In this work, low-intensity ultrasonication (58.3 and 93.6 W/L) was performed at lag, logarithmic and stationary growth phases of Lactobacillus plantarum in apple juice fermentation, separately. Microbial responses to sonication, including microbial growth, profiles of organic acids profile, amino acids, phenolics, and antioxidant capacity, were examined. The results revealed that obvious responses were made by Lactobacillus plantarum to ultrasonication at lag and logarithmic phases, whereas sonication at stationary phase had a negligible impact. Sonication at lag and logarithmic phases promoted microbial growth and intensified biotransformation of malic acid to lactic acid. For example, after sonication at lag phase for 0.5 h, microbial count and lactic acid content in the ultrasound-treated samples at 58.3 W/L reached 7.91 ± 0.01 Log CFU/mL and 133.70 ± 7.39 mg/L, which were significantly higher than that in the non-sonicated samples. However, the ultrasonic effect on microbial growth and metabolism of organic acids attenuated with fermentation. Moreover, ultrasonication at lag and logarithmic phases had complex influences on the metabolism of apple phenolics such as chlorogenic acid, caffeic acid, procyanidin B2, catechin and gallic acid. Ultrasound could positively affect the hydrolysis of chlorogenic acid to caffeic acid, the transformation of procyanidin B2 and decarboxylation of gallic acid. The metabolism of organic acids and free amino acids in the sonicated samples was statistically correlated with phenolic metabolism, implying that ultrasound may benefit phenolic derivation by improving the microbial metabolism of organic acids and amino acids.
The (R)-3-hydroxyacyl-ACP-CoA transferase catalyses the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA derivatives, which serves as the ultimate precursor for polyhydroxyalkanoate (PHA) polymerisation from unrelated substrates in pseudomonads. PhaG was found to be responsible for channelling precursors for polyhydroxyalkanoate (PHA) synthase from a de novo fatty acid biosynthesis pathway when cultured on carbohydrates, such as glucose or gluconate. The phaG gene was cloned from Pseudomonas sp. USM 4-55 using a homologous probe. The gene was located in a 3660 bp Sal I fragment (GenBank accession number EU305558). The open reading frame (ORF) was 885 bp long and encoded a 295 amino acid protein. The predicted molecular weight was 33251 Da, and it showed a 62% identity to the PhaG of Pseudomonas aeruginosa. The function of the cloned phaG of Pseudomonas sp. USM 4-55 was confirmed by complementation studies. Plasmid pBCS39, which harboured the 3660 bp Sal I fragment, was found to complement the PhaG-mutant heterologous host cell, Pseudomonas putida PhaGN-21. P. putida PhaGN-21, which harboured pBCS39, accumulated PHA that accounted for up to 18% of its cellular dry weight (CDW). P. putida PhaGN-21, which harboured the vector alone (PBBR1MCS-2), accumulated only 0.6% CDW of PHA.
Thiamine is known to be an important compound in human diet and it is a cofactor required for vital metabolic processes such as acetyl-CoA biosynthesis, amino acid biosynthesis, Krebs and Calvin cycle. Besides that, thiamine has been shown to be involved in plant protection against stress. In this study, the level of expression of THIC and THI1/THI4, the genes for the first two enzymes in the thiamine biosynthesis pathway were observed when oil palm (Elaeis guineensis) was subjected to oxidative stress. Primers were designed based on the consensus sequence of thiamine biosynthesis genes obtained from Arabidopsis thaliana, Zea mays, Oryza sativa, and Alnus glutinosa. Oxidative stress were induced with various concentrations of paraquat and samplings were done at various time points post-stress induction. The expression of THIC and THI1/THI4 genes were observed via RT-PCR and qPCR analysis. The expression of THIC was increased 2-fold, while THI1/THI4 gene transcript was increased 4-fold upon induction of oxidative stress. These findings showed that oil palm responded to oxidative stress by over-expressing the genes involved in thiamine biosynthesis. These findings support the suggestion that thiamine may play an important role in plant protection against stress.
Genes related to carbohydrate metabolism have evolved rapidly in eusocial bees, including honey bees. However, the characterisation of carbohydrate metabolism genes has not been reported in Apis andreniformis or Apis cerana indica. This study aimed to characterise phosphofructokinase (PFK) and pyruvate kinase (PK) genes in these honey bee species and to analyse the evolution of the genus Apis using these genes. This study found the first data regarding A. andreniformis PFK and PK-like nucleotide sequences. A BLAST-n algorithm-based search showed that A. andreniformis and A. c. indica PFK and PK genes were homologous with those of Apis florea and Apis cerana cerana from Korea, respectively. Multiple alignments of PFKs from five Apis species showed many exon gains and losses, but only one among the PKs. Thus, the exon-intron organisation of the PK genes may be more conserved compare with that of the PFKs. Another evolutionary pattern indicated that more nucleotide substitutions occurred in Apis' PK than PFK genes. Deduced PFK amino acid sequences revealed a PFK consensus pattern of 19 amino acids, while the deduced PK amino acid sequences were predicted to have barrel and alpha/beta domains. Based on these two metabolism-related genes, The Neighbour-joining and Maximum likelihood phylogenetic trees are congruent and revealed that the A. andreniformis and A. florea group were in the basal position. Apis mellifera, A. cerana, and Apis dorsata formed a monophyletic clade, although the positions of A. mellifera and A. dorsata were different in the nucleotide- and amino acid-based phylogenetic trees.
Malaysia is one of the countries that are loaded with mega biodiversity which includes microbial communities. Phages constitute the major component in the microbial communities and yet the numbers of discovered phages are just a minute fraction of its population in the biosphere. Taking into account of a huge numbers of waiting to be discovered phages, a new bacteriophage designated as Escherichia phage YD-2008.s was successfully isolated using Escherichia coli ATCC 11303 as the host. Phage YD-2008.s poses icosahedral head measured at 57nm in diameter with a long non-contractile flexible tail measured at 107nm; proving the phage as one of the members of Siphoviridae family under the order of Caudovirales. Genomic sequence analyses revealed phage YD-2008.s genome as linear dsDNA of 44,613 base pairs with 54.6% G+C content. Sixty-two open reading frames (ORFs) were identified on phage YD-2008.s full genome, using bioinformatics annotation software; Rapid Annotation using Subsystem Technology (RAST). Among the ORFs, twenty-eight of them code for functional proteins. Thirty two are classified as hypothetical proteins and there are two unidentified proteins. Even though majority of the coded putative proteins have high amino acids similarities to phages from the genus Hk578likevirus of the Siphoviridae family, yet phage YD-2008.s stands with its' own distinctiveness. Therefore, this is another new finding to Siphoviridae family as well as to the growing list of viruses in International Committee on Taxonomy of Viruses (ICTV) database.
Malaysia is one of the countries that are loaded with mega biodiversity which
includes microbial communities. Phages constitute the major component in the microbial
communities and yet the numbers of discovered phages are just a minute fraction of
its population in the biosphere. Taking into account of a huge numbers of waiting to be
discovered phages, a new bacteriophage designated as Escherichia phage YD-2008.s
was successfully isolated using Escherichia coli ATCC 11303 as the host. Phage YD-2008.s poses icosahedral head measured at 57nm in diameter with a long non-contractile
flexible tail measured at 107nm; proving the phage as one of the members of Siphoviridae
family under the order of Caudovirales. Genomic sequence analyses revealed phage
YD-2008.s genome as linear dsDNA of 44,613 base pairs with 54.6% G+C content.
Sixty-two open reading frames (ORFs) were identified on phage YD-2008.s full genome,
using bioinformatics annotation software; Rapid Annotation using Subsystem Technology
(RAST). Among the ORFs, twenty-eight of them code for functional proteins. Thirty two are
classified as hypothetical proteins and there are two unidentified proteins. Even though
majority of the coded putative proteins have high amino acids similarities to phages from the
genus Hk578likevirus of the Siphoviridae family, yet phage YD-2008.s stands with its’ own
distinctiveness. Therefore, this is another new finding to Siphoviridae family as well as to the
growing list of viruses in International Committee on Taxonomy of Viruses (ICTV) database.
Nowadays the exploration and utilisation of food and feed from marine origin is becoming more important with the increase of human population. Macroalgae are rich in nutritious compounds, which can directly be used in human and animal feed industries. The current study presents the screening of chemical components of eight macroalgae species, Sargassum boveanum, Sirophysalis trinodis, Hypnea caroides, Palisda perforata, Galaxaura rugosa, Caulerpa racemose, Caulerpa sertularioides and Bryopsis corticolans from the Persian Gulf. The results revealed that the eight studied algal species possess high protein (14.46% to 38.20%), lipid (1.27% to 9.13%) and ash (15.50% to 49.14%) contents. The fatty acids and amino acids profile showed the presence of essential fatty acids and amino acids with high nutritional value. Phaeophyta species, S. boveanum and S. trinodis, showed the highest value of ash content and polyunsaturated fatty acids while Chlorophyta species, C. racemose, C. sertularioides and B. corticolans, showed the highest level of lipid and protein contents. Rhodophyta species, G. rugosa and P. perforata, showed the highest essential amino acid content. In conclusion, this study demonstrates the potential of the studied marine species as a nutritional source for human and animal uses.
Simian betaretrovirus serotype-2 (SRV-2) is an important pathogenic agent in Asian macaques. It is a potential confounding variable in biomedical research. SRV-2 also provides a valuable viral model compared to other retroviruses which can be used for understanding many aspects of retroviral-host interactions and immunosuppression, infection mechanism, retroviral structure, antiretroviral and vaccine development. In this study, we isolated the gene encoding reverse transcriptase enzyme (RT) of SRV-2 that infected Indonesian cynomolgus monkey (Mf ET1006) and predicted the three dimensional structure model using the iterative threading assembly refinement (I-TASSER) computational programme. This SRV-2 RT Mf ET1006 consisted of 547 amino acids at nucleotide position 3284-4925 of whole genome SRV-2. The polymerase active site located in the finger/palm subdomain characterised by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165 and Asp166. We estimated that this SRV-2 RT Mf ET1006 structure has the accuracy of template modelling score (TM-score 0.90 ± 0.06) and root mean square deviation (RMSD) 4.7 ± 3.1Å, indicating that this model can be trusted and the accuracy can be seen from the appearance of protein folding in tertiary structure. The superpositionings between SRV-2 RT Mf ET1006 and Human Immunodeficiency Virus-1 (HIV-1) RT were performed to predict the structural in details and to optimise the best fits for illustrations. This SRV-2 RT Mf ET1006 structure model has the highest homology to HIV-1 RT (2B6A.pdb) with estimated accuracy at TM-score 0.911, RMSD 1.85 Å, and coverage of 0.953. This preliminary study of SRV-2 RT Mf ET1006 structure modelling is intriguing and provide some information to explore the molecular characteristic and biochemical mechanism of this enzyme.
Assessment of amino acid decarboxylase activity can be conducted using tubed broth or plated agar. In this study, the test was carried out in microtitre plates containing lysine, ornithine, arginine, tyrosine, tryptophan, phenylalanine or histidine as biogenic amine precursors. Møller decarboxylase base broth (MDB) with or without 1% of a known amino acid were added to wells of a 96 well-microtitre plate. The wells were inoculated with Escherichia coli, Klebsiella pneumoniae, Acinetobacter anitratus or Staphylococcus aureus to the final concentration of 6.0 x 10(7) cfu/ml and incubated at 35ºC. The absorbance of the culture broth was read at 570 nm at 0, 1.0, 2.0, 3.0, 4.0, 5.5, 6.5 and 7.5 hour. Comparison of means of A'(570) between 0 hour and a specified incubation time was determined statistically. Positive decarboxylase activities were detected in the media inoculated with E. coli and K. pneumoniae in less than 6 hours. The current method is suitable for immediate producers of amino acid decarboxylase enzymes. It costs less as it uses less amino acid and it has the potential to be used for screening aliquots of food materials for amino acid decarboxylase activities.
Rabies is a fatal zoonotic disease caused by rabies virus (RABV) and remains a public health problem in Malaysia. Malaysia was declared rabies-free in 2012, however rabies outbreaks occurred at few states in Peninsular Malaysia three years later; and for the first time, in Sarawak (East Malaysia) in 2017 which has caused more than 20 human deaths. This study describes the phylogenetic analysis of the complete nucleoprotein (N) gene of RABV from animal samples in Malaysia from year 2015 to 2018. The N gene of 17 RABVs from Perlis, Kedah and Sarawak were amplified and sequenced. The nucleotide and deduced amino acid similarities of N gene analysis indicated that there is high similarity among the local RABVs. Phylogenetic analysis of the N gene revealed that all Malaysia RABVs belonged to the Asian clade. Among these, RABVs from Peninsular Malaysia were clustered together with RABVs from Thailand, Vietnam and other Southeast Asia countries except Indonesia. However, RABVs from Sarawak were grouped together with Indonesian strains from Kalimantan. Our study provides baseline genetic information of the potential origins of the circulating RABVs in Malaysia. This crucial information helped the authority in policies making and strategies to be taken in outbreak control. Continuous surveillance program to monitor the disease trend, strict border control, vaccination of dog and cat population and public awareness are important steps to control the spread of the RABV.
This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts.
Beta-amyloid precursor protein cleavage enzyme 1 (BACE1) and beta-amyloid precursor protein cleavage enzyme 2 (BACE2), members of aspartyl protease family, are close homologues and have high similarity in their protein crystal structures. However, their enzymatic properties differ leading to disparate clinical consequences. In order to identify the residues that are responsible for such differences, we used evolutionary trace (ET) method to compare the amino acid conservation patterns of BACE1 and BACE2 in several mammalian species. We found that, in BACE1 and BACE2 structures, most of the ligand binding sites are conserved which indicate their enzymatic property of aspartyl protease family members. The other conserved residues are more or less randomly localized in other parts of the structures. Four group-specific residues were identified at the ligand binding site of BACE1 and BACE2. We postulated that these residues would be essential for selectivity of BACE1 and BACE2 biological functions and could be sites of interest for the design of selective inhibitors targeting either BACE1 or BACE2.
Two strains of Pleurotus giganteus (commercial and wild) were tested for their ability to induce neurite outgrowth in rat pheochromocytoma (PC12) and mouse neuroblastoma-2a (N2a) cells. Treatment with the mushroom extracts resulted in neuronal differentiation and neuronal elongation, but not nerve growth factor (NGF) production. Linoleic acid (4.5-5.0%, w/w) which is a major fatty acid present in the ethanol extract promoted NGF biosynthesis when augmented with low concentration of NGF (5 ng/mL). The two strains of mushroom were found to be high in protein (154-192 g kg(-1)), total polysaccharides, phenolics, and flavonoids as well as vitamins B1, B2, and B3. The total phenolics present in the mushroom extracts were positively correlated to the antioxidant activity (free radical scavenging, ferric reducing power, and lipid peroxidation inhibition). To conclude, P. giganteus could potentially be used in well-balanced diet and as a source of dietary antioxidant to promote neuronal health.
The Malaysian level of health care has greatly improved so that many of the infectious diseases are now under control. However, perinatal death or death due to unknown childhood diseases remains high (10.3%) being second on the list of causes of death amongst Malaysians. Could inborn metabolic diseases be the main cause of death among these children? Recently, with our success in the development of confirmatory techniques for amino acid disorders using high performance liquid chromatography (HPLC), we have examined 404 samples received from all over the country in 1993. Each specimen with abnormal findings from screening tests by one-dimensional thin layer chromatography was confirmed using HPLC. 41% had generalized aminoacidurias and 4.2% had maple syrup urine disease (MSUD). Patients were aged between 11 days to 6 years. Most of them were Malay males and presented with a history suggestive of MSUD. With this preliminary finding, further studies will be carried out in order to have an investigation and management protocol for the diseases and more importantly to formulate a strategy of screening for the country.
High performance liquid chromatography (HPLC) with phenylisothiocyanate (PITC) is recently used for confirming the diagnosis of inborn errors of metabolism (IEM) especially amino acid disorders in Malaysian children. The method of HPLC used is a precolumn derivatization of amino acids with phenylisothiocyanate and is separated by reversed phase chromatography using 3.9 x 300 mm free amino acid columns and is detected by a UV/Vis detector. The samples are obtained from cases suspected of inborn errors of metabolism, especially of amino acid disorders, which are detected clinically by pediatricians. Initially, samples from patients suspected of inborn errors of metabolism, either urine or serum, are run on one-dimensional thin layer chromatography and supplementary chemical tests to detect the abnormal bands and associated abnormalities respectively. Positive samples are further run on HPLC to determine the specific amino acids abnormality. An examples of a case of maple syrup urine disease is discussed, based on the thin layer chromatography findings and HPLC findings.
The increasing emphasis on the development of green replacements to traditional organic solvents and ionic liquids (ILs) can be attributed to the rising concerns over human health and detrimental impacts of conventional solvents towards the environment. A new generation of solvents inspired by nature and extracted from plant bioresources has evolved over the last few years, and are referred to as natural deep eutectic solvents (NADES). NADES are mixtures of natural constituents like sugars, polyalcohols, sugar-based alcohols, amino acids and organic acids. Interest in NADES has exponentially grown over the last eight years, which is evident from an upsurge in the number of research projects undertaken. NADES are highly biocompatible as they can be biosynthesized and metabolized by nearly all living organisms. These solvents pose several noteworthy advantages, such as easy synthesis, tuneable physico-chemical properties, low toxicity, high biodegradability, solute sustainability and stabilization and low melting point. Research on the applicability of NADES in diverse areas is gaining momentum, which includes as - media for chemical and enzymatic reactions; extraction media for essential oils; anti-inflammatory and antimicrobial agent; extraction of bioactive composites; as chromatographic media; preservatives for labile compounds and in drug synthesis. This review gives a complete overview of the properties, biodegradability and toxicity of NADES which we propose can assist in further knowledge generation on their significance in biological systems and usage in green and sustainable chemistry. Information on applications of NADES in biomedical, therapeutic and pharma-biotechnology fields is also highlighted in the current article along with the recent progress and future perspectives in novel applications of NADES.
Feather waste is a potential renewable source to recover valuable products because it is being a rich source of keratin proteins and amino acids. It can be used to make feather meal, fertilizer and yarn sizing agent. Various treatments have been used to recover the protein from chicken feathers as the keratinous feathers cannot be easily degraded due to its tough structure. This paper reviews the existing treatment methods used to hydrolyze chicken feathers. The treatment methods for feather hydrolysis such as physical, chemical, biological and combined treatments as well as their advantages and challenges are highlighted. The effects of these treatments on feather hydrolysis are complex and vary in regards to the performance of feather hydrolysis and product yielded. Hence, it is important to choose an appropriate treatment method since the type of treatment applied affects the product yielded qualitatively and quantitatively. In addition, the economic assessment and environmental impact of the choice of treatment should be considered also.
Ficus carica L. or fig is the oldest fruit tree that being cultivated by man. Grouped under genus Ficus, this species is grown widely in Mediterranean region and now being cultivated in an area with temperate or sub-temperate climate. Fig planting in Malaysia is still new, which was brought by a man as a hobby at first. Fig is a unique fruit tree as some variety can produce fruits without pollination. Contain lots of carbohydrates, essential amino acids, vitamins and minerals such potassium, fibre, calcium, iron compared to other fruits, fig have become an important source of diet to people especially in Mediterranean region since ancient time.
Vitamin E deficiency has been found to impair bone calcification. This study was done to determine the effects of vitamin E deficiency and supplementation on parathyroid hormone, i.e. the hormone involved in bone regulation. Female Sprague-Dawley rats were divided into 4 groups: 1) normal rat chow (RC), 2) vitamin E deficiency (VED), vitamin E deficient rats supplemented with 3) 60 mg/kg alpha-tocotrienol (ATT) and 4) 60 mg/kg (alpha-tocopherol (ATF). Treatment was carried out for 3 months. Vitamin E deficiency caused hypocalcaemia during the first month of the treatment period, increased the parathyroid hormone level in the second month and decreased the bone calcium content in the 4th lumbar bone at the end of the treatment. Vitamin E supplementation (ATT and ATF) failed to improve these conditions. The bone formation marker, osteocalcin, and the bone resorption marker, deoxypyridinoline did not change throughout the study period. In conclusion vitamin E deficiency impaired bone calcium homeostasis with subsequent secondary hyperparathyroidism and vertebral bone loss. Replacing the vitamin E with pure ATF or pure ATT alone failed to correct the changes seen.