We previously identified an expressed sequence tag clone, Der f 22, showing 41% amino acid identity to published Der f 2, and show that both genes are possible paralogues. The objective of this study was to characterize the genomic, proteomic and immunological functions Der f 22 and Der f 2. The full-length sequence of Der f 2 and Der f 22 coded for mature proteins of 129 and 135 amino acids respectively, both containing 6 cysteine residues. Phylogenetic analysis of known group 2 allergens and their homologues from our expressed sequence tag library showed that Der f 22 is a paralogue of Der f 2. Both Der f 2 and Der f 22 were single gene products with one intron. Both allergens showed specific IgE-binding to over 40% of the atopic patients, with limited of cross-reactivity. Both allergens were detected at the gut region of D. farinae by immunostaining. Der f 22 is an important allergen with significant IgE reactivity among the atopic population, and should be considered in the diagnostic panel and evaluated as future hypoallergen vaccine therapeutic target.
As the largest genus in Actinobacteria family, Streptomyces species have the ability to synthesize numerous compounds of diverse structures with bioactivities. Streptomyces mangrovisoli MUSC 149T was previously isolated as a novel streptomycete from mangrove forest in east coast of Peninsular Malaysia. The high quality draft genome of MUSC 149T comprises 9,165,825bp with G+C content of 72.5%. Through bioinformatics analysis, 21 gene clusters identified in the genome were associated with the production of bioactive secondary metabolites. The presence of these biosynthetic gene clusters in MUSC 149T suggests the potential exploitation of the strain for production of medically important compounds.
Vitellibacter aquimaris D-24T (=KCTC 42708T=DSM 101732T), a halophilic marine bacterium, was isolated from seawater collected from Desaru beach, Malaysia. Here, we present the draft genome sequence of D-24T with a genome size of approximately 3.1Mbp and G+C content of 39.93%. The genome of D-24T contains genes involved in reducing a potent greenhouse gas (N2O) in the environment and the degradation of proteinaceous compounds. Genome availability will provide insights into potential biotechnological and environmental applications of this bacterium.
Enterovirus A71 (EV-A71) is a neurotropic enterovirus that uses heparan sulfate as an attachment receptor. The molecular determinants of EV-A71-heparan sulfate interaction are unknown. With In silico heparin docking and mutagenesis of all possible lysine residues in VP1, we identified that K162, K242 and K244 are responsible for heparin interaction and inhibition. EV-A71 mutants with K242A and K244A rapidly acquired compensatory mutations, T100K or E98A, and Q145R-T237N respectively, which restored the heparin-binding phenotype. Both VP1-98 and VP1-145 modulates heparin binding. Heparin-binding phenotype was completely abolished with VP1-E98-E145, but was restored by an E98K or E145Q substitution. During cell culture adaptation, EV-A71 rapidly acquired K98 or Q/G145 to restore the heparin-binding phenotype. Together with next-generation sequencing analysis, our results implied that EV-A71 has high genetic plasticity by modulating positively-charged residues at the five-fold axis during in vitro heparin adaptation. Our finding has impact on EV-A71 vaccine production, evolutionary studies and pathogenesis.
Transcriptome analyses based on high-throughput RNA sequencing (RNA-Seq) provide powerful and quantitative characterization of cell types and in-depth understanding of biological systems in health and disease. In this study, we present a comprehensive transcriptome profile of human primary monocytes, a crucial component of the innate immune system. We performed deep RNA-Seq of monocytes from six healthy subjects and integrated our data with 10 other publicly available RNA-Seq datasets of human monocytes. A total of 1.9 billion reads were generated, which allowed us to capture most of the genes transcribed in human monocytes, including 11,994 protein-coding genes, 5558 noncoding genes (including long noncoding RNAs, precursor miRNAs, and others), 2819 pseudogenes, and 7034 putative novel transcripts. In addition, we profiled the expression pattern of 1155 transcription factors (TFs) in human monocytes, which are the main molecules in controlling the gene transcription. An interaction network was constructed among the top expressed TFs and their targeted genes, which revealed the potential key regulatory genes in biological function of human monocytes. The gene catalog of human primary monocytes provided in this study offers significant promise and future potential clinical applications in the fields of precision medicine, systems diagnostics, immunogenomics, and the development of innovative biomarkers and therapeutic monitoring strategies.
Thelohanellus kitauei is a freshwater myxosporean parasite causing intestinal giant cystic disease of common carp. To clarify the life cycle of T. kitauei, we investigated the oligochaete populations in China and Hungary. This study confirms two distinct aurantiactinomyxon morphotypes (Aurantiactinomyxon type 1 and Aurantiactinomyxon type 2) from Branchiura sowerbyi as developmental stages of the life cycle of T. kitauei. The morphological characteristics and DNA sequences of these two types are described here. Based on 18S rDNA sequence analysis, Aurantiactinomyxon type 1 (2048 bp) and Aurantiactinomyxon type 2 (2031 bp) share 99.2-99.4 %, 99.8-100 % similarity to the published sequences of T. kitauei, respectively. The 18S rDNA sequences of these two aurantiactinomyxon morphotypes share 99.4 % similarity, suggesting intraspecific variation within the taxon, possibly due to geographic origin. Phylogenetic analyses demonstrate the two aurantiactinomyxon types clustered with T. kitauei. Regardless, based on 18S rDNA synonymy, it is likely that Aurantiactinomyxon type 1 and 2 are conspecific with T. kitauei. This is the fourth elucidated two-host life cycle of Thelohanellus species and the first record of T. kitauei in Europe.
The earliest settlers in Peninsular Malaysia are the Orang Asli population, namely Semang, Senoi and Proto Malays. In the present study, we typed the HLA-A, -B and -DRB1 loci of the Kensiu and Semai Orang Asli sub-groups. Sequence-based HLA typing was performed on 59 individuals from two Orang Asli sub-groups. A total of 11, 18 and 14 HLA-A, -B and -DRB1 alleles were identified, respectively. These data are available in the Allele Frequencies Net Database under the population name "Malaysia Kedah Kensiu" and "Malaysia Pahang Semai".
We present the results of a laboratory-based surveillance of dengue in Taiwan in 2014. A total of 240 imported dengue cases were identified. The patients had arrived from 16 countries, and Malaysia, Indonesia, the Philippines, and China were the most frequent importing countries. Phylogenetic analyses showed that genotype I of dengue virus type 1 (DENV-1) and the cosmopolitan genotype of DENV-2 were the predominant DENV strains circulating in southeast Asia. The 2014 dengue epidemic was the largest ever to occur in Taiwan since World War II, and there were 15,492 laboratory-confirmed indigenous dengue cases. Phylogenetic analysis showed that the explosive dengue epidemic in southern Taiwan was caused by a DENV-1 strain of genotype I imported from Indonesia. There were several possible causes of this outbreak, including delayed notification of the outbreak, limited staff and resources for control measures, abnormal weather conditions, and a serious gas pipeline explosion in the dengue hot spot areas in Kaohsiung City. However, the results of this surveillance indicated that both active and passive surveillance systems should be strengthened so appropriate public health measures can be taken promptly to prevent large-scale dengue outbreaks.
As a health-beneficial fruit, litchi is widely accepted by people in subtropical and tropical regions. However, the critical chemicals responsible for the health benefits are not clear yet. As a large amount of polysaccharides are present in litchi, they might play an important role in the health benefits. In this work, the main water-soluble polysaccharide (LPPBa) was purified from litchi pulp. The chemical structure was characterized as arabinogalactan by gas chromatography and nuclear magnetic resonance spectrometry (NMR). NMR data revealed the glycosidic linkages and their locations in backbone and branches. The precise structure was putatively identified as below, and it was different to those commonly occurred arabinogalactans. The molecular weight was determined to be 2.4 × 10(6)Da by gel permeation chromatography.
The taxonomic position of 26 filamentous actinobacteria isolated from a hyper-arid Atacama Desert soil and 2 from an arid Australian composite soil was established using a polyphasic approach. All of the isolates gave the diagnostic amplification product using 16S rRNA oligonucleotide primers specific for the genus Amycolatopsis. Representative isolates had chemotaxonomic and morphological properties typical of members of the genus Amycolatopsis. 16S rRNA gene analyses showed that all of the isolates belong to the Amycolatopsis methanolica 16S rRNA gene clade. The Atacama Desert isolates were assigned to one or other of two recognised species, namely Amycolatopsis ruanii and Amycolatopsis thermalba, based on 16S rRNA gene sequence, DNA:DNA relatedness and phenotypic data; emended descriptions are given for these species. In contrast, the two strains from the arid Australian composite soil, isolates GY024(T) and GY142, formed a distinct branch at the periphery of the A. methanolica 16S rRNA phyletic line, a taxon that was supported by all of the tree-making algorithms and by a 100 % bootstrap value. These strains shared a high degree of DNA:DNA relatedness and have many phenotypic properties in common, some of which distinguished them from all of the constituent species classified in the A. methanolica 16S rRNA clade. Isolates GY024(T) and GY142 merit recognition as a new species within the A. methanolica group of thermophilic strains. The name proposed for the new species is Amycolatopsis deserti sp. nov.; the type strain is GY024(T) (=NCIMB 14972(T) = NRRL B-65266(T)).
Pandoraea thiooxydans DSM 25325(T) is a thiosulfate-oxidizing bacterium isolated from rhizosphere soils of a sesame plant. Here, we present the first complete genome of P. thiooxydans DSM 25325(T). Several genes involved in thiosulfate oxidation and biodegradation of aromatic compounds were identified.
Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.
The gene encoding for a novel cold-adapted enzyme from family II of bacterial classification (GDSL family) was cloned from the genomic DNA of Photobacterium sp. strain J15 in an Escherichia coli system, yielding a recombinant 36 kDa J15 GDSL esterase which was purified in two steps with a final yield and purification of 38.6 and 15.3 respectively. Characterization of the biochemical properties showed the J15 GDSL esterase had maximum activity at 20 °C and pH 8.0, was stable at 10 °C for 3 h and retained 50 % of its activity after a 6 h incubation at 10 °C. The enzyme was activated by Tween-20, -60 and Triton-X100 and inhibited by 1 mM Sodium dodecyl sulphate (SDS), while β-mercaptoethanol and Dithiothreitol (DTT) enhanced activity by 4.3 and 5.4 fold respectively. These results showed the J15 GDSL esterase was a novel cold-adapted enzyme from family II of lipolytic enzymes. A structural model constructed using autotransporter EstA from Pseudomonas aeruginosa as a template revealed the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine which was verified with site directed mutagenesis on active serine.
Antioxidant and α-amylase inhibitor peptides were successfully extracted from Pinto bean protein isolate (PBPI) using Protamex. A factorial design experiment was conducted and the effects of extraction time, pH and temperature were studied. pH 7.5, extraction time of 1h, S/E ratio of 10 (w/w) and temperature of 50 °C gave the highest antioxidant activities (i.e., ABTS scavenging activity (53.3%) and FRAP value (3.71 mM)), whereas pH 6.5 with the same extraction time, S/E ratio and temperature, gave the highest α-amylase inhibitory activity (57.5%). It was then fractioned using membrane ultrafiltration with molecular weight cutoffs of 100, 50, 30, 10 and 3 kDa. Peptide fraction <3 kDa, which exhibited the highest antioxidant activities (i.e., ABTS (42.2%) and FRAP (0.81 mM)) and α-amylase inhibitory activity (62.1%), was then subjected to LCMS and MS/MS analyses. Six sequences were identified for antioxidant peptides, whereas seven peptides for α-amylase inhibitor.
The mitochondrial genome sequence of the stone crab, Myomenippe fornasinii, second of the superfamily Eriphioidea is documented. Myomenippe fornasinii has a mitogenome of 15,658 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the M. fornasinii mitogenome is 36.10% for T, 18.52% for C, 35.48% for A, and 9.90% for G, with an AT bias of 71.58%. The mitogenome gene order conforms to what is the standard arrangement for brachyuran crabs.
The complete mitogenome of the ray Pastinachus atrus was recovered from a partial genome scan using the HiSeq sequencing system. The P. atrus mitogenome has 18,162 base pairs (61% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a 2516 bp non-coding AT-rich region. This mitogenome sequence is the first for a ray from Australian waters, the first for the Genus Pastinachus, and the 6th for the family Dasyatidae.
Samples of 100 random healthy unrelated Iraqi male persons from the Arab ethnic group of Iraqi population were collected for mtDNA coding region sequencing by using the Sanger technique and to establish the degree of variation characteristic of a fragment. Portion of coding region encompassing positions 11,719-12,184 was amplified in accordance with the Anderson reference sequence. PCR products were purified by EZ-10 spin column then sequenced and detected by using the ABI 3130xL DNA Analyzer. This is to intend the detection of polymorphisms of mtDNA. Four new polymorphic positions 11,741, 11,756, 11,878, and 12,133 are described which may be suitable in the future to be the sources for human identification purpose in Iraq. The obtained data can be used to identify variable nucleotide positions characterized by frequent occurrence most promising for identification variants. The calculated value GD = 0.95 and RMP = 0.048 of the genetic diversity should be understood as high in the context of coding function of the analysed DNA fragment. The relatively high gene diversity and a relatively low random match probability were observed in this study.
The complete mitochondrial genome of the moon crab Ashtoret lunaris was obtained from a partial genome scan using the MiSeq sequencing system. The Ashtoret lunaris mitogenome is 15,807 base pairs in length (70% A + T content) and made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a putative 956 bp non-coding AT-rich region. This A. lunaris mitogenome sequence is the first for the genus, as well as the family Matutidae and superfamily Calappoidea.
The commercial freshwater crayfish Cherax quadricarinatus complete mitochondrial genome was recovered from partial genome sequencing using the MiSeq Personal Sequencer. The mitogenome has 15,869 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The base composition of C. quadricarinatus is 32.16% for T, 23.39% for C, 33.26% for A, and 11.19% for G, with an AT bias of 65.42%.
Three new and one previously described species of Physalacria (Physalacriaceae, Agaricales) are reported from China. Specimens of two additional species described from Malaysia and North America were studied for comparison. Placements of these species were corroborated based on morphological observations and molecular evidence from partial sequences of the nuc rDNA internal transcribed spacer regions (ITS) and the 28S D1-D3 region, and genes for translation elongation factor 1-α (tef1α) and the second largest subunit of RNA polymerase II (rpb2). These new species of Physalacria distributed in subtropical China were found on rotten wood of broadleaf trees or bamboo and possess stipitate-capitate basidiomata with four-spored basidia, clamp connections and smooth, inamyloid basidiospores. To facilitate studies of the genus in Asia, a key is provided for all Physalacria species reported from this region.