Performance of commercially available anti-HDV enzyme-linked immunosorbent assays in Taiwan.

BACKGROUND: Hepatitis D virus (HDV) infection is a major global health issue around the world. There are approximately 15-20 million individuals infected with HDV worldwide. HDV infection usually causes increased mortality compared with infection with hepatitis B virus (HBV) alone. However, testing for the detection of HDV is not widely available in Taiwan. Therefore, the General Biologicals Corporation (GB) HDV Ab kit was developed for detecting anti-HDV antibodies. METHODS: A total of 913 serum and 462 EDTA-treated plasma samples were obtained from HBsAg-positive individuals in three hospitals in Taiwan from June 2014 to November 2017. We used three commercially available ELISA kits, DiaPro HDV Ab, DiaSorin ETI-AB-DELTAK-2 and GB HDV Ab, which were utilized strictly according to the instructions of the manufacturers. RESULTS: A comparative study of the results from the GB HDV Ab kit and the other commercial ELISA kits (DiaPro and DiaSorin) was performed to determine their efficacy for anti-HDV detection. The results indicated that the sensitivity of the GB HDV Ab kit for serum and EDTA samples was 100% compared to that of the DiaPro and DiaSorin kits, whereas the specificity for serum and EDTA samples was 99.3 and 98.1%, respectively. In addition, the overall agreement of the results of the GB HDV Ab kit for the serum and EDTA samples was 99.3 and 98.3%, respectively. It is worth noting that the performance of the GB HDV Ab kit was not affected by interference from triglyceride, bilirubin, hemoglobin, or human anti-mouse antibody. The limit of detection of the GB HDV Ab kit is approximately 100-fold lower than that of the other two commercial kits. CONCLUSIONS: The GB HDV Ab kit, which presented equivalent sensitivity and specificity compared to both certified anti-HDV kits, would be a suitable kit for HDV diagnosis in Taiwan.

Multiple promoters driving the expression of astaxanthin biosynthesis genes can enhance free-form astaxanthin production.

Astaxanthin possesses various biological properties and is used in the animal and fish feed, food, and beverage industries. In this study, we derived zeaxanthin biosynthesis genes (crtE, crtB, crtI, crtY, and crtZ) from Erwinia uredovora and crtW from Agrobacterium aurantiacum. We fused inducible and constitutive promoters to astaxanthin biosynthesis genes to construct a novel plasmid (dubbed PTP3-6) that can effectively enhance free-form astaxanthin (FFAX) production. The PTP3-6 plasmid contains one T7 promoter, driving IPTG inducible crtW expression, and three constitutive promoters (isolated from E. uredovora) driving expression of the other zeaxanthin biosynthesis genes. Escherichia coli BL21 (DE3) cells carrying the PTP3-6 plasmid produced 8.3 mg/g dry cell weight astaxanthin, which is 69.4-fold higher than has been previously reported. Using multiple promoter fusions of astaxanthin biosynthesis genes could be applied in other hosts to enhance astaxanthin production. FFAX was identified in recombinant E. coli cells through ultra-performance liquid chromatography-mass spectrometry.

Blue light decreases tanshinone IIA content in Salvia miltiorrhiza hairy roots via genes regulation.

The effect of light-emitting diodes (LEDs) on the production of secondary metabolites in medicinal plants and hairy roots is receiving much attention. The roots and rhizomes of the traditional Chinese medicinal plant Salvia miltiorrhiza Bunge are widely used for treating cardiovascular and cerebrovascular diseases. The main components are liposoluble tanshinones and hydrophilic phenolic acids. Moreover, hairy root culture of S. miltiorrhiza has been used in research of valuable plant-derived secondary metabolites. In this study, we examined the effect of LEDs with different combinations of wavelengths on the content of the main components in hairy roots of S. miltiorrhiza. Tanshinone IIA (TSIIA) content in hairy roots was significantly decreased with all light treatments containing blue light by >60% and was 9 times lower with LED treatment duration changed from 1 week to 3 weeks. HMGR, DXS2, DXR, GGPPS, CPS and CYP76AH1 genes involved in the tanshinone biosynthesis pathway were downregulated by blue light. Furthermore, light quality treatments have different effect on the accumulation of phenolic acids in hairy roots of S. miltiorrhiza. The light treatments 6R3B, 6B3IR, 7RGB and 2R6BUV for 3 weeks could increase rosmarinic acid (RA) content slightly but not salvianolic acid B (SAB) content. Different secondary metabolite contents could be regulated by different wavelength combinations of LEDs. Blue light could reduce TSIIA content in hairy roots of S. miltiorrhiza via gene regulation.

Chilling susceptibility in mungbean varieties is associated with their differentially expressed genes.

BACKGROUND: Mungbean (Vigna radiata L. Wilczek) is an economically important legume of high nutritional value, however, its cultivation is limited by susceptibility to chilling. Varieties NM94 and VC1973A, with differential susceptibility to stress, serve as good materials for uncovering how they differ in chilling tolerance. This study aimed to identify the ultrastructural, physiological and molecular changes to provide new insights on the differential susceptibility to chilling between varieties VC1973A and NM94. RESULTS: Chilling stress caused a greater reduction in relative growth rate, a more significant decrease in maximum photochemical efficiency of PSII and DPPH scavenging activity and more-pronounced ultrastructural changes in VC1973A than in NM94 seedlings. Comparative analyses of transcriptional profiles in NM94 and VC1973A revealed that the higher expression of chilling regulated genes (CORs) in NM94. The transcript levels of lipid transfer protein (LTP), dehydrin (DHN) and plant defensin (PDF) in NM94 seedlings after 72 h at 4 °C was higher than that in its parental lines VC1973A, 6601 and VC2768A. CONCLUSIONS: Our results suggested that LTP, DHN and PDF may mediate chilling tolerance in NM94 seedlings.

Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene.

Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives.

Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

Genomic and transcriptomic comparison of nucleotide variations for insights into bruchid resistance of mungbean (Vigna radiata [L.] R. Wilczek).

BACKGROUND: Mungbean (Vigna radiata [L.] R. Wilczek) is an important legume crop with high nutritional value in South and Southeast Asia. The crop plant is susceptible to a storage pest caused by bruchids (Callosobruchus spp.). Some wild and cultivated mungbean accessions show resistance to bruchids. Genomic and transcriptomic comparison of bruchid-resistant and -susceptible mungbean could reveal bruchid-resistant genes (Br) for this pest and give insights into the bruchid resistance of mungbean. RESULTS: Flow cytometry showed that the genome size varied by 61 Mb (mega base pairs) among the tested mungbean accessions. Next generation sequencing followed by de novo assembly of the genome of the bruchid-resistant recombinant inbred line 59 (RIL59) revealed more than 42,000 genes. Transcriptomic comparison of bruchid-resistant and -susceptible parental lines and their offspring identified 91 differentially expressed genes (DEGs) classified into 17 major and 74 minor bruchid-resistance-associated genes. We found 408 nucleotide variations (NVs) between bruchid-resistant and -susceptible lines in regions spanning 2 kb (kilo base pairs) of the promoters of 68 DEGs. Furthermore, 282 NVs were identified on exons of 148 sequence-changed-protein genes (SCPs). DEGs and SCPs comprised genes involved in resistant-related, transposable elements (TEs) and conserved metabolic pathways. A large number of these genes were mapped to a region on chromosome 5. Molecular markers designed for variants of putative bruchid-resistance-associated genes were highly diagnostic for the bruchid-resistant genotype. CONCLUSIONS: In addition to identifying bruchid-resistance-associated genes, we found that conserved metabolism and TEs may be modifier factors for bruchid resistance of mungbean. The genome sequence of a bruchid-resistant inbred line, candidate genes and sequence variations in promoter regions and exons putatively conditioning resistance as well as markers detecting these variants could be used for development of bruchid-resistant mungbean varieties.

A Novel Recombinant Chlorophyllase1 from Chlamydomonas reinhardtii for the Production of Chlorophyllide Derivatives.

Natural chlorophyll metabolites have exhibited physiological activity in vitro. In this study, a recombinant chlorophyllase1 gene from Chlamydomonas reinhardtii (CrCLH1) was isolated and characterized. Recombinant CrCLH1 can perform chlorophyll dephytylation and produce chlorophyllide and phytol. In a transient assay, the subcellular localization of CrCLH1-green fluorescent protein was determined to be outside the chloroplast. Biochemical analyses of the activity of recombinant CrCLH1 indicated that its optimal pH value and temperature are 6.0 and 40 °C, respectively. Enzyme kinetic data revealed that the recombinant CrCLH1 had a higher catalytic efficiency for chlorophyll a than for chlorophyll b and bacteriochlorophyll a. According to high-performance liquid chromatography analysis of chlorophyll hydrolysis, recombinant CrCLH1 catalyzed the conversion of chlorophyll a to pheophorbide a at pH 5. Therefore, recombinant CrCLH1 can be used as a biocatalyst to produce chlorophyllide derivatives.

Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymnosperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

Purification and immobilization of the recombinant Brassica oleracea Chlorophyllase 1 (BoCLH1) on DIAION®CR11 as potential biocatalyst for the production of chlorophyllide and phytol.

Recombinant Brassica oleracea chlorophyllase 1 (BoCLH1) with a protein molecular weight of 38.63 kDa was successfully expressed in E. coli and could catalyze chlorophyll (Chl) hydrolysis to chlorophyllide and phytol in vitro. In this study, we used DIAION®CR11, a highly porous cross-linked polystyrene divinylbenzene-based metal chelator, for purifying and immobilizing the poly (His)-tagged enzyme. The Cu(II) showed the highest protein adsorption (9.2 ± 0.43 mg/g gel) and enzyme activity (46.3 ± 3.14 U/g gel) for the immobilization of the poly (His)-tagged recombinant BoCLH1 compared with other metal chelators. Biochemical analysis of the immobilized enzyme showed higher chlorophyllase activity for Chl a hydrolysis in a weak base environment (pH 8.0), and activity above 70% was in a high-temperature environment, compared with the free enzyme. In addition, compared with free BoCLH1, the enzyme half-life (t1/2) of the immobilized BoCLH1 increased from 25.42 to 54.35 min (approximately two-fold) at 60 °C. The immobilized enzyme retained a residual activity of approximately 60% after 17 cycles in a repeated-batch operation. Therefore, DIAION®CR11Cu(II)-immobilized recombinant BoCLH1 can be repeatedly used to lower the cost and is potentially useful for the industrial production of chlorophyllide and phytol.

Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana.

The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1) encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+)-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC)/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+)-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

Arabidopsis ENDO2: its catalytic role and requirement of N-glycosylation for function.

The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.

Characterization of a novel Y2K-type dehydrin VrDhn1 from Vigna radiata.

A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y(2)K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. The VrDhn1 mRNA level was extremely high in mature seeds and decreased to ∼30% at 1 DAI, and was not detectable at ~7 DAI. Tissue dehydration, salinity and exogenous ABA markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity for non-specific interaction with DNA using electrophoretic mobility shift assays (EMSAs), and the exogenous addition of Zn(2+) or Ni(2+) stimulated interaction. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y(2)K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.

Functional characterization of three ethylene response factor genes from Bupleurum kaoi indicates that BkERFs mediate resistance to Botrytis cinerea.

Three novel ethylene response factor (ERF) genes, BkERF1, BkERF2.1 and BkERF2.2, were isolated from a medicinal plant, Bupleurum kaoi. The deduced BkERFs contain a canonical nuclear localization signal and an ERF/AP2 DNA binding domain. RNA gel blot analysis revealed that BkERF1 and BkERF2.1 were ubiquitously expressed at low levels in all parts of mature plants, and that BkERF2.2 was expressed at moderate levels in vegetative tissues. Exogenous application of methyl jasmonate induced BkERF1/2.1/2.2 transcripts. BkERF2.2 transcript levels were slightly increased by addition of ethephon and salicylic acid. BkERFs were localized in the plant nucleus and functioned as transcriptional activators. In B. kaoi cells overexpressing BKERFs, inoculation with Botrytis cinerea increased expression of some defense genes which are associated with enhanced disease resistance. Similarly, overexpression of BkERFs in transgenic Arabidopsis thaliana resulted in elevated mRNA levels of the defense gene PDF1.2, and in enhanced resistance to B. cinerea. Collectively, these results provide evidence that BkERFs mediate the expression of defense-related genes in plants.