The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure

AbstractWe have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five α helices and a β-hairpin. The dimer interface consists of a continuous four-stranded β-sheet superposed by two long α helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the β-sheet and the α helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology

Serologic and Molecular Biologic Methods for SARS-associated Coronavirus Infection, Taiwan

Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase–polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR . With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus–based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates

The localization and biochemical analysis of Arabidopsis thaliana APYRASE1 through 7

textNTPDases (Apyrases) (EC 3.6.1.5) require divalent cations (Mg2+, Ca2+) for hydrolysis of di- and triphosphate nucleotides, but do not hydrolyze monophosphate nucleotides. They are insensitive to inhibitors of F-type, P-type, and V-type ATPases and are categorized as E-type ATPases. They are grouped in the GDA_CD39 superfamily. Seven NTPDases (AtAPY1-7) have been cloned from Arabidopsis. In this work, AtAPY1 or AtAPY2 tagged with C-terminal green fluorescence protein (GFP) and driven by their respective native promoter displayed Golgi apparatus localization. These GFP constructs can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, which indicates their accuracy and functionality in localization studies. Furthermore, both AtAPY1 and AtAPY2 can complement the Saccharomyces cerevisiae Golgi-localized GDA1 mutant by rescuing its aberrant protein glycosylation phenotype. The GFP tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity in the mutants confirming that they both also have functional competency. Loss-of-function apy1, apy2 and APY1RNAi apy2 mutants showed higher levels of galactose in the cell wall monosaccharide analysis. However, the efficiency of the galactose transport was not altered APY1RNAi apy2 mutants. AtAPY3 through 7 all displayed intracellular localization by transiently expressed C-terminal tagged YFP in the onion epidermal cells. AtAPY3 showed a subcellular localization distinct from the others. Biochemical analyses showed that AtAPY3 prefers to hydrolyze NTP more than NDP. AtAPY4 resides in the cis-Golgi. It has fairly weak NTPDase activity but can still rescue some part of the phenotypic defects in Golgi luminal NTPDases mutants. AtAPY5 is a strong NDPase and has a broad spectrum of substrate preferences. It can fully restore phenotypic defects in Golgi luminal NTPDases in yeast. AtAPY6 and AtAPY7 are ER and Golgi associated. However, the expression of these two enzymes cannot be detected in the Saccharomyces cerevisiae host, which prevents further analysis. Taken together these results reveal that the current seven APYRASE members are intracellulary associated with Golgi/ER or unknown vesicles. They all display typical NTPDase enzyme activities that can hydrolyze di- or triphosphate nucleotides in the cells.Plant Biolog

Developmental biology of Exacum Styer Group pollen as related to haploid induction

Exacum Styer Group, a group of interspecific hybrids, possesses several valuable traits for ornamental use. However, current production techniques are limited to asexual reproduction of selected genotypes. Typically, inbred line development is highly desirable for commercial introduction of F1 hybrids as well as to facilitate genetic research. Unfortunately, this group suffers from severe inbreeding depression, precluding sexually derived inbred lines. In order to avoid inbreeding depression, double haploid plant development is one alternative approach. Therefore, this research developed fundamental information related to the successful application of this technology. Initially, fourteen genotypes were evaluated for pollen viability ranging from 0% to 80.5%. Three genotypes, with different levels of pollen viability, were selected and characterized for microsporogenesis, microgametogenesis, pollen morphology, and chromosome number. Microsporogenesis was normal among all three selected genotypes. However, the low fertility genotype and sterile genotype did not complete normal microgametogenesis and were found to have abnormal exine structure, perhaps indicating a dysfunctional tapetum layer. In contrast, the fertile genotype completed microgametogenesis, produced normal exine, and produced functional pollen grains. At anthesis, pollen was shed at the bi-nucleate stage. The chromosome numbers of the three genotypes evaluated ranged from 50 (fertile genotype) to 66 (low pollen viability). The individual chromosomes ranged in size from 0.3 μm (dot-shaped) to 2.65 μm (rod-shaped) with all three genotypes containing each form. Following characterization of pollen development, reprogramming treatments (e.g., temperature, mannitol, media composition and plant growth regulators) to induce haploid embryogenesis were applied at the mid uninucleate to early binucleate stage. After cold treatment (10°C) for 7 days, microspores maintained normal nuclei development without symmetrical divisions observed, suggesting that no androgenic switch occurred. Furthermore, heat treatment (35°C) for 4 days induced nuclei degradation and microspore non-viability. In addition to temperature treatments, mannitol treatments did not induce symmetrical divisions. When anthers were cultured under common temperature treatments, regardless of media composition, nuclei displayed a similar response indicating temperature was more effective in influencing nuclei development than media composition. Three different auxin and cytokinin combinations were evaluated on androgenic callilembryo induction. However, none of the combinations successfully induced calli/embryo formation.Land and Food Systems, Faculty ofGraduat

The Current Population Structure of the Demersal Hairtail (Trichiurus japonicus) in the Western North Pacific was Shaped by the Taiwan Strait, as Revealed by Mitochondrial DNA

We sought to evaluate the genetic variability of largehead hairtail (Trichiurus japonicus) in the western North Pacific for the purpose of determining natural management units applicable to the fisheries regulation and ecological conservation. To achieve this, we analyzed two homologous segments in each fish's mitochondrial DNA using the AMOVA statistical model. In total, 180 DNA samples were collected from the continental shelves of Japan, the Yellow Sea (YS), the East China Seas (ECS), the Taiwan Strait (TS), and the South China Sea (SCS). In general, the haplotype diversity in the Cyt-b gene and D-loop region was high (>0.92), while the nucleotide diversity was moderate (<0.011). Our AMOVA model showed statistically significant genetic difference between the SCS and pan-ECS (Japan+YS+ECS+TS) populations. These subpopulations likely diverged as a result of the last geological sea-level recession event that created an outcrop within the Taiwan Strait, closing the waterway and gene flows between the ECS and SCS populations. Currently, sedentary life, limited dispersal ability, and modern monsoon-influenced marine flow patterns are the major forces that maintain the separation of these two hairtail populations. Our results support the creation of two independent management units for Trichiurus japonicus in the East China Sea and the South China Sea to protect the diversity of this species

Argentine Shortfin Squid (Illex argentinus) Stock Assessment in the Southwest Atlantic Using Geostatistical Techniques

The spatial and temporal variation in Argentine shortfin squid Illex argentinus abundance distribution was examined over its fishing phase on the Patagonia Shelf and shelf break, Southwest Atlantic (SWA), using Taiwanese jigger’s fishery data. Geostatistical techniques were applied to characterize the spatial and temporal variability in the squid abundance and its relation to seawater temperature. The experimental semivariograms indicated that the abundance of Argentine shortfin squid was spatially structured in the SWA, with various abundance levels. The spherical models for all years, except 2010, explained most spatial information from the annual squid abundance distribution patterns. The linear regression analysis confirmed a negative relationship between the annual squid abundance and seawater temperature in the studied years. High squid abundance was estimated using Kriging interpolations along the 200-m isobath from 40°S to as far south as 50°S. The elliptical isopleth lines extended a longer distance from the 200-m isobath to the shelf side in high abundance years, such as 1999 and 2007. Scattered patches of low values were observed in the very low-abundance year of 2004. The integrated total squid biomass using Kriging interpolation upheld a healthy stock status in the SWA fishing ground. Our research showed that the geostatistical procedure is effective in describing the annual spatial pattern, and the parameters resulting from stationary modeling are valuable and useful in estimating the annual total biomass in the realized fishing ground

Significant enhancement of catalytic properties in mononuclear yttrium complexes by nitrophenolate-type ligands: Synthesis, structure, and catalysis for lactide polymerization

The syntheses, structures, and catalytic properties for lactones polymerization of eight novel yttrium complexes containing an amine-bis(benzotriazole phenolate) ( C1NNBiBTP) ligand are reported. A series of nitrophenolate (NP)-type of ligands possessing R substituents with variable electronic properties (R = NO2, Cl, H, CH3) on ortho and/or para position attached to the phenolate rings have been selected and further reacted with C1NNBiBTP-H2 proligand and YCl3·6H2O. Two series of complexes, [Y( C1NNBiBTP)(TNP)(MeOH)2] (3), [Y( C1NNBiBTP)(2,4-DNP)(MeOH)2] (4) and [Y( C1NNBiBTP)(2,5-DNP)(MeOH)2] (5) with two MeOH molecules as initiators as well as [Y( C1NNBiBTP-H)(CNP)2] (6), [Y( C1NNBiBTP-H)(2-NP)2] (7) and [Y( C1NNBiBTP-H)(MNP)2] (8) with two NP derivatives, were synthesized. Their ring-opening polymerizations of L-lactide (L-LA) were investigated for all complexes in order to further understand the correlations between the inductive effect of substitutions and catalytic properties. Particularly, the activity and controllability of yttrium complexes 3 and 5 were improved dramatically comparing with the literature complex with the similar coordination environment, [Y( C1NNBiBTP)(NO3)(MeOH)2], which can be a successful example to enhance the catalytic properties by exchanging coordinate molecules. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 2038–204

Application of Upper Extremity Radionuclide Venography as a Diagnostic Approach for Port-A Catheter Thrombosis

To investigate the role of upper extremity radionuclide venography as a potential diagnostic modality in the assessment of venous thrombosis associated with a Port-A catheter. Methods: Fourteen symptomatic patients who had received Port-A catheter implantation were enrolled. A dynamic nuclear medicine flow study was performed with intravenous administration of Technetium-99m macroaggregated albumin to both upper extremities. Imaging patterns of the venous system were categorized as patency, partial obstruction, and total occlusion. Results: The findings of the dynamic images clearly demonstrated clinical problems. Three patients were free of a definite venous flow change. Three patients had partial obstruction of venous return. A significant cut-off of venous return was demonstrated in 8 patients, and total occlusions were hence diagnosed. All patients underwent this procedure smoothly without any complication. Conclusion: These results suggest that upper extremity radionuclide venography is an easily performed and effective method for diagnosing Port-A catheter thrombosis in clinical practice