[Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway].

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.

Disinfection effect of hexadecyl pyridinium chloride on SARS-CoV-2 in vitro.

The novel coronavirus (COVID-19 or 2019-nCoV) is a respiratory virus that can exist in the mouth and saliva of patients and spreads through aerosol dispersion. Therefore, stomatological hospitals and departments have become high-infection-risk environments. Accordingly, oral disinfectants that can effectively inactivate the virus have become a highly active area of research. Hexadecyl pyridinium chloride, povidone-iodine, and other common oral disinfectants are the natural primary choices for stomatological hospitals. Therefore, this study investigated the inhibitory effect of hexadecyl pyridinium chloride on SARS-CoV-2 in vitro. Vero cells infected with SARS-CoV-2 were used to determine the disinfection effect; the CCK-8 method was used to determine cytotoxicity, and viral load was determined by real-time PCR. The results showed that hexadecyl pyridinium chloride has no obvious cytotoxic effect on Vero cells in the concentration range 0.0125-0.05 mg/mL. The in vitro experiments showed that hexadecyl pyridinium chloride significantly inhibits the virus at concentrations of 0.1 mg/mL or above at 2 min of action. Thus, the results provide experimental support for the use of hexadecyl pyridinium chloride in stomatological hospitals.

[Mechanism of Shaofu Zhuyu Decoction in treatment of endometriosis-associated dysmenorrhea with syndrome of cold coagulation and blood stasis based on MSK1/2].

This study aims to decipher the mechanism underlying the effect of Shaofu Zhuyu Decoction on endometriosis(EMT)-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis based on mitogen-and stress-activated protein kinase 1/2(MSK1/2).We employed a random number table to randomly assign SPF female non-pregnant rats into the sham group, and treated the rest rats with autologous transplantation+refrigerator freezing for the modeling of the syndrome of cold coagulation and blood stasis.The modeled rats were then randomly assigned into the control group and high-, medium-and low-dose Shaofu Zhuyu Decoction groups.The rats in the low-, medium-, and high-dose decoction groups were respectively administrated with 9, 4.5, and 2.3 g·kg~(-1) decoction through gavage once a day for 2 consecutive weeks, and those in the control group were administrated with 0.24 mg·kg~(-1) gestrinone through gavage once every 3 days for 2 weeks.After that, the size of ectopic focus in each rat was measured via laparotomy.Enzyme-linked immunosorbent assay(ELISA) was adopted to determine the expression of interleukin(IL)-6, IL-10, prostaglandin E2(PGE2), tumor necrosis factor-α(TNF-α).Western blot was employed to determine the protein levels of MSK1/2 and dual-specificity phosphatase 1(DUSP1) and real-time quantitative polymerase chain reaction(RT-PCR) to determine the mRNA levels of the two genes in rat eutopic endometrial tissue.Compared with the sham group, the model group showed increased levels of IL-6, PGE2, and TNF-α while decrease level of IL-10 in the serum(P<0.01).Compared with the model group, the high-and medium-dose decoction groups and the gestrinone group had declined levels of IL-6, PGE2, and TNF-α while risen level of IL-10 in the serum(P<0.01).The model group had lower protein levels and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the sham group(P<0.01). The high-and medium-dose decoction groups and the gestrinone group had higher protein and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the model group(P<0.01).The results indicated that Shaofu Zhuyu Decoction can regulate the abnormal expression of pro-inflammatory cytokines TNF-α, IL-6, and PGE2 and anti-inflammatory cytokines IL-10 and DUSP1 via MSK1/2 to alleviate EMT-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis.

[Mechanism of Shaofu Zhuyu Decoction in treatment of EMT induced dysmenorrhea based on network pharmacology and molecular docking].

Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM) were searched for the effective components and targets of Shaofu Zhuyu Decoction. The relevant targets for endometriosis(EMT) and dysmenorrhea were retrieved from the Comparative Toxicogenomics Database(CTD), Therapeutic Target Database(TTD), GeneCards, and DisGeNET with the terms of "endometriosis" and "dysmenorrhea". Cytoscape 3.8.0 was employed to construct the drug-active component-therapeutic target network. A protein-protein interaction(PPI) network was established by STRING 11.0. Analyze Network, the plug-in in the Cytoscape 3.8.0, was used to calculate the topological parameters of the nodes and screen out the critical proteins in the network. The potential therapeutic targets were imported into RStudio and subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses with clusterProfiler package. Finally, the AutoDock Vina(Vina) platform was used for molecular docking to predict the binding degree of the main active components of Shaofu Zhuyu Decoction to key targets. As revealed by the screening results, 136 active components and 380 targets of Shaofu Zhuyu Decoction were obtained. Additionally, there were 1 627 targets related to EMT and 142 targets related to dysmenorrhea with 107 common targets, and 42 potential therapeutic targets of Shaofu Zhuyu Decoction for the treatment of EMT-induced dysmenorrhea. The targets such as interleukin 6(IL6) and prostaglandi-nendoperoxide synthase-2(PTGS2) were pivotal in the biological network of Shaofu Zhuyu Decoction intervention in EMT-induced dysmenorrhea, which involved multiple signaling pathways, including inflammation, hormones, and those promoted cell proliferation [e.g., mitogen-activated protein kinase(MAPK) and phosphatidylinositol-3 kinase(PI3 K)-protein kinase B(AKT)]. The results of molecular docking showed that the active components of Shaofu Zhuyu Decoction had good binding capacities to key targets such as IL6 and PTGS2. The findings of this study demonstrated that Shaofu Zhuyu Decoction could treat EMT-induced dysmenorrhea through multiple targets and multiple pathways, which could provide new ideas for investigating the underlying mechanism of Shaofu Zhuyu Decoction in the treatment of EMT-induced dysmenorrhea.

[Life history traits of an Echinochloa crus-galli var. crus-galli population with extreme adaptations to rice planting systems]. / 特异性适应稻作系统的稗草种群生活史特性.

Long-term herbicide application may facilitate the adaptive evolution of weed populations. With Echinochloa crus-galli var. crus-galli population A from a rice field used for the experiment of effectiveness of herbicide in Nanling County, Anhui Province, we conducted common garden experi-ments with seeds of population A and three control populations collected from normal rice fields. Compared with the three control populations, population A had significantly lower seed production for individual plant, but higher 1000-seed weight. Population A had faster in seedling growth, higher in number of reproductive tillers, shorter life span, lower in height and biomass of individual plant, as well as lower in sensitivity to herbicide penoxsulam. Individuals from population A survived from 2× label dose (60 g·hm-2) of penoxsulam treatment at the three- to four-leaf stage showed significantly reduction in plant height, biomass, and mature seed production (1066 seeds per plant), but no difference in heading period, number of reproductive tillers, number of seeds per raceme and 1000-seed weight. The short lifespan, heavy seeds, dwarf architecture, more reproductive tillers and penoxsulam resistance made E. crus-galli var. crus-galli population A extremely adapting to rice planting systems, which should be prevented to spread to normal rice fields.

Serological and molecular epidemiology of Japanese Encephalitis in Zhejiang, China, 2015-2018.

BACKGROUND: Shifts have occurred in the epidemiological characteristics of Japanese encephalitis (JE), extending from the molecular level to the population level. The aim of this study was to investigate the seroprevalence of JE neutralizing antibodies in healthy populations from different age groups in Zhejiang Province, and to conduct mosquito monitoring to evaluate the infection rate of Japanese encephalitis virus (JEV) among vectors, as well as the molecular characteristics of the E gene of isolated JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1190 sera samples were screened by a microseroneutralization test, including 429 infants (28d-11m) and 761 participants (2y-82y). For those under 1 year old, the geometric mean titers (GMTs) of the JE neutralizing antibody was 9.49 at birth and significantly declined as the age of month increased (r = -0.225, P<0.001). For those above 1-year old, seropositive proportions were higher in subjects aged 1-3 years old as well as ≥25 years old (65%-75%), and relatively lower in subjects aged between 4-25 years old (22%-55%). Four or more years after the 2nd dose of JEV-L (first dose administered at 8 months and the second at 2 years of age), the seropositive proportion decreased to 32.5%, and GMTs decreased to 8.08. A total of 87,201 mosquitoes were collected from livestock sheds in 6 surveillance sites during 2015-2018, from which 139 E gene sequences were successfully amplified. The annual infection rate according to bias-corrected maximum likelihood estimation of JEV in Culex tritaeniorhynchus was 1.56, 2.36, 5.65 and 1.77 per 1000, respectively. JEV strains isolated during 2015-2018 all belonged to Genotype I. The E gene of amplified 139 samples differed from the JEV-L vaccine strain at fourteen amino acid residues, including the eight key residues related to virulence and virus attenuation. No divergence was observed at the sites related to antigenicity. CONCLUSIONS/SIGNIFICANCE: Zhejiang Province was at a high risk of JE exposure due to relatively lower neutralizing antibody levels among the younger-aged population and higher infection rates of JEV in mosquitoes. Continuous, timely and full coverage of JE vaccination are essential, as well as the separation of human living areas and livestock shed areas. In addition, annual mosquito surveillance and periodic antibody level monitoring are important for providing evidence for improvement in JE vaccines and immunization schedules.

Patient with severe fever with thrombocytopenia syndrome virus infection and central nervous system disturbance in Dongyang, Zhejiang Province, China, 2017.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever that was first described in China in 2011. We report a patient who died of Severe fever with thrombocytopenia syndrome virus (SFTSV) infection, with a rapidly progressive central nervous system (CNS) disturbance, in Dongyang, Zhejiang Province, China, in 2017. CASE PRESENTATION: A 64-year-old man was admitted to hospital after 4 days of fever. SFTSV was detected 1 day after the patient was admitted to hospital. The patient presented with CNS disturbance and died 4 days after admission. Detailed clinical and epidemiological investigations and laboratory tests were conducted. Reduced platelet, white blood cell, lymphocyte, and neutrophil counts, elevated lactate dehydrogenase, creatine kinase, aspartate aminotransferaseand alanine aminotransferase concentrations, and an increased activated partial thromboplastin time were observed. In a phylogenetic analysis, the isolate clustered close to a strain derived from South Korea. CONCLUSIONS: This is the first case of SFTSV infection with CNS disturbance in Dongyang, Zhejiang Province, China. The surveillance of suspected cases of SFTS is important in SFTSV endemic regions.

Reducing Allergenicity to Arginine Kinase from Mud Crab Using Site-Directed Mutagenesis and Peptide Aptamers.

The mud crab ( Scylla paramamosain) is widely consumed but can cause a severe food allergic reaction. To reduce allergenicity to arginine kinase (AK), site-directed mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of conformational epitopes. Three hypoallergenic mutant AKs (mAK1, mAK2, and mAK3) were generated, with the immunoreactivity decreasing by 54.2, 40.1, and 71.4%, respectively. In comparison to recombinant AK (rAK), the structure of mAKs was clearly changed. Additionally, antisense peptides were designed on the basis of linear epitopes and pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking and then analyzed by the immunoglobulin E inhibition enzyme-linked immunosorbent assay and human Laboratory of Allergic Diseases 2 mast cell degranulation assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs, and the inhibitory effect of peptide aptamer 3 was slightly better than the others. These results provide synergistic methods to reduce allergenicity to AK, which could be applied to other shellfish allergens.

Risk factors for avian influenza virus in backyard poultry flocks and environments in Zhejiang Province, China: a cross-sectional study.

BACKGROUND: Human infection of avian influenza virus (AIV) remains a great concern. Although live poultry markets are believed to be associated with human infections, ever more infections have been reported in rural areas with backyard poultry, especially in the fifth epidemic of H7N9. However, limited information is available on backyard poultry infection and surrounding environmental contamination. METHODS: Two surveillance systems and a field survey were used to collect data and samples in Zhejiang Province. In total, 4538 samples were collected by surveillance systems and 3171 from the field survey between May 2015 and May 2017, while 352 backyard poultry owners were interviewed in May 2017 by questionnaire to investigate factors influencing the prevalence of avian influenza A virus and other AIV subtypes. RT-PCR was used to test the nucleic acids of viruses. ArcGIS 10.1 software was used to generate maps. Univariate and logistic regression analyses were conducted to identify risk factors for AIV infection. RESULTS: Of the 428 poultry premises observed by the surveillance system, 53 (12.38%) were positive for influenza A virus. Of the 352 samples from poultry premises observed by field survey, 13 (3.39%) were positive for influenza A virus. The prevalence of AIV was unevenly distributed and the dominant subtype differed among cities. Eastern (Shaoxing and Ningbo) and southern (Wenzhou) cities exhibited a higher prevalence of AIV (16.33, 8.94, and 7.30% respectively). Contamination of AIV subtypes was most severe in January, especially in 2016 (23.26%, 70/301). The positive rate of subtype H5/H7/H9 was 2.53% (115/4538). Subtype H5 was the least prevalent, while subtypes H7 and H9 had similar positivity rates (1.50 and 1.32% respectively). Poultry flocks and environmental samples had a similar prevalence of AIV (4.46% vs 5.06%). The type of live birds was a risk factor and the sanitary condition of the setting was a protective factor against influenza A contamination. CONCLUSIONS: AIV subtypes were prevalent in backyard poultry flocks and surrounding environments in Zhejiang Province. The types of live birds and sanitary conditions of the environment were associated with influenza A contamination. These findings shine a light on the characteristics of contamination of AIV subtypes and emphasize the importance of reducing AIV circulation in backyard poultry settings.

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang, an Eastern Province of China.

BACKGROUND: Sporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 570 serum specimens were sampled from community population aged from 0 to 92 years old in Xianju county of Zhejiang Province in 2013-2014. Microseroneutralization test results were analyzed to estimate the population immunity and to observe antibody dynamics in vaccinated children. E genes of 28 JEV strains isolated in Zhejiang Province were sequenced for phylogenetic tree construction and molecular characteristics analysis with other selected strains. Positive JE neutralizing antibody rates were higher in residents ≥35 years old (81%~98%) and lower in residents <35 years old (0~57%). 7 or 8 years after the 2nd live attenuated vaccine dose, the antibodies against for 4 different strains with microseroneutralization test were decreased by 55%~73% on seropositive rates and by 25%~38% on GMTs respectively. JEV strains isolated in recent years were all grouped into GI, while those isolated in the 1980s belonged to GIII. On important amino acid sites related to antigenicity, there was no divergence between the Zhejiang JE virus strains and the vaccine strain (SA14-14-2). CONCLUSION/SIGNIFICANCES: JE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.

Expression of high-mobility group box-1 (HMGB1) in the basilar artery after experimental subarachnoid hemorrhage.

It has been suggested that inflammatory damage may be involved in the pathogenesis of cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). High-mobility group box-1 protein (HMGB1) has been identified as a potent proinflammatory mediator, and may trigger increases in other inflammatory cytokines. However, little is known about the role of HMGB1 in SAH-induced cerebrovascular inflammation. In this study, 48 male rats were assigned randomly to four groups: a control group, or SAH day 3, day 5, or day 7 groups. The animals in SAH day 3, day 5, and day 7 groups were subjected to injection of autologous blood into the cisterna magna twice, on day 0 and day 2, and were killed on days 3, 5, and 7, respectively. Cross-sectional area of the basilar artery was measured and the HMGB1 expression was assessed by immunohistochemistry and western blot analysis. The mRNA level of HMGB1 was also determined by reverse transcription polymerase chain reaction. The basilar arteries exhibited vasospasm after SAH which was most severe on day 3 and 5. Elevated expression of HMGB1 was detected after SAH and was highest on day 3 and 5. HMGB1 is increasingly expressed in parallel to the development of CVS in this rat experimental model of SAH. These results suggest that HMGB1 may be related to the CVS observed after SAH and HMGB1 may play a key role in the inflammatory response in CVS after SAH.

Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.

Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability.

[Genomic sequences of human infection of avian-origin influenza A(H7N9)virus in Zhejiang province].

OBJECTIVE: To analyze the etiology and genomic sequences of human infection of avian-origin influenza A(H7N9)virus from Zhejiang province. METHODS: Viral RNA was extracted from patients of suspected H7N9 influenza virus infection and real-time RT-PCR was conducted for detection of viral RNA. All 8 segments of influenza virus were amplified by one-step RT-PCR and genomic sequences were assembled using the sequencing data. All the currently available HA and NA genes of the novel H7N9 virus, some other HAs from H7 subtype and NAs from N9 subtype were downloaded from public database for phylogenetic analysis, using the Mega 5.1 software. Mutations and variations were analyzed, using the genomic sequence data. RESULTS: Reactions for influenza type A, subtype H7 and subtype N9 were all positive and all the genomic fragments were amplified for sequencing. After alignment, sequences were subjected for phylogenetic analysis. The results revealed highest homology with A/duck/Zhejiang/12/2011(H7N3)in HA gene and with A/wild bird/Korea/ A14/2011(H7N9)in NA gene of the H7N9 influenza virus. All 6 genes coding for internal proteins shared highest identities with H9N2 avian influenza which were circulated in the Chinese mainland, in the last two years. The sequenced virus showed Q226L mutation in HA protein, but E627K was not presented in PB2 protein of this virus. The E627K mutation was shared by all the other novel H7N9 viruses resulted in human infections through analysis on the currently available sequences. CONCLUSION: Using the clinical samples, both detection of the viral genes and amplification of all 8 segments of the novel H7N9 influenza virus were accomplished. High homology of the novel H7N9 influenza viruses was observed by phylogenetic test, using the currently available sequences. The virus showed Q226L mutation on HA protein but E627K did not present on PB2 protein of this virus.

Purification, physicochemical and immunological characterization of arginine kinase, an allergen of crayfish (Procambarus clarkii).

Arginine kinase (AK) has attracted considerable attention because it has been identified as a shellfish allergen. However, little information is available about AK in crayfish (Procambarus clarkii). In this study, crayfish AK was purified and cloned. Its physicochemical properties, processing stability, and immunological characteristics were analyzed. Crayfish AK was purified by column chromatography, which revealed a single band with molecular mass of 40 kDa; this result was further confirmed by mass spectrometry. The full-length gene sequence of crayfish AK was 1462 bp and encoded a protein of 357 amino acid residues. The results of this study revealed that crayfish AK is a glycoprotein with an isoelectric point of approximately 6.5. Thermal stability assays revealed that crayfish AK easily forms aggregates at temperatures >44°C and was stable at pH 4.0-8.0. SDS-PAGE and dot blotting were used to assess processing stability of purified AK. The results revealed that the IgE-binding activity of crayfish AK is reduced after boiling.

[Analysis of genetic variation of hemagglutinin and three internal genes of influenza B virus isolated in Zhejiang province from 1999 to 2012].

OBJECTIVE: To explore the characteristics of the genetic variation of hemagglutinin( HA) and three internal genes coding for the nucleoprotein ( NP) , matrix protein ( M) and nonstructural protein ( NS) of influenza B virus. METHODS: A total of 31 strains of influenza B virus were isolated in Zhejiang province from 1999 to 2012, and then were amplified and sequenced the genes of HAl , NP, M and NS. The phylogenetic tree was constructed, the nucleotide substitution rate of the above individual gene was estimated and the variation sites of amino acids were analyzed. RESULTS: The 31 isolated strains of influenza B virus were divided into two distinct lineages Victoria and Yamagata in the phylogenetic tree of HAl gene,represented by B/Victoria/2/87 and B/Yamagata/16/88. Phylogenetic analysis of the NP gene showed that the NP gene of Victoria-like influenza B strains which were isolated after 2010 was highly homologous with Yamagata-like isolates, and thereby they were found to be on the same branch of the phylogenetic tree of the NP gene. Nucleotide substitution rates of HAl , NP, M and NS genes were estimated to be 2. 29 x 10 -3 ,1. 39 X 10-3 ,1. 78 X 10-3 ,1. 30 X 10-3 /site per year, respectively. Variations of amino acid of HAl domain of Victoria-like isolates mainly included K48E ,L58P ,N75K,K80R,K129N/S,N165K,S172P ,Sl97N/D and A202V; while those in Yamagata-like isolates were R48K, S1501, N166Y, N203S, G230D and D233N. Determined amino acid sequences of NP of Victoria-like influenza B isolates were similar to Yamagata-like isolates after 2010 and variations happened on four characteristic amino acid sites, naming A60D, I233V, N513S and V5341, compared with previous Victoria-like influenza B isolates. CONCLUSION: Significant variation was found among influenza B strains isolated in Zhejiang province from 1999 to 2012. The surface HAl gene evolved more rapidly than internal genes. Gene reassortment and gene mutation were the main evolutionary mechanism of influenza B virus.

Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain.

Arginine kinase (AK) is reported to be the pan-allergen of shellfish. However, there is limited information on its IgE epitopes and structural characteristics. In this study, AK from Scylla paramamosain was purified and characterized. The purified AK is a glycoprotein with the molecular weight of 40 kDa and it demonstrates cross-reactivity with the related allergens present in other shellfish. The cDNA of S. paramamosain AK was cloned, which encodes 357 amino acid residues. Nine linear epitopes and seven conformational epitopes were predicted following bioinformatics analysis. In addition, the entire recombinant AK (rAK) and three partial recombinant AKs (rAK1, rAK2, and rAK3) were successfully expressed in Escherichia coli BL21 (DE3). The proteins of rAK1, rAK2 and rAK have strong IgE reactivity with the pooled sera from crab allergic patients, while rAK3 has significantly weaker IgE reactivity, which indicates that the IgE epitopes of AK are mainly distributed in the regions of rAK1 and rAK2. Furthermore, three experimental linear epitopes (epitope 1: AA 127-141, epitope 2: AA 141-155, and epitope 3: AA 211-225) were discovered in the region of rAK1 and rAK2 using synthetized overlapping peptides. The experimental linear epitopes were mapped onto the protein homology model of AK. Meanwhile, in the IgE-binding assays of the sera from nine crab allergic patients, only three sera reacted with the denatured, linear AK as shown by Western-blotting, eight sera reacted with the native, folded AK by both dot-blotting and ELISA, which indicates that the conformational IgE epitopes of S. paramamosain AK may be more predominant.

[Molecular evolution of two lineages related to influenza B virus based on HA1 gene].

OBJECTIVE: To study the evolutionary characteristics and rules of two lineages on influenza B virus. METHODS: A total of 126 HA1 sequences of strains isolated during 1940 to 2012 were downloaded from the GenBank. Time of the most recent common ancestor (TMRCA) and divergence of the two lineages were calculated based on the data from phylogenetic analysis of HA1 gene, using Bayesian Markov Chain Monte Carlo (Bayesian-MCMC) and molecular clock method. RESULTS: The average amino acid variant ratios were ranged from 5.4% to 10.2% within the strains of influenza B virus isolated during 1978 to 2010. Compared with the Victoria-like strains, all Yamagata-like strains showed an amino acid deletion at 163(th) site, while some of them showing a deletion at position 166. HA1 gene of influenza B virus seemed not have been affected by positive selection except a few sites. The evolutionary average rate on HA1 gene was 2.138×10(-3) substitutions/site/year (95% HPD: 1.833×10(-3) - 2.437×10(-3) substitutions/site/year). The estimated dates for TMRCA of the two lineages of influenza B virus could be dated back to 1971 (95% HPD: 1969 - 1972), while the divergence times of the two lineages were 1973 (95% HPD: 1971 - 1974) and 1977 (95% HPD: 1975 - 1978) respectively. CONCLUSION: Significant differences were found on HA1 gene between earlier and recent identified strains of Victoria and Yamagata lineage. Differences between the two lineages increased and showing the potential of dividing themselves into different subtypes in the future. More attention should be paid to these trends and the related epidemiological significance.

Purification and characterisation of sarcoplasmic calcium-binding protein, a novel allergen of red swamp crayfish (Procambarus clarkii).

Crayfish sarcoplasmic calcium-binding protein (SCP) was purified. The physicochemical and polymorphic characterisations were also analysed. SCP was purified by column chromatography to reveal a single band with molecular mass of 22 kDa and further confirmed by mass spectrometry. The results of physicochemical characterisation showed that SCP was stable in the processes of thermal or acid/alkali treatment, and could be digested by simulate gastrointestinal fluid. Importantly, the comparison of SCP polymorphism using sera from crustacean-allergic patients demonstrated SCP-II had a weaker IgE-binding activity. The isoelectric points of SCP subunits a, b and c were 4.6, 4.7, and 4.8, respectively, as determined by two-dimensional electrophoresis and IgE immunoblotting analysis showed that patients' sera reacted to three subunits of SCP. Finally, it can be concluded that SCP is a stable polymorphic allergen in crayfish, and all of its isotypes and subunits have allergenicity.

Purification, cloning, and immunological characterization of arginine kinase, a novel allergen of Octopus fangsiao.

Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.