[Genetic study of cardiovascular disease subtypes defined by International Classification of Diseases].

OBJECTIVE: To study the molecular connection among cardiovascular diseases (CVD) subtypes defined by the International Classification of Diseases (ICD) version 10 (ICD-10). METHODS: Both phenotypic data and genotypic data used in this study were obtained from the UK Biobank. A total of 380 083 participants aged between 40 and 69 years were included. Those without any cardiovascular disease (either no ICD-10 code at all or no ICD-10 code containing letter I) were assigned to the control group. The five CVD subtypes were: ischaemic heart diseases (IHD), pulmonary heart disease and diseases of pulmonary circulation (PHD), cerebrovascular diseases (CRB), diseases of arteries, arterioles and capillaries (AAC), diseases of veins, lymphatic vessels and lymph nodes, and diseases not elsewhere classified (VLL). We first performed a genome-wide association study (GWAS) for each of the five subtypes. We summarized novel loci using genome-wide significance threshold P=5×10-8. Next, we used linkage disequilibrium score regression (LDSC) method to assess genetic correlation among the five subtypes. Lastly, we applied mendelian randomization (MR) approach to assess the causal relationship among the subtypes. The particular software that we used was generalised summary-data-based mendelian randomisation (GSMR). RESULTS: Through GWAS, we identified hundreds of genome-wide significant SNPs: 672 for IHD, 241 for PHD, 31 for CRB, 48 for AAC, and 193 for VLL. By comparing with published literature, we found 28 novel loci, for PHD (n=14), CRB (n =7) and AAC (n =7). Eight of these 28 loci were rare, where the lead SNP had minor allele frequency (MAF) less than 1%. LDSC analyses indicated IHD had significant genetic correlation with VLL (P=2.52×10-7), PHD (P=3.77×10-3) and AAC (P=4.90×10-3), respectively. Bidrectional GSMR analyses showed that IHD had a positive causal relationship with VLL (P=7.40×10-5) and AAC (P=1.50×10-3), while reverse causality was not supported. CONCLUSION: This study adopted an innovative approach to study the molecular connection among CVD subtypes that are defined by ICD. We identified potentially positive genetic correlation and causal effects among some of these subtypes. Research along this line will provide scientific insights and serve as a guidance for future ICD standards.

Analysis of hemorrhagic fever with renal syndrome and its pathogenic gene sequence based on geographic information system.

This study analyzed the temporal-spatial distribution characteristics, epidemiological characteristics and gene sequences of hemorrhagic fever with renal syndrome (HFRS) in Guangxi, with the intention of providing a theoretical and technical support for the prevention of HFRS. A map of the incidence of HFRS of different cities in Guangxi was drawn up using the Geographic Information System (GIS) to investigate the epidemiological characteristics and infection source of HFRS between 2013 and 2016. Guangxi has a low incidence of HFRS, and autumn and winter are the main high-incidence seasons. Cases of HFRS were reported in all regions in Guangxi except Laibin city between 2013 and 2016. The distribution of cases in the four years suggested that Guilin, Nanning, Hechi and Wuzhou were the main infected regions, especially the local areas in the north of Guilin. The nucleotide and amino acid of S fragment and M fragment of Hantaviruses (HV) detected were highly homologous, and no obvious variation was found. Through analyzing the space-time characteristics, epidemiological characteristics and gene sequence of HFRS in Guangxi, it was found that areas rich in water, grass and moisture, such as paddy fields, are the main active areas for the host of HFRS.

Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway.

Role of salubrinal in protecting cardiomyocytes from doxorubicin-induced apoptosis.

We determined whether salubrinal can protect cardio-myocytes from doxorubicin-induced apoptosis and explored the related mechanisms to provide experimental evidence for exploring novel drug candidates to decrease cardiac toxicity. Neonatal rat cardiomyocytes were isolated, cultured in vitro, and pretreated with salubrinal (10, 20, or 40 µM) to observe their response to doxorubicin-induced cell apoptosis. Lactate dehydrogenase assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and flow cytometry were used to assess the extent of cardiomyocyte apoptosis. Fluorescent probes conjugated with 2',7'-dichlorofluorescein diacetate and a chemiluminescence assay were used to detect the pro-duction of reactive oxygen species. Western blotting was employed to quantify expression levels of cleaved caspase-3, cytosolic cytochrome c, and B-cell lymphoma-extra large (Bcl-xL). The mechanisms of salubrinal-related functions were also explored. Salubrinal effectively inhibited doxorubicin-induced reactive oxygen species production and nicotinamide adenine dinucleotide phosphate oxidase activation, decreased the levels of cleaved caspase-3 and cytosol cytochrome c, and increased Bcl-xL expression, thereby protecting cardiomyocytes from doxorubicin-induced apoptosis. Furthermore, salubrinal was found to protect cardiomyocytes by decreasing the dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Salubrinal can protect cardiomyocytes from doxorubicin-induced apoptosis through its effects on eIF2α. It possibly ameliorates cardiac toxicity and can be used in clinical practice.

Genetic diversity in intraspecific hybrid populations of Eucommia ulmoides Oliver evaluated from ISSR and SRAP molecular marker analysis.

Eucommia ulmoides Oliver, the only extant species of Eucommiaceae, is a second-category state-protected endangered plant in China. Evaluation of genetic diversity among some intraspecific hybrid populations of E. ulmoides Oliver is vital for breeding programs and further conservation of this rare species. We studied the genetic diversity of 130 accessions from 13 E. ulmoides intraspecific hybrid populations using inter-simple sequence related (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Of the 100 ISSR primers and 100 SRAP primer combinations screened, eight ISSRs and eight SRAPs were used to evaluate the level of polymorphism and discriminating capacity. A total number of 65 bands were amplified using eight ISSR primers, in which 50 bands (76.9%) were polymorphic, with an average of 8.1 polymorphic fragments per primer. Alternatively, another 244 bands were observed using eight SRAP primer combinations, and 163 (66.8%) of them were polymorphic, with an average of 30.5 polymorphic fragments per primer. The unweighted pair-group method (UPGMA) analysis showed that these 13 populations could be classified into three groups by the ISSR marker and two groups by the SRAP marker. Principal coordinate analysis using SRAP was completely identical to the UPGMA-based clustering, although this was partly confirmed by the results of UPGMA cluster analysis using the ISSR marker. This study provides insights into the genetic background of E. ulmoides intraspecific hybrids. The progenies of the variations "Huazhong-3", "big fruit", "Yanci", and "smooth bark" present high genetic diversity and offer great potential for E. ulmoides breeding and conservation.

Genetic diversity and population structure of Eucommia ulmoides Oliver, an endangered medicinal plant in China.

Eucommia ulmoides Oliver, one of the tertiary relict species found only in China, is the only extant species of Eucommiaceae. Using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers, we studied the genetic diversity and population genetic structure of 187 accessions from 17 E. ulmoides populations throughout its main distribution in China. A total of 65 bands were amplified using eight ISSR primers, of which 50 bands (76.9%) were polymorphic. Meanwhile, another 244 bands were observed using eight SRAP primer combinations and 163 (66.8%) of these were polymorphic. The analysis of molecular variation (AMOVA) indicated that 88.8 and 92.4% of the total variation resided within populations based on ISSR and SRAP analysis, respectively. Moreover, we found that the E. ulmoides populations were clustered into six distinct groups using ISSR and SRAP markers via the unweighted pair-group method (UPGMA). Furthermore, STRUCTURE analysis showed that these 17 populations could be classified into four groups using ISSR markers, but only two groups using SRAP markers. No significant relevancy was observed between genetic and geographic distances among the sampled populations. The results of this study support the view that exchange of seeds among local farmers plays an important role in shaping the present genetic distribution pattern. "Core collection" is suggested for genetic diversity conservation of E. ulmoides in China.

Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues.

Nelumbo nucifera is widely used as food, as an ornamental, in medicine, and as packing material; it is also reported to have anti-HIV effects and antioxidant capacity. We sought an improved method for extracting high-quality total RNA from different tissues of N. nucifera. Four methods for RNA extraction were assessed for their ability to recover high-quality RNA applicable for evaluation of polyphenol oxidase (PPO) gene expression profiles. The recovery and quality of the RNA obtained from five different tissues by the best CTAB-LiCl method were evaluated through UV light absorbance. Both A(260)/A(280) and A(260)/A(230) absorbance ratios were more than 2.0; the yield ranged from 59.87 to 163.75 µg/g fresh weight. The brightness of the 28S band was approximately twice that of 18S; the latter was also considered as high-quality RNA. The PPO gene fragment (606 bp) was successfully amplified by RT-PCR, demonstrating the integrity of the isolated RNA. The relative expression levels of the PPO gene based on RT-PCR in five tissues of lotus were: rhizome buds (2.66), young leaves (2.42), fresh cut rhizome (2.02), petals (1.80), and petiole (1.65), using housekeeping gene ß-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.

Photosynthesis, water use efficiency and stable carbon isotope composition are associated with anatomical properties of leaf and xylem in six poplar species.

Although fast-growing Populus species consume a large amount of water for biomass production, there are considerable variations in water use efficiency (WUE) across different poplar species. To compare differences in growth, WUE and anatomical properties of leaf and xylem and to examine the relationship between photosynthesis/WUE and anatomical properties of leaf and xylem, cuttings of six poplar species were grown in a botanical garden. The growth performance, photosynthesis, intrinsic WUE (WUE(i) ), stable carbon isotope composition (δ(13) C) and anatomical properties of leaf and xylem were analysed in these poplar plants. Significant differences were found in growth, photosynthesis, WUE(i) and anatomical properties among the examined species. Populus cathayana was the clone with the fastest growth and the lowest WUE(i) /δ(13) C, whereas P. × euramericana had a considerable growth increment and the highest WUE(i) /δ(13) C. Among the analysed poplar species, the highest total stomatal density in P. cathayana was correlated with its highest stomatal conductance (g(s) ) and lowest WUE(i) /δ(13) C. Moreover, significant correlations were observed between WUE(i) and abaxial stomatal density and stem vessel lumen area. These data suggest that photosynthesis, WUE(i) and δ(13) C are associated with leaf and xylem anatomy and there are tradeoffs between growth and WUE(i) . It is anticipated that some poplar species, e.g. P. × euramericana, are better candidates for water-limited regions and others, e.g. P. cathayana, may be better for water-abundant areas.

Transient population bypassed by polio vaccination programmes in Yunnan Province, China.

PIP: Four wild strains of polioviruses were isolated between 1995 and April 1996 around the Myanmar border with China. This may have occurred because immunization programs missed certain groups of children who migrated with their parents when they were searching for work. Market tradespeople, in particular, travel with their children. Since it is illegal for persons to live where they are not registered and for parents to have more than the number of children permitted by the family planning law, these children may not participate in national immunization days (NIDs) out of fear of being identified. In September and October of 1996, four markets in Yunnan Province were surveyed to determine the proportion of transients and the prevalence of oral polio vaccine (OPV) immunization; parents and guardians of 127 children, who appeared to be under the age of 5 years, were interviewed. The analysis included 91 children (45 boys and 46 girls), aged 1-3 years, who had complete information. Children were grouped as follows: 1) 37 nontransients who lived where their families were registered; 2) 15 transients from within Yunnan Province; 3) 32 transients from other provinces; and 4) 7 foreign transients. The following information was obtained for each child: 1) whether the child had received a routine OPV; 2) whether the child had a BCG scar; 3) whether the child had ever participated in a NID; and 4) whether the child had participated in the 9 supplementary OPV campaigns since 1990. There were no significant differences in sex or age between groups. As shown in a table of survey results, all indices of immunization were lower in transients. Of the nontransient children, 94.6% had received an OPV; 86.5% had a BCG scar; 89.2% had participated in a NID; and 89.2% had received OPV supplementation. The same figures for all transients as a group were 44.4%, 50.0%, 37.0%, and 38.9%, respectively. For the intraprovincial group, the figures were 66.7%, 46.7%, 60.0%, and 46.7%, respectively. For the extraprovincial group, the figures were 34.4%, 53.1%, 21.9%, and 37.5%, respectively. For foreign transients, the figures were 42.9%, 42.9%, 57.1%, and 28.6%, respectively.^ieng

[Correlation between HBV pre-C/C gene mutations and advanced pathologic activity in chronic hepatitis B].

To investigate the relationship of the HBV preC/C gene mutations with the disease persistence and exacerbation, the sequence of this region was analysed with direct sequencing by using the polymerase chain reaction product of the DNAs extracted from 24 such cases. A mutant with preC stop codon at the site 28 was detected in all the 11 anti-HBe-positive patients with different severity. This stop 28 mutant also coexisted with wild virus in seven of 18 HBeAg-positive patients. C gene mutations clustered in a small segment of codon 84-101, of those especially codon 97 was substituted in 11 of 15 mutants. There were 2, 6 and 20 codon mutations in 8 mild, 6 moderate and 10 severe cases respectively. In addition, the deletion of 144bp was also found in this region in a moderate case and that of 96bp in a severe case. Follow up of six patients revealed a close correlation between increase of genetic variation and advance of pathology.

Etiological investigation of acute post-transfusion non-A, non-B hepatitis in China.

The etiology of acute post-transfusion hepatitis (PTH) non-A, non-B (NANB) in China was investigated with the combination of advanced techniques, including a second generation of enzyme immunoassay for detection of the antibodies to hepatitis C virus (anti-HCV), a reverse transcription and nested polymerase chain reaction (RT-nPCR) for HCV RNA, and a PCR for hepatitis B virus DNA. Of the 57 patients who were diagnosed as acute PTH-NANB, 46 (80.7%) were positive for anti-HCV and 41 (71.9%) had HCV RNA. Combining together, 53 (93.0%) were seropositive for anti-HCV and/or HCV RNA. Surprisingly, 18 of these with HCV markers were also positive for HBV DNA, although they were negative for HBsAg, suggesting that a portion of the patients with acute PTH NANB were coinfected by both HCV and HBV. In addition, 4 (7%) of the patients with acute PTH-NANB had no detectable HCV and HBV markers with the use of the above-mentioned techniques. These results indicate that etiologic agents(s) other than HCV and HBV may also cause acute PTH-NANB or that the current techniques may still not be sensitive enough to detect trace levels of HCV and HBV markers.

Correlation between the prevalence of serum HBV DNA and immunoserologic HBV markers in the subjects with or without hepatitis.

The prevalence of serum HBV DNA, detected by polymerase chain reaction, and that of immunoserologic HBV markers (HBsAg, HBeAg, anti-HBs, and anti-HBc), determined by immunoassays, were compared among three groups of subjects: (A) chronic active hepatitis B patients, (B) chronic asymptomatic HBV carriers, and (C) normal individuals. Except five of the normal individuals, all of the subjects are positive for anti-HBc while some of them were also positive for other immunoserologic HBV markers, such as anti-HBs, HBsAg, and HBeAg. Serum HBV DNA were detected in 81% in group A, 52% in group B, and 20% in group C. In both group A and B, serum HBV DNA were detected in all the subjects with anti-HBc+/HBsAg+/HBeAg+. However, the percentage of seropositive HBV DNA in the subjects with anti-HBc+/HBsAg+ in Group A was much higher than that in Group B. Interestingly, the percent of serum HBV DNA+ in the individuals with anti-HBc+ only was markedly higher than that in the subjects with anti-HBc+/anti-HBs+ in both Group A and C, suggesting that anti-HBs may play a role in the inhibition of HBV replication and clearance of HBV virion from blood. Above serological profiles will provide important information concerning the significance of serum HBV DNA detection in judgement of HBV replication in the individuals with or without HBV infection. Cautions should be taken to clarify those so called normal individuals who have no symptoms of hepatitis B, no HBsAg in the sera and normal transaminase, but have HBV replication in their bodies.

Hepatitis B virus DNA in sera of virus carriers positive exclusively for antibodies to the hepatitis B core antigen.

The prevalence of serum HBV DNA in individuals positive for anti-HBc alone was determined by the polymerase chain reaction in two groups with endemic HBV infection from Canton (group A) and Hainan (group B), provinces of China. Twenty-one out of 294 individuals in group A (7.2%) and 193 out of 1995 in group B (9.7%) were positive for anti-HBc but negative for other markers of ongoing or past HBV infection (HBsAg and anti-HBs). HBV DNA was detected in 6/21 sera in group A (28.6%) and 68/193 in group B (35.2%) in their initial serum specimen. One of the six HBV-DNA-positive individuals in group A became negative after 6 months and four of the 58 positive in group B became negative at 4 years of follow-up. All of the individuals remained positive for anti-HBc and negative for anti-HBs, but one of them became positive for HBsAg on follow-up. None of the anti-HBc- and HBV-DNA-positive subjects had symptoms of liver diseases. They were, therefore, defined as chronic asymptomatic HBV carriers with undetectable HBsAg. This type of carrier should be added to the typical HBsAg-positive carrier, who constitutes about 10-15% of the general Chinese population, to give a more complete estimate of asymptomatic HBV carriers in China.

[Identification of HBV infection in HBsAg-negative chronic active hepatitis with polymerase chain reaction].

To explore the etiological agent(s) of HBsAg negative chronic active hepatitis, serum HBV DNA was tested in thirty-six of such cases with polymerase chain reaction. In total, viral DNA was detected in 24 cases (67%), including 11 of the 12 cases with positive anti-HBc alone, four of the nine cases with positive anti-HBs and nine of the 15 cases without any HBV markers. The results suggest that in cases of HBsAg-negative chronic active hepatitis, the etiology may still be hepatitis B virus, especially in cases with positive anti-HBc. Furthermore, the cause of activated histology might be due to viral replication.