VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.

The trapping problem of the weighted scale-free treelike networks for two kinds of biased walks.

It has been recently reported that trapping problem can characterize various dynamical processes taking place on complex networks. However, most works focused on the case of binary networks, and dynamical processes on weighted networks are poorly understood. In this paper, we study two kinds of biased walks including standard weight-dependent walk and mixed weight-dependent walk on the weighted scale-free treelike networks with a trap at the central node. Mixed weight-dependent walk including non-nearest neighbor jump appears in many real situations, but related studies are much less. By the construction of studied networks in this paper, we determine all the eigenvalues of the fundamental matrix for two kinds of biased walks and show that the largest eigenvalue has an identical dominant scaling as that of the average trapping time (ATT). Thus, we can obtain the leading scaling of ATT by a more convenient method and avoid the tedious calculation. The obtained results show that the weight factor has a significant effect on the ATT, and the smaller the value of the weight factor, the more efficient the trapping process is. Comparing the standard weight-dependent walk with mixed weight-dependent walk, although next-nearest-neighbor jumps have no main effect on the trapping process, they can modify the coefficient of the dominant term for the ATT.

Shear stress blunts tubuloglomerular feedback partially mediated by primary cilia and nitric oxide at the macula densa.

The present study tested whether primary cilia on macula densa serve as a flow sensor to enhance nitric oxide synthase 1 (NOS1) activity and inhibit tubuloglomerular feedback (TGF). Isolated perfused macula densa was loaded with calcein red and 4,5-diaminofluorescein diacetate to monitor cell volume and nitric oxide (NO) generation. An increase in tubular flow rate from 0 to 40 nl/min enhanced NO production by 40.0 ± 1.2%. The flow-induced NO generation was blocked by an inhibitor of NOS1 but not by inhibition of the Na/K/2Cl cotransporter or the removal of electrolytes from the perfusate. NO generation increased from 174.8 ± 21 to 276.1 ± 24 units/min in cultured MMDD1 cells when shear stress was increased from 0.5 to 5.0 dynes/cm(2). The shear stress-induced NO generation was abolished in MMDD1 cells in which the cilia were disrupted using a siRNA to ift88. Increasing the NaCl concentration of the tubular perfusate from 10 to 80 mM NaCl in the isolated perfused juxtaglomerular preparation reduced the diameter of the afferent arteriole by 3.8 ± 0.1 µm. This response was significantly blunted to 2.5 ± 0.2 µm when dextran was added to the perfusate to increase the viscosity and shear stress. Inhibition of NOS1 blocked the effect of dextran on TGF response. In vitro, the effects of raising perfusate viscosity with dextran on tubular hydraulic pressure were minimized by reducing the outflow resistance to avoid stretching of tubular cells. These results suggest that shear stress stimulates primary cilia on the macula densa to enhance NO generation and inhibit TGF responsiveness.

Conserved Residues Adjacent to ß-Barrel and Loop Intersection among Enterovirus VP1 Affect Viral Replication: Potential Target for Anti-Enteroviral Development.

Enterovirus genus has over one hundred genotypes and could cause several kinds of severe animal and human diseases. Understanding the role of conserved residues in the VP1 capsid protein among the enterovirus genus may lead to anti-enteroviral drug development. The highly conserved residues were found to be located at the loop and ß-barrel intersections. To elucidate the role of these VP1 residues among the enterovirus genus, alanine substitution reverse genetics (rg) variants were generated, and virus properties were investigated for their impact. Six highly conserved residues were identified as located near the inside of the canyon, and four of them were close to the ß-barrel and loop intersection. The variants rgVP1-R86A, rgVP1-P193A, rgVP1-G231A, and rgVP1-K256A were unable to be obtained, which may be due to disruption in the virus replication process. In contrast, rgVP1-E134A and rgVP1-P157A replicated well and rgVP1-P157A showed smaller plaque size, lower viral growth kinetics, and thermal instability at 39.5°C when compared to the rg wild type virus. These findings showed that the conserved residues located at the ß-barrel and loop junction play roles in modulating viral replication, which may provide a pivotal role for pan-enteroviral inhibitor candidate.

Auxin concentration and xylem production of Pinus massoniana in a subtropical forest in south China.

Auxin is involved in various developmental processes of plants, including cell division in cambium and xylem differentiation. However, most studies linking auxin and xylem cell production are performed in environments with a strong seasonality (i.e., temperate and boreal climates). The temporal dynamics of auxin and cambial activity of subtropical trees remain basically unknown. In this study, we sampled four microcores weekly in three individuals of Chinese red pine (Pinus massoniana Lamb.) from February to December 2015-16 to compare xylem formation with auxin concentration in subtropical China. During the entire period of sampling, the number of cambial cells varied from 2 to 7, while the number of cells in the enlarging zone ranged from 1 to 4 and from 1 to 5 in the wall-thickening zone. In 2015, the average auxin concentration was 3.46 ng g-1, with 33 xylem cells being produced at the end of the year. In 2016, a lower auxin concentration (2.59 ng g-1) corresponded to a reduced annual xylem production (13.7 cells). No significant relationship between auxin concentration and number of xylem cells in differentiation was found at the weekly scale. Unlike in boreal and temperate forests, the lack of wood formation seasonality in subtropical forests makes it more difficult to reveal the relationship between auxin concentration and number of xylem cells in differentiation at the intra-annual scale. The frequent and repeated samplings might have reduced auxin concentration in the developing cambium and xylem, resulting in a lower xylem cell production.

Ecosystem carbon storage in forest fragments of differing patch size.

Forest fragmentation threatens the ecosystem carbon (C) storage. The distribution patterns of ecosystem C density are poorly documented for fragmented forests of differing patch size. The objectives of this study were to examine C density in these forest ecosystems and the influence of edge effects on C density. Allometric equations were used to quantify aboveground biomass. Carbon density was estimated by analyzing the C concentration of each component. We found that ecosystem carbon density ranged from 173.9 Mg ha-1 in the small sized forest fragments, to 341.1 Mg ha-1 in the contiguous evergreen sub-tropical forest. Trees (46.5%) and mineral soil (50.2%) were the two largest contributors to the total ecosystem C pool in all fragments. Both C and nitrogen (N) in soil and fine roots were highly heterogeneous among the different fragment sizes and soil depths. We concluded that ecosystem C density of forest fragments were significantly influenced by patch size and edge effects. The fragmented forests in southern China play an important role in the C budget, and need urgent conservation. These results are likely to be further integrated into forest management plans and generalized into other contexts, to evaluate C stocks at the landscape scale.

Expression of androgen receptor in coronary artery in the cases of sudden coronary death.

To study the expression of androgen receptor (AR) in the cases of sudden death caused by coronary heart disease (CHD) and relationship between AR and sudden coronary death (SCD) to explore the mechanism of the development of coronary atherosclerosis and provide references for the prevent and treatment of CHD and medicolegal identification of SCD. 53 cases selected from the autopsied cases in our department from 2011 to 2012 were divided into 3 groups: 18 case of SCD, including 11 males and 7 females, as experimental group, another 18 cases, including 11 males and 7 females, with CHD but died of mechanical injuries and poisoning as control group I, and 17 cases without CHD who also died of mechanical injuries and poisoning including 10 males and 7 females, as control group II. After HE-stained and immunohistochemistry-stained (SP) for the slices, the expression of AR in coronary arteries were observed and the average optical density (AOD) of positive signal in each case were detected by using Image-Pro Plus 6.0 software. We found strongly positive expression of AR in control group II, weakly positive expression in control group I and a little weaker in the experimental group. The expression of AR in coronary artery was reduced in CHD patients, which suggested that androgen may have favorable effects on the cardiovascular system.

Single nucleotide polymorphisms of ERß and coronary atherosclerotic disease in Chinese Han women.

BACKGROUND: Growing evidence has shown that with the increase of age, the incidence of coronary atherosclerotic disease (CAD) in women increases to equal that of men. Several studies on the single nucleotide polymorphisms [SNPs] seem to provide evidence in support of the protective role estrogen receptor ß (ERß) has in reducing the risk of CAD. OBJECTIVE: To determine the association of ERß SNPs rs1256049 RsaI 1082 A > G and rs4986938 AluI 1730 G > A with coronary atherosclerotic disease in Chinese Han women. METHODS: We designed a nested case-control research, in which 120 case women and 30 control women were selected from the Forensic Medicine Department of Tongji Medical College, HUST. We isolated DNA from their lung paraffin blocks, and then screened for these two SNPs for each DNA sample. Post-statistical analysis of their genotypes and haplotypes was used to figure out the targeted association. RESULTS: We found no significant difference between the genotypes or haplotypes of the two SNPs and the risk of CAD. However, the rs4986938 heterozygote AG variant was correlated with a significantly lower risk for CAD than did homozygote GG variant in the group of less than 40 years old. Haplotype AA of the two SNPs was correlated with a higher risk for CAD in the same group. CONCLUSION: The rs4986938 AluI 1730 G > A seems to be quite involved in the genetic basis of the disease and needs more attention in future studies. Meanwhile, this very association made between CAD and the mentioned SNP seems to be affected quite a bit by age.

[The enhancement effect of emulsion in flame atomic absorption spectrometry].

A enhancement method of FAAS with emulsion as enhancement agent has been developed. The enhancement effect of emulsion made of three organic solvents (benzene, benzene-propanone, xylene), one organic reagent (dibutyl phthalate) and three emulsifiers (Tween-80, Triton X-100, OP) for iron, nickel, zinc, manganese and lead was studied. The results indicated that the enhancement is satisfactory. The emulsion with maximum enhancement percentage are respectively emulsion of benzene-OP-dibutyl phthalate with 89%, emulsion of xylene-Trition-100-dibutyl phthal with 34%, emulsion of benzene-Trition-100 with 121%, emulsion of benzene-Trition-100-dibutyl phthalate with 38% and 69% in sequence of the above elements.