[Notch1 inhibits the mechanistic role of STING signaling to regulate hepatocyte lipophagy in nonalcoholic steatohepatitis].

Objective: To study the mechanistic role of myeloid-specific Notch1 knockout inhibiting STING signaling to regulate hepatocyte lipophagy. Methods: A mouse model of nonalcoholic steatohepatitis (NASH) was established using a high-fat diet (HFD) and mouse bone marrow-derived macrophages (BMMs). Primary hepatocytes were isolated to construct a co-culture system. Twelve Notch1(FL/FL) mice were randomly divided into two groups: the Notch1(FL/FL) + normal diet (NCD) and the Notch1(FL/FL) + HFD group. Further, 12 Notch1(M-KO) mice were randomly divided into two groups: Notch1(M-KO) + NCD, and Notch1(M-KO) + HFD group.Serum alanine aminotransferase (sALT), total cholesterol (TC) and triglyceride (TG) were collected from mice serum samples. Liver tissue samples were collected for H&E staining, immunofluorescence (IF), Western blot and qRT-PCR. Tumor necrosis factor (TNF)-α was detected in the supernatant by enzyme-linked immunosorbent assay (ELISA). The comparison of inter group data was conducted using a t-test. Results: The mouse NASH model, mouse BMMs co-culture system, and primary hepatocytes were successfully constructed. Compared with the Notch1(FL/FL) + HFD group, the Notch1(M-KO) + HFD group showed a significant increase in serum ALT [(250.02 ± 58.21) U/L vs (370.70 ± 54.57) U/L, t = 3.705, P = 0.004], TG [(29.90 ± 3.54) mg/g vs (43.83 ± 8.56) mg/g, t = 3.685, P = 0.004], and TC [(33.70 ± 8.43) mg/g vs (90.53 ± 12.53) mg/g, t = 9.917, P < 0.001]. HE staining of liver tissue showed remarkable balloon-like alterations in liver cells, while IF staining demonstrated increased macrophage infiltration (t = 7.346, P < 0.001). Compared with the hepatocyte group co-cultured with Notch1(FL/FL) BMMs, the BODIPY probe showed a significant increase in lipid droplet (LDs) deposition in liver cells in the Notch1(M-KO) group (t = 3.835, P < 0.001). The co-localization of lysosomal associated membrane protein 1 (LAMP1), LDs (t = 7.103, P < 0.001), microtubule-associated protein light chain 3 (LC3) -II/LC3-I (t = 5.0, P = 0.007), and autophagy associated gene 12 (Atg12) (t = 28.36, P < 0.001) had decreased expression, while P-62 had increased expression (t = 3.253, P = 0.03), indicating a decrease in autophagic flow. Additionally, LC3 and LDs colocalization decreased (t = 5.24, P = 0.0003), indicating reduced lipophagy. Compared with the Notch1(FL/FL) group, the Notch1(M-KO) BMMS mouse group showed an increase in the expression of p-STING (t = 5.318, P = 0.006), p-TANK1 binding kinase 1 (TKB1) (t = 6.467, P = 0.002), p-interferon regulatory factor 3 (IRF3) (t = 14.61, P < 0.001), and p-P65 (t = 12.7, P = 0.002) protein, accompanied by mRNA expression of the inflammatory mediators interferon (IFN)-ß (t = 7.978, P < 0.001), TNFα (t = 8.496, P = 0.001), interleukin-1 ß (IL-1 ß) (t = 4.7, P < 0.001), and CXCL-10 (t = 4.428, P = 0.001). The STING gene was knocked out in the BMMs Notch1(M-KO) mice using CRISPR/Cas9. Compared with the CRISPR-Control group, the expression of P-TKB1 (t = 2.909, P = 0.044), p-IRF3 (t = 10.96, P < 0.001), p-IRF3 (t = 10.96, P < 0.001), and p-P65 (t = 7.091, P = 0.002) proteins was lower in the STING-KO BMMs group. The release of TNF-α in the supernatant was decreased (732.3 ± 129.35 pg/ml vs. 398.17 ± 47.15 pg/ml, t = 4.204, P = 0.014). However, in hepatocytes co-cultured with STING-KO BMMs, LC3-II/LC3-I (t = 7.546, P = 0.001) increased, p-62 (t = 10.96, P < 0.001) expression decreased, autophagic flow increased, and the colocalization of LC3 and LDs increased, lipophagy increased, and LDs deposition decreased. Conclusion: Myeloid-specific Notch1 knockout can activate macrophages STING signaling, increase the expression of inflammatory mediator genes, inhibit the occurrence of autophagy flow and lipophagy in hepatocyte cells, and aggravate LDs deposition and NASH progression.

[Macrophage heterogeneity role in NAFLD and NASH disease progression].

Nonalcoholic fatty liver disease (NAFLD) is a type of metabolic stress liver injury that is closely associated with insulin resistance and genetic susceptibility. The continuum of liver injury in NAFLD can range from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and even lead to cirrhosis and liver cancer. The pathogenesis of NAFLD is complicated. Pro-inflammatory cytokines, lipotoxicity, and gut bacterial metabolites play a key role in activating liver-resident macrophages (Kupffer cells, KCs) and recruiting circulating monocyte-derived macrophages (MoDMacs) to deposit fat in the liver. With the application of single-cell RNA-sequencing, significant heterogeneity in hepatic macrophages has been revealed, suggesting that KCs and MoDMacs located in the liver exert distinct functions in regulating liver inflammation and NASH progression. This study focuses on the role of macrophage heterogeneity in the development and occurrence of NAFLD and NASH, in view of the fact that innate immunity plays a key role in the development of NAFLD.

[Association between obesity with the diversity and genus of gut microbiota in school-aged children].

Objective: To analyze the diversity differences of gut microbiota between school-aged obese children and normal-weight children and identify the characteristic flora in obese children to provide some evidence for the subsequent study of the following study-related mechanisms and the prevention and treatment of obesity in school-aged children. Methods: This study was based on a cohort established in a primary school in Jiading district, Shanghai, 2016. Sixty-three children, including 43 boys and 20 girls, who were obese during the three years from 2016 to 2018, were enrolled in the obesity group. Among children who were average weight for three years, a total of 63 were selected as the control group, with matching with the obese ones in a 1 to 1 way according to age, sex and class. The primary characteristics, diet status, breastfeeding, and other information of children were collected by questionnaire. Fecal samples of participants in both groups were collected, and 16S rDNA sequencing was performed. Operable taxon units clustering according to 97% similarity and species annotation were based on the quality-optimized sequences. The difference in the diversity and genius of gut microbiota among children in the obesity and control groups were analyzed. The α diversity indices, including Ace, Chao1, Shannon, and Simpson index, were calculated. The ß diversity was presented based on unweighted Unifrac distance and weighted Unifrac space by principal coordinate analysis. The differences in ß diversity between the two groups were compared by similarity analysis (ANOSIM).The differences in genus distribution between the two groups were selected by STAMP software. The association of obesity with the α diversity and genus with significant differences were analyzed by the generalized linear model (GLM). Results: The Ace and Chao1 index in the obesity group was significantly lower than those in the control group (The P values were 0.026 and 0.039, respectively). There was no significant difference in Shannon and Simpson index between the two groups (The P values were 0.879 and 0.922, respectively). The results of ANOSIM showed differences in gut microbiota between the two groups (R>0), but the group contribution was not significant (unweighted Unifrac distance: R=0.006, P=0.223; weighted Unifrac distance: R=0.010, P=0.134). Among the obese group, the relative abundance of Prevotella and Sarcina was highest in the endemic genus. The STAMP results showed that the relative lots of 15 genera were significantly different between the two groups (P<0.05). The results of GLM showed that, compared with the control group, the obesity group had a lower level of the relative abundance in Akkermansia muciniphila, while a higher level in Coprococcus_3, Ruminococcus, Agathobacter and Collinsella. After stratification by sex, the Chao1 index was also lower in the obese boys than that in the normal-weight boys. However, the obese girls only had a higher level in Coprococcus_3 than the ordinary weight girls. Conclusions: Compared with children with average weight, obese children had lower α diversity of gut microbiota and lower relative abundance of dominant probiotics but had a higher relative lot of genus associated with metabolic disorders, inflammation promotion, and metabolism rate reduction.

[Incidence of unintended pregnancy within 2 years after delivery and its influencing factors in China].

Objective: To investigate the present situation of unintended pregnancy within two years postpartum and its influencing factors in China. Methods: Participants who delivered a live birth at 60 hospitals in 15 provinces in the eastern, central and western regions of China during July 2015 to June 2016 were interviewed by using structured questionnaire. Information on occurrence of unintended pregnancy within 2 years after delivery, postpartum contraceptive use, sexual resumption, breastfeeding, and women's socio-demographic characteristics, and so on, were collected. Life-table analysis, cluster log-rank tests and a 2-level Cox regression model were used for data analysis. Results: A total of 18 045 postpartum women were investigated. The cumulative 1- and 2-year unintended pregnancy rates after delivery were 5.3% (95%CI: 4.5%－6.1%) and 13.1% (95%CI: 11.3%－14.8%), respectively. Cox regression model analysis showed that the risk of unintended pregnancy within 2 years postpartum were increased in younger women, ethnic minorities, women with abortion history, and those who had a vaginal delivery with short lactation time and late postpartum contraceptive initiation (all P<0.01). The risk of postpartum unintended pregnancy was not associated with geographic regions and hospitals where women gave a birth (all P>0.05). Conclusions: In China, the risk of unintended pregnancy within 2 years after delivery is relatively high. Service institutions and service providers should improve the quality of postpartum family planning services, promote the use of high effect contraceptive methods, and educate women to use a method at the time of their sexual resumption or even before.

Prevalence and factors associated with motoric cognitive risk syndrome in community-dwelling older Chinese: a cross-sectional study.

BACKGROUND AND PURPOSE: A recently proposed pre-dementia syndrome, motoric cognitive risk (MCR) syndrome, is characterized by cognitive complaints and slow gait, and increases the risk of dementia and mortality. The aim of the present study was to explore the prevalence of and factors associated with MCR syndrome in elderly community-dwelling Chinese subjects. METHODS: The Ningbo Community Study on Aging recruited 953 Chinese community-dwelling participants aged ≥ 65 years from November 2016 to March 2017. Handgrip, Five-Times-Sit-to-Stand (FTSS) test time and body composition, as well as comprehensive geriatric evaluation, were measured as potentially independent factors associated with MCR syndrome. RESULTS: The prevalence of MCR syndrome was 12.8% in men and 12.6% in women, and high prevalence of MCR syndrome was not associated with age or sex. Multiple logistic regression analysis by sex showed that a 1-SD increase in FTSS test time in males and females was associated with 45% (95% confidence intervals, 19-76; P < 0.01) and 20% (95% confidence intervals, 9-33; P < 0.01) higher risk of having MCR syndrome, respectively, whereas handgrip strength was inversely correlated with MCR syndrome in males [odds ratio (OR), 0.91; P = 0.02] but not females (P = 0.06). Moreover, the relationship of arm fat mass and MCR syndrome was statistically significant in both sexes (OR, 1.69-1.77), but leg fat mass was only associated with MCR syndrome (OR, 1.56; P = 0.02) in men. CONCLUSIONS: Handgrip, FTSS test time and body composition were associated in a sex-specific manner with MCR syndrome in elderly community-dwelling Chinese subjects. Our results on MCR syndrome are novel and should be considered as important information in future studies.

Influences of miR-320a on proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis through targeting MAPK-ERK1/2.

OBJECTIVE: To study the expression of micro ribonucleic acid (miR)-320a in synovial tissues of patients with rheumatoid arthritis (RA) and explore the influences of miR-320a on the proliferation and apoptosis of fibroblast-like synoviocytes (FLSs) in RA and its mechanism. PATIENTS AND METHODS: The expression level of miR-320a in synovial tissues of 40 healthy people and 32 RA patients was detected via reverse transcription-polymerase chain reaction (RT-PCR). The FLSs were isolated from RA patients, cultured in vitro and divided into Control group and miR-320a mimic group. The proliferation and apoptosis of FLSs in each group were observed. Finally, the expression level of mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) 1/2 in each group was detected via Western blotting. RESULTS: The expression level of miR-320a in synovial tissues of RA patients was significantly lower than that in healthy people (p < 0.05). After miR-320a mimic was transfected into FLSs cultured in vitro, EdU staining and flow cytometry analysis were performed. The results revealed that the proportion of EdU-positive cells significantly declined in miR-320a mimic group, the proportion of cells in G0/G1 phase was increased, while the cells in G2/M and S phases were significantly decreased (p < 0.05). Above data indicated that the cell proliferation ability was significantly inhibited. In addition, the results of flow cytometry also showed that the apoptosis rate of FLSs in miR-320a mimic group was significantly higher than that in Control group (p < 0.05). The results of Western blotting manifested that the Bcl-2 associated X protein (Bax)/Bcl-2 ratio in miR-320a mimic group was also obviously increased (p < 0.05). According to further studies, the phosphorylation level of ERK1/2 in miR-320a mimic group was remarkably inhibited (p < 0.05). CONCLUSIONS: The expression level of miR-320a significantly declined in synovial tissues of RA patients. MiR-320a attenuated proliferation and promoted apoptosis of FLSs through inhibiting the activation of the MAPK-ERK1/2 signaling pathway.

MiR-601 inhibits the proliferation and metastasis of esophageal squamous cell carcinoma (ESCC) by targeting HDAC6.

OBJECTIVE: The aim of this study was to explore the role of microRNA-601 (miR-601) in the proliferation and invasion of esophageal squamous cell carcinoma (ESCC) cells, thereby providing new thoughts for prognosis evaluation and targeted therapy of ESCC. PATIENTS AND METHODS: 23 pairs of ESCC tissue samples and adjacent normal tissues were collected, and the expression level of miR-601 was detected. Biological information analysis and Luciferase report gene assay were used to verify the potential target genes of miR-601. Then, three groups were established in ESCC cell line (TE-1) to perform similar experiments, including the miR-NC group, the miR-601 mimics group and the mimics + HDAC6 group. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation ability. Meanwhile, transwell assay and scratch-wound assay were applied to observe the effect of miR-601 on cell invasion and migration. Quantitative reverse transcription Polymerase Chain Reaction (qPCR) and Western blot assay were applied to determine the mRNA and protein expression changes after transfection. RESULTS: Compared with normal adjacent tissues and normal esophageal epithelial cells, the expression of miR-601 was significantly decreased in ESCC tissues and cells. HDAC6 was identified as a target gene of miR-601. The expression of HDAC6 in esophageal carcinoma cells transfected with miR-601 mimics was significantly down-regulated. The negative correlation between miR-601 and HDAC6 expression was assessed by qPCR and Western blot (WB) assay. Furthermore, miR-601 remarkably suppressed the proliferation of ESCC cells. Meanwhile, cell invasion and migration were also found markedly restricted after transfection of miR-601 mimics. However, the overexpression of HDAC6 significantly counteracted the effects of miR-601. CONCLUSIONS: MiR-601 suppressed the proliferation, invasion and migration of esophagus carcinoma cells by down-regulating HDAC6 expression.

A Cross-Sectional Study of the Association between Arterial Stiffness and Sarcopenia in Chinese Community-Dwelling Elderly Using the Asian Working Group for Sarcopenia Criteria.

BACKGROUND: Several previous researches had found artery stiffness associated skeletal muscle mass, but not considering muscle strength and physical performance, which also were compositions of sarcopenia. This study aims to reveal the relationship of artery stiffness and sarcopenia using the Asian Working Group for Sarcopenia criteria. METHODS: Study was performed on 1002 Chinese community dwelling participants aged ≥65 years from November 2016 to March 2017. Body composition, muscle strength, physical performance, and brachial-ankle pulse wave velocity (baPWV) considering as artery stiffness index were measured. RESULTS: In multiple regression analysis, baPWV was associated with handgrip (ß=-0.13, P=0.04) and Relative skeletal muscle mass index (ASM/Ht2) (ß=-0.02, P<0.01), but not with 4-meter velocity (P=0.21). Multiple logistic regression analysis showed that 1-SD (3.50m/s) increased in baPWV was still associated with a 11% (CI, 4%-20%; P<0.01) higher odds of being sarcopenia. In the gender subgroup analysis, the relationship of baPWV and sarcopenia remain significant in men (OR, 1.23; 95% CI, 1.07-1.42, P<0.01), but not in women (P=0.07). CONCLUSIONS: High brachial-ankle pulse wave velocity is associated with sarcopenia in Chinese community-dwelling elderly, with gender differences.

[Diagnosis and treatment of traumatic optic neuropathy with internal carotid artery trauma].

Objective: To summarize our experience in the diagnosis of internal carotid artery trauma in patients with traumatic optic neuropathy, and to make recommendations for the treatment. Methods: The clinic data of 6 cases who had traumatic optic neuropathy with internal carotid artery trauma and who were admited in Department of Otorhinolaryngology, the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from Jan. 2013 to Dec. 2015 were analyzed retrospectively. Results: All 6 cases were monocular blindness. Four cases did not undergo nasal endoscopic optic nerve decompression because of the diagnoses of internal carotid artery trauma. One case was diagnosed after nasal endoscopic optic nerve decompression because of fatal bleeding during the operation. One case was diagnosed because of late-onset recurrent epistaxis. Among the 6 cases with internal carotid artery trauma, 3 cases were successfully treated with endovascular interventional treatment (stent embolization was used in one case, Coil embolization was used in two cases), and 3 patients refused treatment. Conclusions: The patients with traumatic optic neuropathy have the possibility of severe carotid artery trauma. Endoscopic optic nerve decompression is not suitable for these cases. It should pay more attention to patients with traumatic optic neuropathy. For suspected cases, vascular-enhanced computed tomography screening and digital subtraction angiography should be recommended and patients should be treated by endovascular intervention in a timely manner.

The relevance of Tim-3 polymorphisms and F protein to the outcomes of HCV infection.

Hepatitis C virus (HCV) is one of the major causes of liver inflammation. The aim of this study was to investigate the associations of T-cell immunoglobulin and mucin domain-3 (Tim-3) polymorphisms and the alternate reading frame protein (F protein) with the outcomes of HCV infection. Three single-nucleotide polymorphisms (SNPs; rs10053538, rs12186731, and rs13170556) of Tim-3 were genotyped in this study, which included 203 healthy controls, 558 hepatitis C anti-F-positive patients, and 163 hepatitis C anti-F-negative patients. The results revealed that the rs12186731 CT and rs13170556 TC and CC genotypes were significantly less frequent in the anti-F-positive patients [odds ratio (OR) = 0.54, 95 % confidence interval (CI) = 0.35-0.83, p = 0.005; OR = 0.26, 95 % CI = 0.18-0.39, p < 0.001; and OR = 0.19, 95 % CI = 0.10-0.35, p < 0.001, respectively), and the rs13170556 TC genotype was more frequent in the chronic HCV (CHC) patients (OR = 1.70, 95 % CI = 1.20-2.40, p = 0.002). The combined analysis of the rs12186731 CT and rs13170556 TC/CC genotypes revealed a locus-dosage protective effect in the anti-F-positive patients (OR = 0.22, 95 % CI = 0.14-0.33, p trend < 0.001). Stratified analyses revealed that the frequencies of the rs12186731 (CT + TT) genotypes were significantly lower in the older (OR = 0.31, 95 % CI = 0.15-0.65, p = 0.002) and female (OR = 0.30, 95 % CI = 0.17-0.52, p < 0.001) subgroups, and rs13170556 (TC + CC) genotypes exhibited the same effect in all subgroups (all p < 0.001) in the anti-F antibody generations. Moreover, the rs13170556 (TC + CC) genotypes were significantly more frequent in the younger (OR = 1.86, 95 % CI = 1.18-2.94, p = 0.007) and female (OR = 2.38, 95 % CI = 1.48-3.83, p < 0.001) subgroups of CHC patients. These findings suggest that the rs12186731 CT and rs13170556 TC/CC genotypes of Tim-3 provide potential protective effects with the F protein in the outcomes of HCV infection and that these effects are related to sex and age.

TBX21 polymorphisms are associated with virus persistence in hepatitis C virus infection patients from a high-risk Chinese population.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and the varied outcomes of the infection depend on both viral and host factors. We have demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias which is involved in virus persistence in chronic hepatitis C (CHC) patients. The purpose of this study was to test the hypothesis that genetic variants of TBX21 (T cell specific T-box transcription factor) were associated with the outcomes of HCV infection and F protein generation. Three single nucleotide polymorphisms (SNPs) (rs17250932, rs2074190, rs4794067) in the TBX21 gene were genotyped in a case-control study in a cohort of a high-risk group, including 354 healthy controls and 747 CHC patients (190 anti-F protein antibody seronegative patients and 557 anti-F protein antibody seropositive patients). Results showed that the rs4794067 C allele in the TBX21 promoter was significantly more common in CHC patients (OR = 1.335, 95% CI = 1.058-1.684, P = 0.015), exceptionally in anti-F protein seropositive patients (OR = 1.547, 95% CI = 1.140-2.101, P = 0.005), compared with healthy controls. And the risk effect was also significantly high in patients with HCV 1b genotype and mild fibrosis (P = 0.021, P = 0.010, respectively). Compared with the most frequent haplotype TAT, haplotype analysis showed that the distribution of TAC was significantly different between the chronic HCV carrier group and the healthy group, and so was the anti-F antibody seronegativity group and the anti-F antibody seronegativity group (all P < 0.001). Our results suggested that TBX21 variants may be involved in the etiology of this disease.

Genetic diversity and relationship of chicory (Cichorium intybus L.) using sequence-related amplified polymorphism markers.

Chicory is a crop with economically important roles and is cultivated worldwide. The genetic diversity and relationship of 80 accessions of chicories and endives were evaluated by sequence-related amplified polymorphism (SRAP) markers to provide a theoretical basis for future breeding programs in China. The polymorphic rate was 96.83%, and the average polymorphic information content was 0.323, suggesting the rich genetic diversity of chicory. The genetic diversity degree of chicory was higher (GS = 0.677) than that of endive (GS = 0.701). The accessions with the highest genetic diversity (effective number of alleles, NE = 1.609; Nei's genetic diversity, H = 0.372; Shannon information index, I = 0.556) were from Italy. The richest genetic diversity was revealed in a chicory line (NE = 1.478, H = 0.289, I = 0.443) among the 3 types (line, wild, and cultivar). The chicory genetic structure of 8 geographical groups showed that the genetic differentiation coefficient (GST) was 14.20% and the number of immigrants per generation (Nm) was 3.020. A GST of 6.80% and an Nm of 6.853 were obtained from different types. This observation suggests that these chicory lines, especially those from the Mediterranean region, have potential for providing rich genetic resources for further breeding programs, that the chicory genetic structure among different countries obviously differs with a certain amount of gene flow, and that SRAP markers could be applied to analyze genetic relationships and classifications of Cichorium intybus and C. endivia.

Analysis of diversity and relationships among orchardgrass (Dactylis glomerata L.) accessions using start codon-targeted markers.

Orchardgrass, or cocksfoot, is an important perennial forage grass worldwide. The comprehensive understanding of orchardgrass accessions will benefit germplasm collection and breeding progress, and it will enhance efforts to improve forage yield and quality. Therefore, 24 novel, simple, polymorphic, and reliable start codon-targeted (SCoT) markers were used to analyze the diversity and genetic relationships among 95 orchardgrass accessions. In total, 273 polymorphic bands were detected with an average of 11.4 bands per primer. The average polymorphic rate for the species was 83.4%, suggesting a high discriminating ability of the SCoT technique for orchardgrass. The molecular variance analysis revealed that 69.13 and 30.87% of variation resided within and among groups, respectively, demonstrating that the orchardgrass germplasms had a higher level of genetic diversity within groups than among geographical regions and distributions. The distinct geographical divergence of orchardgrass was revealed between North America and Oceania. The unweighted pair-group method with arithmetic mean dendrogram revealed a separation of 7 main clusters between 95 accessions according to the geographical origin. Furthermore, each cluster was divided into subgroups mainly according to the origin of its state. The genetic divergence of orchardgrass might be influenced by the ecogeographical conditions, climatic types, breeding systems and gene flow with variations in cultures, bird migration, and breeder selection. These results could facilitate orchardgrass germplasm collection, management, and breeding worldwide.

Identification of orchardgrass (Dactylis glomerata L.) cultivars by using simple sequence repeat markers.

The accurate identification of orchardgrass (Dactylis glomerata L.) cultivars is necessary to ensure purity for consumers, the effective utilization of cultivars, and to protect the intellectual property for breeders. Therefore, this study aimed to use SSR to construct DNA fingerprinting of orchardgrass cultivars. The genetic diversity of 32 orchardgrass cultivars originated from 21 countries, but grown in China, was assessed using a set of 29 SSR markers distributed across 9 linkage groups of the orchardgrass genome. A total of 229 bands were detected, with an average of 7.9 bands per marker. The average polymorphic rate for the species was 92.1%. The polymorphism information content ranged from 0.771 to 0.893. The genetic similarity ranged from 0.55 to 0.84, which confirmed a high level of genetic diversity among orchardgrass cultivars. The unweighted pair-group method, in combination with the arithmetic mean algorithm (UPGMA) dendrogram and principal coordinate analysis, showed a separation of 6 major clusters among 32 cultivars. The number of distinguishable cultivars ranged from 3 to 23, with an average of 12.1 per primer. Moreover, 11 bands that showed stable and repeatable SSR patterns were amplified by A01E14, A01K14, and D02K13. These bands were used to develop the DNA fingerprints for 32 orchardgrass cultivars. In the DNA fingerprints constructed, each cultivar had a unique fingerprinting pattern that was easily distinguished from the others. These results indicate that the SSR marker was polymorphic, and reliable for use in potential large-scale DNA fingerprinting of orchardgrass cultivars.

Analysis of antigen epitopes and molecular pathogenic characteristics of the 2009 H1N1 pandemic influenza A virus in China.

In order to further predict the epidemic trend and develop vaccines for 2009 H1N1 virus, we monitored its epitopes and molecular pathogenic characteristics during the epidemic process. We also analyzed the similarity of antigenic and genetic characteristics among the novel 2009 H1N1, representative seasonal H1N1 strains, and vaccine strains. 2009 H1N1 isolates had high similarity of hemagglutinin (HA) antigenic sites with H1N1 viruses isolated before 1940 and up to 80.0% similarity with 1918 H1N1. The elderly people born before 1940 have relatively low 2009 H1N1 infection rate, which might be responsible for their previous infection with either 1918 H1N1 virus or an early progeny. Compared to seasonal H1N1 vaccine strains from 1999 to 2010, the HA, neuraminidase (NA), and nucleoprotein (NP) proteins of the isolates had highly conserved CTL epitopes (60.5-65.8%, 69.6-82.6%, and 76.7%, respectively). The seriousness and mortality rate of 2009 H1N1 infections were similar to seasonal influenza, which may be related to the molecular characteristics of low toxicity of 2009 H1N1 and cross-T-cell immunity, due to vaccination or exposure to seasonal H1N1 virus. Some strains of 2009 H1N1 acquired mutations at antigenic and glycosylation sites. It is of particular interest that Haishu/SWL110/10 and Beijing/SE2649/09, isolated after November 2009, gained a new glycosylation site at the position 179 of HA protein, near the RBD. Thus, in the future, vaccination with glycosylated 2009 H1N1 virus may prevent the seasonal epidemic caused by strains with glycosylation site mutation near the receptor binding domain (RBD).

Molecular characterization of the surface glycoprotein genes of an H5N1 influenza virus isolated from a human in Guangdong, China.

In March 2006, a human H5N1-infected case was found in Guangdong province, China. Here, we molecularly characterized the hemagglutinin (HA) and neuraminidase (NA) genes of the A/China/GD01/06 (GD01) strain causing the infection. The phylogenetic analyses suggested that the HA and NA genes of GD01 and recent human H5N1 viruses from different provinces of China were probably derived from a common ancestor and the H5N1 human infection was acquired directly from affected poultry. At the cleavage site of HA, GD01 contained multiple basic amino acids, a feature characteristic of highly pathogenic avian influenza A viruses. The virus possessed Gln222, Gly224, Ser223, Asn182, Gln192 residues adjacent to the receptor-binding site, preferential for recognizing SAalpha2, 3Gal. In addition, the GD01 NA amino acid sequence possessed Asn344 and Phe466, which might be related to the low-pH stability of the sialidase activity and gastrointestinal symptoms of the patient.