Bacterial community structure was investigated in five tropical rainforests in Sarawak, Malaysia and one temperate forest in Kyoto, Japan. A hierarchical sampling approach was employed, in which soil samples were collected from five sampling-sites within each forest. Pyrosequencing was performed to analyze a total of 493,790 16S rRNA amplicons. Despite differences in aboveground conditions, the composition of bacterial groups was similar across all sampling-sites and forests, with Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes and Bacteroidetes accounting for 90% of all Phyla detected. At higher taxonomic levels, the same taxa were predominant, although there was significant heterogeneity in relative abundance of specific taxa across sampling-sites within one forest or across different forests. In all forests, the level of bacterial diversity, estimated using the Chao1 index, was on the order of 1,000, suggesting that tropical rainforests did not necessarily have a large soil bacterial diversity. The average number of reads per species (OTUs) per sampling-site was 8.0, and more than 40-50% of species were singletons, indicating that most bacterial species occurred infrequently and that few bacterial species achieved high predominance. Approximately 30% of species were specific to one sampling-site within a forest, and 40-60% of species were uniquely detected in one of the six forests studied here. Only 0.2% of species were detected in all forests, while on average 32.1% of species were detected in all sampling-sites within a forest. The results suggested that bacterial communities adapted to specific micro- and macro-environments, but macro-environmental diversity made a larger contribution to total bacterial diversity in forest soil.
The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.
Co-inheritance of alpha-thalassemia with homozygosity or compound heterozygosity for beta-thalassemia may ameliorate beta-thalassemia major. A wide range of clinical phenotypes is produced depending on the number of alpha-thalassemia alleles (-alpha/alphaalpha --/alphaalpha, --/-alpha). The co-inheritance of beta-thalassemia with alpha-thalassemia with a single gene deletion (-alpha/alphaalpha) is usually associated with thalassemia major. In contrast, the co-inheritance of beta-thalassemia with two alpha-genes deleted in cis or trans (--/alphaalpha or -alpha/-alpha) generally produces beta-thalassemia intermedia. In Southeast Asia, the most common defect responsible for alpha-thalassemia is the Southeast Asian (SEA) deletion of 20.5 kilobases. The presence of the SEA deletion with Hb Constant Spring (HbCS) produces HbH-CS disease. Co-inheritance of HbH-CS with compound heterozygosity for beta-thalassemia is very rare. This study presents a Malay patient with HbH-CS disorder and beta degrees/beta+-thalassemia. The SEA deletion was confirmed in the patient using a duplex-PCR. A Combine-Amplification Refractory Mutation System (C-ARMS) technique to simultaneously detect HbCS and Hb Quong Sze confirmed HbCS in the patient. Compound heterozygosity for CD41/42 and Poly A was confirmed using the ARMS. This is a unique case as the SEA alpha-gene deletion in cis (--SEA/alphaalpha) is generally not present in the Malays, who more commonly possess the two alpha-gene deletion in trans (-alpha/-alpha). In addition, the beta-globin gene mutation at CD41/42 is a common mutation in the Chinese and not in the Malays. The presence of both the SEA deletion and CD41/42 in the mother of the patient suggests the possible introduction of these two defects into the family by marriage with a Chinese.
The genetic variation of Trigonobalanus verticillata, the most recently described genus of Fagaceae, was studied using chloroplast DNA sequences and AFLP fingerprinting. This species has a restricted distribution that is known to include seven localities in tropical lower montane forests in Malaysia and Indonesia. A total of 75 individuals were collected from Bario, Kinabalu, and Fraser's Hill in Malaysia. The sequences of rbcL, matK, and three non-coding regions (atpB-rbcL spacer, trnL intron, and trnL-trnF spacer) were determined for 19 individuals from these populations. We found a total of 30 nucleotide substitutions and four length variations, which allowed identification of three haplotypes characterizing each population. No substitutions were detected within populations, while the tandem repeats in the trnL -trnF spacer had a variable repeat number of a 20-bp motif only in Kinabalu. The differentiation of the populations inferred from the cpDNA molecular clock calibrated with paleontological data was estimated to be 8.3 MYA between Bario and Kinabalu, and 16.7 MYA between Fraser's Hill and the other populations. In AFLP analysis, four selective primer pairs yielded a total of 431 loci, of which 340 (78.9%) were polymorphic. The results showed relatively high gene diversity (H(S) = 0.153 and H(T) = 0.198) and nucleotide diversity (pi(S) = 0.0132 and pi(T) = 0.0168) both within and among the populations. Although the cpDNA data suggest that little or no gene flow occurred between the populations via seeds, the fixation index estimated from AFLP data (F(ST) = 0.153 and N(ST) = 0.214) implies that some gene flow occurs between populations, possibly through pollen transfer.
Geographical variation in soil bacterial community structure in 26 tropical forests in Southeast Asia (Malaysia, Indonesia and Singapore) and two temperate forests in Japan was investigated to elucidate the environmental factors and mechanisms that influence biogeography of soil bacterial diversity and composition. Despite substantial environmental differences, bacterial phyla were represented in similar proportions, with Acidobacteria and Proteobacteria the dominant phyla in all forests except one mangrove forest in Sarawak, although highly significant heterogeneity in frequency of individual phyla was detected among forests. In contrast, species diversity (α-diversity) differed to a much greater extent, being nearly six-fold higher in the mangrove forest (Chao1 index = 6,862) than in forests in Singapore and Sarawak (~1,250). In addition, natural mixed dipterocarp forests had lower species diversity than acacia and oil palm plantations, indicating that aboveground tree composition does not influence soil bacterial diversity. Shannon and Chao1 indices were correlated positively, implying that skewed operational taxonomic unit (OTU) distribution was associated with the abundance of overall and rare (singleton) OTUs. No OTUs were represented in all 28 forests, and forest-specific OTUs accounted for over 70% of all detected OTUs. Forests that were geographically adjacent and/or of the same forest type had similar bacterial species composition, and a positive correlation was detected between species divergence (β-diversity) and direct distance between forests. Both α- and β-diversities were correlated with soil pH. These results suggest that soil bacterial communities in different forests evolve largely independently of each other and that soil bacterial communities adapt to their local environment, modulated by bacterial dispersal (distance effect) and forest type. Therefore, we conclude that the biogeography of soil bacteria communities described here is non-random, reflecting the influences of contemporary environmental factors and evolutionary history.
Soil bacterial community structures of six dominant phyla (Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes, Bacteroidetes and Actinobacteria) and unclassified bacteria detected in tropical Sarawakian and temperate Japanese forests were compared based on 16S rRNA gene sequence variation. The class composition in each phylum was similar among the studied forests; however, significant heterogeneities of class frequencies were detected. Acidobacteria and Proteobacteria were the most dominant phyla in all six forests, but differed in the level of bacterial species diversity, pattern of species occurrence and association pattern of species composition with physicochemical properties in soil. Species diversity among Acidobacteria was approximately half that among Proteobacteria, based on the number of clusters and the Chao1 index, even though a similar number of sequence reads were obtained for these two phyla. In contrast, species diversity within Planctomycetes and Bacteroidetes was nearly as high as within Acidobacteria, despite many fewer sequence reads. The density of species (the number of sequence reads per cluster) correlated negatively with species diversity, and species density within Acidobacteria was approximately twice that within Proteobacteria. Although the percentage of forest-specific species was high for all bacterial groups, sampling site-specific species varied among bacterial groups, indicating limited inter-forest migration and differential movement of bacteria in forest soil. For five of the seven bacterial groups, including Acidobacteria, soil pH appeared to strongly influence species composition, but this association was not observed for Proteobacterial species. Topology of UPGMA trees and pattern of NMDS plots among the forests differed among the bacterial groups, suggesting that each bacterial group has adapted and evolved independently in each forest.
Identifying susceptible genes associated with the pathogenesis of atherosclerosis (ATH) may contribute toward better management of this condition. This preliminary study was aimed at assessing the expression levels of 11 candidate genes, namely tumor protein (TP53), transforming growth factor, beta receptor II (TGFBR2), cysthathionenine-beta-synthase (CBS), insulin receptor substrate 1 (IRS1), lipoprotein lipase (LPL), methylenetetrahydrofolate reductase (MTHFR), thrombomodulin (THBD), lecithin-cholesterol acyltransferase (LCAT), matrix metallopeptidase 9 (MMP9), low density lipoprotein receptor (LDLR), and arachidonate 5-lipoxygenase-activating protein (ALOX5AP) genes associated with ATH. Twelve human coronary artery tissues (HCATs) were obtained from deceased subjects who underwent post-mortem procedures. Six atherosclerotic coronary artery tissue (ACAT) samples representing the cases and non-atherosclerotic coronary artery tissue (NCAT) samples as controls were gathered based on predetermined inclusion and exclusion criteria. Gene expression levels were assessed using the GenomeLab Genetic Analysis System (GeXP). The results showed that LDLR, TP53, and MMP9 expression levels were significantly increased in ACAT compared to NCAT samples (p < 0.05). Thus, LDLR, TP53, and MMP9 genes may play important roles in the development of ATH in a Malaysian study population.
Obtaining high-quality nucleic acid extracted from seaweeds is notoriously difficult due to contamination with polysaccharides and polyphenolic compounds after cell disruption. Specific methods need to be employed for RNA isolation in different seaweed species, and therefore studies of the thiamine biosynthesis pathway have been limited. Two selected Malaysian species which are highly abundant and underutilized, namely Gracilaria sp. and Padina sp., representing the red and brown seaweeds, respectively, were collected to develop optimized total RNA extraction methods. Prior to that, DNA was extracted, and amplification of the 18S rRNA gene and the THIC gene (encoding the first enzyme in the pyrimidine branch of the thiamine biosynthesis pathway) from the DNA template was successful in Gracilaria sp. only. RNA was then extracted from both seaweeds using three different existing methods, with some modifications, using cetyltrimethylammonium bromide, guanidine thiocyanate and sodium dodecyl sulphate. Methods I and III proved to be efficient for Padina sp. and Gracilaria sp., respectively, for the extraction of highly purified RNA, with A260/A280 values of 2.0 and 1.8. However, amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and the THIC gene was successful in only Gracilaria sp. cDNA derived from extracted RNA. Further modifications are required to improve the exploitation of nucleic acids from brown seaweeds, which has been proven to be difficult. This work should pave the way for molecular studies of seaweeds generally and for the elucidation, specifically, of the thiamine biosynthesis pathway.