Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

Background

Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.

Principal Finding

In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10⁶ the tissue culture''s infectious dose (TCID₅₀) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10⁻⁵ was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190.

Conclusion

These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.     

RNA-seq analysis reveals a key role of brassinolide-regulated pathways in NaCl-stressed cotton

Brassinolide (BL) alleviates salt injury in cotton seedlings; however, little is known about the molecular mechanisms of this response. In this study, digital gene expression analysis was performed to better understand the regulatory pathways of BL in NaCl-stressed cotton (Gossypium hirsutum L.). Compared with control plants (CK), a total of 1 162 and 7 659 differentially expressed genes (DEGs) were detected in the leaves and roots of NaCl-treated plants, respectively. Most of the DEGs in NaCl-treated plants, compared to CK, were regulated by BL. Moreover, expression patterns of DEGs in BL+NaCl treated plants were similar to those in CK plants; however, the responses of DEGs in the leaves and roots of NaCl-treated plants to BL differed. In the roots, BL-regulated DEGs were involved in protein biosynthesis, whereas in the leaves, BL promoted photosynthesis in NaCl-stressed cotton. BL treatment also significantly increased the overall biomass, chlorophyll a + b content in leaves, and the protein content in roots in NaCl-stressed cotton. The downregulation of stress-responsive genes in BL+NaCl-stressed leaves was also found. These results suggest that BL can alleviate NaCl injury in cotton plants.     

Abscisic acid-induced hydrogen peroxide is required for anthocyanin accumulation in leaves of rice seedlings

The role of hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced anthocyanin accumulation in detached and intact leaves of rice seedlings was investigated. Treatment with ABA resulted in an accumulation of anthocyanins in detached rice leaves. Dimethylthiourea, a chemical trap for H(2)O(2), was observed to be effective in inhibiting ABA-induced accumulation of anthocyanins. Inhibitors of NADPH oxidase (diphenyleneiodonium chloride and imidazole), phosphatidylinositol 3-kinase (wortmannin and LY 294002), and a donor of nitric oxide (N-tert-butyl-alpha-phenylnitrone), which have previously been shown to prevent ABA-induced H(2)O(2) accumulation in detached rice leaves, inhibited ABA-induced anthocyanin increase. Exogenous application of H(2)O(2), however, was found to increase the anthocyanin content of detached rice leaves. In terms of H(2)O(2) accumulation, intact (attached) leaves of rice seedlings of cultivar Taichung Native 1 (TN1) are ABA sensitive and those of cultivar Tainung 67 (TNG67) are ABA insensitive. Upon treatment with ABA, H(2)O(2) and anthocyanins accumulated in leaves of TN1 seedlings but not in leaves of TNG67. Our results, obtained from detached and intact leaves of rice seedlings, suggest that H(2)O(2) is involved in ABA-induced anthocyanin accumulation in this species.     

Cloning and characterization of a novel human RNA binding protein gene PNO1.

Present work reported the cloning and characterization of a human novel RNA binding gene Partner of NOB1 (PNO1), with a length of 1637bp and a putative open reading frame of 759 bp, isolated from human kidney. It is composed of seven exons and is localized on chromosome 2p14. Western blot showed that the molecular weight of PNO1 is about 35kDa. RT-PCR results in 16 human tissues indicated that PNO1 is expressed mainly in liver, lung, spleen and kidney, slightly in thymus, testis, ovary, respectively, but not in heart, brain, skeletal muscle, placenta, pancreas, prostate, small intestine, colon and peripheral blood leukocytes. GFP fusion expression in mammalian cells exhibited its localization in the nucleus, especially in nucleoli. Subcellular localization of thirteen GFP fusion PNO1 deletion proteins showed that the region of 92-230 aa is solely responsible for its nucleolar retention, and KH domain alone is not sufficient for nucleolar retention. The PNO1 family shows significant conservation in both eukaryotes and prokaryotes.     

Nitrogen and phosphorus addition differentially enhance seed production of dominant species in a temperate steppe

Previous studies have demonstrated changes in plant growth and reproduction in response to nutrient availability, but responses of plant growth and reproduction to multiple levels of nutrient enrichment remain unclear. In this study, a factorial field experiment was performed with manipulation of nitrogen (N) and phosphorus (P) availability to examine seed production of the dominant species, Stipa krylovii, in response to N and P addition in a temperate steppe. There were three levels of N and P addition in this experiment, including no N addition (0 g N m⁻² year⁻¹), low N addition (10 g N m⁻² year⁻¹), and high N addition (40 g N m⁻² year⁻¹) for N addition treatment, and no P addition (0 g P m⁻² year⁻¹), low P addition (5 g P m⁻² year⁻¹), and high P addition (10 g P m⁻² year⁻¹) for P addition treatment. Low N addition enhanced seed production by 814%, 1371%, and 1321% under ambient, low, and high P addition levels, respectively. High N addition increased seed production by 2136%, 3560%, and 3550% under ambient, low, and high P addition levels, respectively. However, P addition did not affect seed production in the absence of N addition, but enhanced it under N addition. N addition enhanced seed production mainly by increasing the tiller number and inflorescence abundance per plant, whereas P addition stimulated it by decreasing the plant density yet stimulating height of plants and their seed number per inflorescence. Our results indicate seed production is not limited by P availability but rather by N availability in the temperate steppe, whereas seed production will be increased by P addition when N availability is improved. These findings enable a better understanding of plant reproduction dynamics in the temperate steppe under intensified nutrient enrichment and can inform their improved management in the future.     

Pax3/7BP is a Pax7- and Pax3-binding protein that regulates the proliferation of muscle precursor cells by an epigenetic mechanism

In mouse skeletal muscles, Pax7 uniquely marks muscle satellite cells and plays some important yet unknown functions at the perinatal stage. To elucidate its in vivo functions, we initiated a yeast two-hybrid screening to look for Pax7-interacting proteins and identified a previously uncharacterized Pax7- and Pax3-binding protein (Pax3/7BP). Pax3/7BP is a ubiquitously expressed nuclear protein, enriched in Pax7+ muscle precursor cells (MPCs), and serves as an indispensable adaptor for Pax7 to recruit the histone 3 lysine 4 (H3K4) methyltransferase (HMT) complex by bridging Pax7 and Wdr5. Knockdown of Pax3/7BP abolished the Pax3/7-associated H3K4 HMT activity and inhibited the proliferation of Pax7+ MPCs from young mice both in culture and in vivo. Id3 and Cdc20 were direct target genes of Pax7 and Pax3/7BP involved in the proliferation of Pax7+ MPCs. Collectively, our work establishes Pax3/7BP as an essential adaptor linking Pax3/7 with the H3K4 HMT to regulate the proliferation of MPCs.     

Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T₉–T₁₀) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.     

Identification of a stable quantitative trait locus for percentage grains with white chalkiness in rice (Oryza sativa)

High chalkiness is a major problem in many rice-producing areas of the world, especially in hybrid rice (Oryza sativa L.) in China. We previously showed a major quantitative trait locus for the percentage of grains with white chalkiness (QTLqPGWC-8) in the interval G1149-R727 on chromosome 8 using a chromosome segment substitution line (CSSL). Here, we selected the line-CSSL50 harboring the QTLqPGWC-8 allele from the CSSLs derived from a cross between Asominori (as a recurrent parent) and IR24 (as a donor parent), which had higher percentage chalkiness, markedly different from that of Asominori. There were also significant differences in starch granules, appearance of amylose content (AAC) and milling qualities between Asominori and CSSL50, but not in grain size or thousand grain weight (TGW). The BC(4) F(2) and BC(4) F(3) populations from a cross between CSSL50 and Asominori were used for fine mapping of qPGWC-8. We narrowed down the location of this QTL to a 142 kb region between Indel markers 8G-7 and 8G-9. QTLqPGWC-8 accounted for 50.9% of the difference in PGWC between the parents. The markers tightly linked to qPGWC-8 should facilitate cloning of the gene underlying this QTL and will be of value for marker-assisted selection in breeding rice varieties with better grain quality.     

抗药性叶螨的种群参数和相对适合度

朱砂叶螨对氧化乐果、三氯杀螨醇、双甲脒和哒螨灵产生抗性后（抗药性系数分别为152．83倍、55．59倍、62．61倍和15．67倍）,繁殖力均显著降低,且发育加速。通过组建各品系生命表得知,该螨抗氧化乐果品系、抗三氯杀螨醇品质、抗双甲脒品系和抗哒螨灵品系的相对适合度分别为0．53、0．62、0．59和0．64,均小于1,具有明显的适合度缺陷。抗药性系数和相对适合度呈直线负相关。