network. However, high transmission overhead arising from sending large coefficients vector as header has been the most important challenge of the RNC. Moreover, due to employing the Gauss-Jordan elimination method, considerable computational complexity can be imposed on peers in decoding the encoded blocks and checking linear dependency among the coefficients vectors. In order to address these challenges, this study introduces MATIN which is a random network coding based framework for efficient P2P video streaming. The MATIN includes a novel coefficients matrix generation method so that there is no linear dependency in the generated coefficients matrix. Using the proposed framework, each peer encapsulates one instead of n coefficients entries into the generated encoded packet which results in very low transmission overhead. It is also possible to obtain the inverted coefficients matrix using a bit number of simple arithmetic operations. In this regard, peers sustain very low computational complexities. As a result, the MATIN permits random network coding to be more efficient in P2P video streaming systems. The results obtained from simulation using OMNET++ show that it substantially outperforms the RNC which uses the Gauss-Jordan elimination method by providing better video quality on peers in terms of the four important performance metrics including video distortion, dependency distortion, End-to-End delay and Initial Startup delay.     

Mutations in prickle orthologs cause seizures in flies, mice, and humans

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.     

Characterization and pathogenicity of Fusarium proliferatum causing stem rot of Hylocereus polyrhizus in Malaysia

During a series of sampling in 2008 and 2009, stem rot disease was detected in Hylocereus polyrhizus plantations in Malaysia, with symptom appeared as circular, brown sunken lesion with orange sporodochia and white mycelium formation on the lesion surface. Eighty‐three isolates of Fusarium were isolated from 20 plantations and were morphologically identified as F. proliferatum based on the variability of colony appearance, pigmentation, growth rate, length of chains, production of bluish sclerotia, concentric ring aerial mycelium and sporodochia. Three species‐specific primers, namely ITS1/proITS‐R, PRO1/2 and Fp3‐F/4‐R successfully produced PCR products and confirmed that the isolates from stem rot of H. polyrhizus were F. proliferatum isolates. From BLAST search of translation elongation factor 1‐alpha (TEF1‐α) sequences, the isolates showed 99–100% similarity with F. proliferatum deposited in GenBank which further confirmed that the isolates were F. proliferatum. The results from amplification of MAT‐allele specific primers indicated that 14.5% of F. proliferatum isolates carried MAT‐1 allele and 85.5% carried MAT‐2. Crossing results showed that all 83 F. proliferatum isolates were male fertile showing positive crosses with the tester strains of MATD‐1 and MATD‐2. Perithecia oozing ascospore were produced. Forty isolates as representative were evaluated for pathogenicity test, produced rot symptoms similar to those observed in the fields which confirmed the isolates as the causal agent of stem rot of H. polyrhizus. To our knowledge, this is the first report of stem rot of H. polyrhizus caused by F. proliferatum in Malaysia.