Substantial Attenuation of Virulence of Tembusu Virus Strain PS Is Determined by an Arginine at Residue 304 of the Envelope Protein.

The Tembusu virus (TMUV) PS strain, derived by several passages and plaque purifications in BHK-21 cells, displays markedly lower virulence in Pekin ducklings relative to a natural isolate of TMUV, but the potential virulence determinants and the in vivo mechanisms for substantial virulence attenuation of the passage variant remain unknown. Here, we constructed a series of chimeric and mutant viruses and assessed their virulence using a 2-day-old Pekin duckling model. We showed that residue 304 in the envelope (E) protein is the molecular determinant of TMUV virulence. Further investigations with mutant and parental viruses demonstrated that acquisition of positive charges at E protein residue 304 plays a critical role in substantial attenuation of neurovirulence and neuroinvasiveness, which is linked to enhanced binding affinity for glycosaminoglycans (GAGs). In Pekin ducklings infected by subcutaneous inoculation, an Arg at residue 304 in the E protein was shown to contribute to more rapid virus clearance from the circulation, markedly reduced viremia, and significantly decreased viral growth in the extraneural tissues and the central nervous system, relative to a Met at the corresponding residue. These findings suggest that the in vivo mechanism of virulence attenuation of the TMUV passage variant closely resembles that proposed previously for GAG-binding variants of other flaviviruses. Overall, our study provides insight into the molecular basis of TMUV virulence and the in vivo consequences of acquisition of a GAG-binding determinant at residue 304 in the E protein of TMUV.IMPORTANCE TMUV-related disease emerged in 2010 and has a significant economic impact on the duck industry. Although the disease was originally recognized to affect adult ducks, increasing evidence has shown that TMUV also causes severe disease of young ducklings. It is, therefore, essential to investigate the pathogenesis of TMUV infection in a young duckling model. The significance of our studies is in identifying E protein residue Arg304 as the molecular determinant for TMUV virulence and in clarifying the crucial role of positive charges at E protein residue 304 in virulence attenuation of a TMUV passage variant. These data will greatly enhance our understanding of the pathogenesis of TMUV infection in ducklings and have implications for development of a safe and efficient vaccine.

Evaluation of interactive effects between paternal alcohol consumption and paternal socioeconomic status and environmental exposures on congenital heart defects.

BACKGROUND: While the maternal risk factors on congenital heart defects (CHDs) have often been assessed, paternal contribution to CHDs, especially the joint effects of paternal risk factors on CHDs remain unknown. This study examined the major impacts of paternal alcohol consumption and its interaction (on multiplicative and additive scales) with paternal socioeconomic status (SES) and environmental exposures on CHDs in China. METHODS: A population-based case-control study involving 4,726 singleton CHDs cases and 4,726 controls (without any malformation and matched on hospital, gender, and gestational age) was conducted in Guangdong, China, 2004-2014. Information on parental demographics, behavioral patterns, disease/medication, and environmental exposures (3 months before pregnancy) was collected through face-to-face interviews. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) while controlling for all parental factors. RESULTS: Paternal alcohol consumption was associated with an increased OR of CHDs (adjusted OR = 2.87, 95% CI: 2.25-3.65). Additionally, paternal smoking, industry occupation, organic solvent contact, virus infection and antibiotic use, living in rural areas, low household income, and migrant status were significantly associated with CHDs (ORs ranged: 1.42-4.44). Significant additive or multiplicative interactions were observed between paternal alcohol consumption and paternal smoking, industrial occupation, and low income on any CHDs (interaction contrast ratio [ICR] = 4.72, 95% CI: 0.96-8.47] and septal defects (ICRs ranged from 2.04 to 2.79, p < .05). CONCLUSIONS: Paternal alcohol consumption and multiple paternal factors were significantly associated with CHDs in China. Paternal smoking and low SES factors modified paternal alcohol consumption-CHDs relationships. Further studies are needed to confirm these findings.

Identification of a Neutralizing Monoclonal Antibody That Recognizes a Unique Epitope on Domain III of the Envelope Protein of Tembusu Virus.

Domain III of the envelope protein (EDIII) is the major target of flavivirus neutralizing antibody. To date, little is known regarding antibody-mediated neutralization of Tembusu virus (TMUV), a novel flavivirus emerging in duck in 2010. Here, a novel monoclonal antibody (MAb), designated 12F11, was prepared by immunization of mice with recombinant EDIII (rEDIII) protein. Using virus neutralization test, 12F11 in undiluted ascites neutralized the TMUV infectivity to induce the development of cytopathic effects in BHK-21 cells, indicating that 12F11 exhibits a neutralizing activity. The neutralizing activity of 12F11 was confirmed by plaque reduction neutralization test, in which 12F11 reduced significantly the number of plaques produced by TMUV in BHK-21 cells. Western blot analyses of a series of truncated rEDIII proteins showed that the epitope recognized by 12F11 includes amino acids between residues 8 and 77 of EDIII protein. Function analysis demonstrated that 12F11 neutralizes TMUV infection at virus adsorption and at a step after adsorption to a certain extent. The present study provides an important step towards elucidating antibody-mediated neutralization of TMUV.

Prolonging and enhancing the protective efficacy of the EtMIC3-C-MAR against eimeria tenella through delivered by attenuated salmonella typhimurium.

The microneme adhesive repeats (MAR) of Eimeria tenella microneme protein 3 (EtMIC3) are associated with binding to and invasion of host cells. Adhesion and invasion-related proteins or domains are often strongly immunogenic, immune responses mounted against these factors that play a key role in blocking invasion. In the present study, an oral live vaccine consisting of attenuated Salmonella typhimurium X4550 carrying two MAR domains fragment (St-X4550-MAR) was constructed and its protective efficacies were evaluated. The results showed that St-X4550-MAR was more immunogenic and conferred a higher degree of protection than recombinant MAR polypeptide as reflected by increased body weight, decreased oocyst shedding and lesion scores, increased serum IgG and cecal sIgA antibody production, and increasing levels of interferon-γ and interleukin-10. Thus, MAR domains are highly immunogenic and St-X4550-MAR had moderate activity against E. tenella infection by stimulating humoral, mucosal and cellular immunity. Chickens immunized with our constructed live vaccine provided considerable protections as early as at 10 d post-immunization (ACI: 155.17), and maintained higher protection levels at 20 d post-immunization (ACI: 173.66), and at 30 d post-immunization (ACI: 162.4). While the protective efficacy of chickens immunized with the recombinant MAR peptides showed a decreased trend as the post immunization time prolonging. Thus, using live-attenuated S. typhimurium X4550 as a vaccine expression and delivery system can significantly improve the protective efficacy and duration of protective immunity of MAR of EtMIC3.

Short-term outcomes of extremely preterm infants at discharge: a multicenter study from Guangdong province during 2008-2017.

BACKGROUND: An increasing number of extremely preterm (EP) infants have survived worldwide. However, few data have been reported from China. This study was designed to investigate the short-term outcomes of EP infants at discharge in Guangdong province. METHODS: A total of 2051 EP infants discharged from 26 neonatal intensive care units during 2008-2017 were enrolled. The data from 2008 to 2012 were collected retrospectively, and from 2013 to 2017 were collected prospectively. Their hospitalization records were reviewed. RESULTS: During 2008-2017, the mean gestational age (GA) was 26.68 ± 1.00 weeks and the mean birth weight (BW) was 935 ± 179 g. The overall survival rate at discharge was 52.5%. There were 321 infants (15.7%) died despite active treatment, and 654 infants (31.9%) died after medical care withdrawal. The survival rates increased with advancing GA and BW (p < 0.001). The annual survival rate improved from 36.2% in 2008 to 59.3% in 2017 (p < 0.001). EP infants discharged from hospitals in Guangzhou and Shenzhen cities had a higher survival rate than in others (p < 0.001). The survival rate of EP infants discharged from general hospitals was lower than in specialist hospitals (p < 0.001). The major complications were neonatal respiratory distress syndrome, 88.0% (1804 of 2051), bronchopulmonary dysplasia, 32.3% (374 of 1158), retinopathy of prematurity (any grade), 45.1% (504 of 1117), necrotizing enterocolitis (any stage), 10.1% (160 of 1588), intraventricular hemorrhages (any grade), 37.4% (535 of 1431), and blood culture-positive nosocomial sepsis, 15.7% (250 of 1588). The multivariate logistic regression analysis indicated that improved survival of EP infants was associated with discharged from specialist hospitals, hospitals located in high-level economic development region, increasing gestational age, increasing birth weight, antenatal steroids use and a history of premature rupture of membranes. However, twins or multiple births, Apgar ≤7 at 5 min, cervical incompetence, and decision to withdraw care were associated with decreased survival. CONCLUSIONS: Our study revealed the short-term outcomes of EP infants at discharge in China. The overall survival rate was lower than the developed countries, and medical care withdrawal was a serious problem. Nonetheless, improvements in care and outcomes have been made annually.

Identification of Key Genes and Circular RNAs in Human Gastric Cancer.

BACKGROUND Globally, gastric cancer (GC) is the third most common source of cancer-associated mortality. The aim of this study was to identify key genes and circular RNAs (circRNAs) in GC diagnosis, prognosis, and therapy and to further explore the potential molecular mechanisms of GC. MATERIAL AND METHODS Differentially expressed genes (DEGs) and circRNAs (DE circRNAs) between GC tissues and adjacent non-tumor tissues were identified from 3 mRNA and 3 circRNA expression profiles. Functional analyses were performed, and protein-protein interaction (PPI) networks were constructed. The significant modules and key genes in the PPI networks were identified. Kaplan-Meier analysis was performed to evaluate the prognostic value of these key genes. Potential miRNA-binding sites of the DE circRNAs and target genes of these miRNAs were predicted and used to construct DE circRNA-miRNA-mRNA networks. RESULTS A total of 196 upregulated and 311 downregulated genes were identified in GC. The results of functional analysis showed that these DEGs were significantly enriched in a variety of functions and pathways, including extracellular matrix-related pathways. Ten hub genes (COL1A1, COL3A1, COL1A2, COL5A2, FN1, THBS1, COL5A1, SPARC, COL18A1, and COL11A1) were identified via PPI network analysis. Kaplan-Meier analysis revealed that 7 of these were associated with a poor overall survival in GC patients. Furthermore, we identified 2 DE circRNAs, hsa_circ_0000332 and hsa_circ_0021087. To reveal the potential molecular mechanisms of circRNAs in GC, DE circRNA-microRNA-mRNA networks were constructed. CONCLUSIONS Key candidate genes and circRNAs were identified, and novel PPI and circRNA-microRNA-mRNA networks in GC were constructed. These may provide useful information for the exploration of potential biomarkers and targets for the diagnosis, prognosis, and therapy of GC.

Identification of the Novel TMEM16A Inhibitor Dehydroandrographolide and Its Anticancer Activity on SW620 Cells.

TMEM16A, a calcium-activated chloride channel (CaCC), is highly amplified and expressed in human cancers and is involved in the growth and metastasis of some malignancies. Inhibition of TMEM16A represents a novel pharmaceutical approach for the treatment of cancers and metastases. The purpose of this study is to identify a new TMEM16A inhibitor, investigate the effects of this inhibitor on the proliferation and metastasis of TMEM16A-amplified SW620 cells, and to elucidate the underlying molecular mechanism in vitro. We identified a novel small-molecule TMEM16A inhibitor dehydroandrographolide (DP). By using patch clamp electrophysiology, we showed that DP inhibited TMEM16A chloride currents in Fisher rat thyroid (FRT) cells that were transfected stably with human TMEM16A and in TMEM16A-overexpressed SW620 cells but did not alter cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents. Further functional studies showed that DP suppressed the proliferation of SW620 cells in a dose- and time-dependent manner using MTT assays. Moreover, DP significantly inhibited migration and invasion of SW620 cells as detected by wound-healing and transwell assays. Further mechanistic study demonstrated that knockdown of human TMEM16A decreased the inhibitory effect of DP on the proliferation of SW620 cells and that TMEM16A-dependent cells (SW620 and HCT116) were more sensitive to DP than TMEM16A-independent cells (SW480 and HCT8). In addition, we found that treatment of SW620 cells with DP led to a decrease in TMEM16A protein levels but had no effect on TMEM16A mRNA levels. The current work reveals that DP, a novel TMEM16A inhibitor, exerts its anticancer activity on SW620 cells partly through a TMEM16A-dependent mechanism, which may introduce a new targeting approach for an antitumour therapy in TMEM16A-amplified cancers.

Evaluation of immune protective efficacies of Eimeria tenella EtMic1 polypeptides with different domain recombination displayed on yeast surface.

In the present study, three different live oral vaccines using the EBY100/pCTCON-2 yeast surface display system with different Eimeria tenella microneme-1 (EtMic1) protein domain recombination were constructed and their protective efficacies against homologous challenge were compared by evaluating the body weight gains, relative growth rate, cecal lesion scores, oocyst output, oocyst decrease ratio, anti-coccidial index, the serum antibody levels and the proliferation ability of blood lymphocytes. The results indicated that all the three constructed live oral vaccines expressing different EtMic1 polypeptides provided excellent protection against homologous challenge by significantly increasing weight gains, reducing cecal lesions, achieving a high ACI, elevating specific antibody response and splenocyte proliferation ability compared with controls. The yeasts displaying the EtMic1 polypeptide-III (expressed TSP-2, TSP-3 and TSP-4 domains) provided better protection against challenge than the yeasts displaying either the EtMic1 polypeptide-I (expressed I-domain, TSP-1 and TSP-2) or polypeptide-II (expressed I-domain and all the five TSP domains) did. Considering the exclusion of antibiotic resistant gene in the system, the strain EBY100 of Saccharomyces cerevisiae may be a better choice for coccidian antigen delivery.

Poly[[[µ-1,1'-(butane-1,4-di-yl)diimidazole-κN:N](µ-cyclo-hexane-1,4-dicarboxyl-ato-κO,O:O,O)nickel(II)] 0.25-hydrate].

In the title coordination polymer, {[Ni(C(8)H(10)O(4))(C(10)H(14)N(4))]·0.25H(2)O}(n), the coordination of the Ni(II) ion is distorted octa-hedral. The 1,1'-(butane-1,4-di-yl)diimidazole ligand and the cyclo-hexane-1,4-dicarboxyl-ate dianion bridge metal centres, forming a two-dimensional (4,4) network. The network is consolidated by O-H⋯O hydrogen bonds between the statistically occupied water molecules and O atoms of the two carboxylate groups.