The ha72 Core Gene of Baculovirus Is Essential for Budded Virus Production and Occlusion-Derived Virus Embedding,and Amino Acid K22 Plays an Important Role in Its Function

ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33.     

Chemical speciation distribution characteristics and ecological risk assessment of heavy metals in soil from Sunan mining area,Anhui Province,China

In this study, the content characteristics, comprehensive pollution assessment, and morphological distribution characteristics of heavy metals (Mn, Cd, Cr, Pb, Ni, Zn, and Cu) were researched based on the processes of field investigation, sample collection, and experimental analysis. Results showed that the mean concentrations of Mn, Pb, Cr, Cu, Cd, Zn, and Ni in surface soils were 522.77, 22.56, 55.10, 25.41, 0.25, 57.02, and 48.47 mg kg^?1, respectively. The surface soil from Sunan mining area was contaminated by Cu, Cd, and Ni in different degrees, and high CV values of Cd, Zn, Pb, and Ni were influenced by local human activities possibly. The evaluation results suggested that the mean I_geo values were in the sequence of Cd (0.657) > Ni (0.052) > Cu (?0.293) > Mn (?0.626) > Zn (?0.761) > Cr (?0.884) > Pb (?0.899). Besides, Cd was the most significant potential risk factor among all elements. Nevertheless, the Cd of bioavailable speciations with higher proportion had stronger migration and toxicity, and was more easier to be absorbed and enriched than other elements by some crops (e.g., vegetables, rice), and being at a relatively higher potential ecological risk in soil.     

Analysis of leaf tissue structures between rust-resistant and rust-susceptible Zoysia grass (<Emphasis Type="Italic">Zoysia japonica</Emphasis>)

The differences of leaf anatomic structures between rust-susceptible and -resistant zoysia (Zoysia japonica) were compared to reveal the physical defense mechanism of rust-resistant zoysia against Puccinia zoysiae. Zoysias were screened in greenhouse and fields of Beijing, Qingdao and Yangzhou from 2009 to 2012. After identified by inoculation test, rust-susceptible and rust-resistant zoysias were transplanted to flowerpots in Shangzhuang test field (Beijing, China) and were divided into indoor and outdoor groups. Leaves in the same position and formed in the same growth period of both rust-susceptible and rust-resistant zoysia strains were chosen for the current study. Wax content, the number and size of stomata, and the thickness of palisade tissue, spongy tissue, upper epidermis and lower epidermis were analyzed. All the anatomic structures of leaves stained with toluidine blue were observed under microscope. Leaf epidermis wax contents of rust-susceptible zoysia in indoor and outdoor groups were 30 and 39% lower than that of rust-resistant zoysia (p?<?0.05), respectively. The number of stomata in rust-resistant zoysia was 68% more than that in susceptible ones (p?<?0.05). Stomata of rust-resistant zoysia were 45% shorter and 65% narrower than those of rust-susceptible zoysia (p?<?0.05). The thickness of spongy parenchyma and palisade tissue in rust-resistant zoysia was significantly higher than that of the susceptible samples (p?<?0.05). Cell structure tightness and cell structure looseness of rust-resistant zoysia were tighter than that of rust-susceptible zoysia. Our study provides a histological understanding of the resistance mechanism of rust-resistant zoysia and might be useful for the identification of resistant varieties.     

DNA vaccine with discontinuous T‐cell epitope insertions into HSP65 scaffold as a potential means to improve immunogenicity of multi‐epitope Mycobacterium tuberculosis vaccine

DNA‐based vaccine is a promising candidate for immunization and induction of a T‐cell‐focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi‐functional T response against multi‐TB antigens, a multi‐epitope DNA vaccine and a ‘protein backbone grafting’ design method is adopted to graft five discontinuous T‐cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T‐cell epitopes derived from ESAT‐6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope‐scaffold construct was significantly more potent to induce IFN‐γ⁺ T response to five T‐cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi‐functional‐CD4⁺ T response. It also enhanced granzyme B⁺ CTL and IL‐2⁺ CD8⁺ T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi‐epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.     

Circulating memory B cells and plasmablasts are associated with the levels of serum immunoglobulin in patients with ulcerative colitis

Humoural immunity is crucial for the pathogenesis of ulcerative colitis (UC), but the precise perturbation of B cell immunity is poorly understood. This study is aimed at evaluating the numbers of different subsets of circulating memory B cells, plasmablasts, and the levels of serum immunoglobulin in UC patients. Total of 23 patients with active UC and 14 healthy controls (HC) were examined for the numbers of different subsets of circulating memory B cells and plasmablasts before and after treatment with mesalazine for 8–12 weeks by flow cytometry. Disease activity was evaluated by the Mayo clinic score. The levels of serum immunoglobulin, C‐reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured in individual subjects. In comparison with that in HC, significantly reduced numbers of IgG⁺ IgD^? CD27⁺ CD19⁺ memory B cells, increased numbers of CD20^? CD19⁺ plasmablast subsets, and higher serum IgG levels were detected in UC patients. The concentrations of serum IgG, the numbers of CD138⁺ CD38⁺ CD20^? CD19⁺, and IgG⁺ CD38⁺ CD20^? CD19⁺ plasmablasts were negatively associated with the numbers of IgG⁺ IgD^? CD27⁺ CD19⁺ memory B cells. Furthermore, the values of Mayo clinic score, CRP, or ESR in UC patients were negatively correlated with the numbers of IgG⁺ IgD^? CD27⁺ CD19⁺ memory B cells, while positively correlated with the serum IgG levels and the numbers of plasmablast subsets. Following treatment with mesalazine, the numbers of circulating IgG⁺ IgD^? CD27⁺ CD19⁺ memory B cells were significantly increased, while the numbers of CD138⁺ CD38⁺ CD20^? CD19⁺ and IgG⁺ CD38⁺ CD20^? CD19⁺ plasmablasts were reduced in UC patients. These decreased IgG⁺ IgD^? CD27⁺ CD19⁺ memory B cells and increased plasmablasts may be involved in the pathogenic process of UC.     

Autophagy is involved in ethanol-induced cardia bifida during chick cardiogenesis

Excess alcohol consumption during pregnancy has been acknowledged to increase the incidence of congenital disorders, especially the cardiovascular system. However, the mechanism involved in ethanol-induced cardiac malformation in prenatal fetus is still unknown. We demonstrated that ethanol exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Previously, we reported that autophagy was involved in heart tube formation. In this context, we demonstrated that ethanol exposure increased ATG7 and LC3 expression. mTOR was found to be inhibited by ethanol exposure. We activated autophagy using exogenous rapamycin (RAPA) and observed that it induced cardiac bifida and increased GATA5 expression. RAPA beads implantation experiments revealed that RAPA restricted ventricular myosin heavy chain (VMHC) expression. In vitro explant cultures of anterior primitive streak demonstrated that both ethanol and RAPA treatments could reduce cell differentiation and the spontaneous beating of cardiac precursor cells. In addition, the bead experiments showed that RAPA inhibited GATA5 expression during heart tube formation. Semiquantitative RT-PCR analysis indicated that BMP2 expression was increased while GATA4 expression was suppressed. In the embryos exposed to excess ethanol, BMP2, GATA4 and FGF8 expression was repressed. These genes are associated with cardiomyocyte differentiation, while heart tube fusion is associated with increased Wnt3a but reduced VEGF and Slit2 expression. Furthermore, the ethanol exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. In sum, our results revealed that disrupting autophagy and excess ROS generation are responsible for inducing abnormal cardiogenesis in ethanol-treated chick embryos.     

An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus

Japanese encephalitis virus(JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus–host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope(E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.     

Fiber Mediated Receptor Masking in Non-Infected Bystander Cells Restricts Adenovirus Cell Killing Effect but Promotes Adenovirus Host Co-Existence

The basic concept of conditionally replicating adenoviruses (CRAD) as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI), and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses.