[Pathological study on the relationship between nucleic acid oxidative stress and heart failure with preserved ejection fraction in patients aged over 85 years].

Objective: To investigate the level of nucleic acid oxidation in myocardial tissue of patients aged over 85 with heart failure with preserved ejection fraction (HFpEF) and the correlation with myocardial amyloid deposition. Methods: This was a retrospective case-control study. Data of patients≥85 years old who underwent systematic pathological autopsy in Beijing Hospital from 2003 to 2017 were retrospectively collected. Twenty-six patients were included in the HFpEF group and 13 age-and sex-matched patients who had not been diagnosed with heart failure and died of non-cardiovascular diseases served as the control group. The left ventricular myocardium slices of both groups were semi-quantitatively analyzed using immunohistochemical staining of 8-oxidized guanine riboside (8-oxo-G) and 8-oxidized guanine deoxyriboside (8-oxo-dG) to evaluate the oxidation of RNA and DNA in cardiomyocytes. Using the median of the mean absorbance value of 8-oxo-G immunohistochemical staining as the cut-off value, patients were divided into high-absorbance group and low-absorbance group. Congo red staining was used to compare myocardial amyloid deposition between the two groups. Results: The mean age of patients in HFpEF group was (91.8±3.7) years, 24 (92.3%) were males. The mean age of patients in control group was (91.7±3.7) years old, 11 (84.6%) were males. The median mean optical absorbance value of 8-oxo-G immunohistochemical staining of myocardium was significantly higher in HFpEF patients than in control group (0.313 8 (0.302 2, 0.340 6) vs. 0.289 2 (0.276 7, 0.299 4), Z=-3.245, P=0.001). The median mean absorbance value of 8-oxo-dG immunohistochemical staining of myocardial tissue was similar between the two groups (0.300 0 (0.290 0, 0.322 5) vs. 0.300 0 (0.290 0, 0.320 0), Z=-0.454, P=0.661). Proportion of patients with moderate and severe cardiac amyloid deposition was significantly higher in the high-absorbance group than in the low-absorbance group ((85.0%, 17/20) vs. (31.6%, 6/19), P=0.001). Conclusion: The RNA oxidation degree of myocardium in HFpEF patients is higher than that in elderly people without heart failure. Degree of myocardial amyloid deposits is higher in patients with high levels of RNA oxidation.

[Study on the clinical significance of miR-100 expression in the invasion and metastasis of hepatocellular carcinoma].

Objective: To study the miR-100 expression levels in the tissues of hepatocellular carcinoma patients, and to further explore the correlation between miR-100 and the invasion and metastasis of hepatocellular carcinoma cells and its effect on patients' prognostic survival. Methods: Clinicopathological data of 70 cases that underwent hepatectomy from December 2013 to December 2016 in the Department of Hepatobiliary and Pancreatic Surgery of Henan Provincial People's Hospital were retrospectively analyzed. Real-time fluorescent quantitative PCR was used to detect the different miR-100 expression levels in cancerous and adjacent tissues. The expression of miR-100 with different clinicopathological features was compared, and the prognostic factors of patients with hepatocellular carcinoma were comprehensively analyzed. The correlation between miR-100 and patients' clinicopathological features was tested by χ(2). Kaplan-Meier method was used to draw the survival curve. Log-rank test was used to examine the survival rate difference in each subgroup. Cox regression model was used to analyze the multivariate prognosis. Results: miR-100 expression was down-regulated to a different degree in hepatocellular carcinoma tissues than the corresponding adjacent tissues. Among them, the down-regulated expression of miR-100 in hepatocellular carcinoma tissues accounted for 82.9% (58/70, P < 0.05) of all cases when compared to corresponding paracancerous tissues. miR-100 expression level was significantly correlated to high Edmondson's grade, high TNM stage and intrahepatic metastasis (P < 0.05). The overall survival time of miR-100 positive expression was significantly higher than that of miR-100 negative expression (Log-rank χ(2) = 8.257, P < 0.05). Univariate survival analysis results revealed that the miR-100 expression level, tumor size, TNM stage, Edmondson's grade, and presence or absence of venous tumor thrombosis had a poor prognosis (P < 0.05). Cox multivariate regression analysis showed that the tumor size, Edmondson's grade, and miR-100 expression level were independent factors affecting the prognostic survival in hepatocellular carcinoma patients. In addition, patients with low positive expression rate of miR-100, large tumors and high Edmondson's grade had a poor prognosis. Conclusion: The level of miR-100 expression in hepatocellular carcinoma cells is low, so it is closely related to the invasion and metastasis and affects the prognostic survival of hepatocellular carcinoma patients.

[The prognostic value of circulating microRNA-29b in left ventricular hypertrophy in patients with hypertension].

Objective: To analyze the relationship between the level of microRNA-29b in circulation and left ventricular hypertrophy in hypertensive patients. Methods: A total of 240 subjects from Henan Province People's Hospital from June 2015 to June 2018 were included in the present study. Among them, 160 were hospitalized patients, and were divided into two groups. Patients with simple hypertension and had no left ventricular hypertrophy (80 cases) were in the simple hypertension group (HBP-NLVH), and patients with hypertension combined with left ventricular hypertrophy (80 cases) were in the high blood pressure with left ventricular hypertrophy group (HBP-LVH). Normal control subjects (80 cases) were those with no hypertension and randomly selected from the medical center of Henan Province People's Hospital. Serum microRNA-29b expressions were detected by real time fluorescence quantitative PCR. The thickness of interventricular septum (IVSD) and left ventricular posterior wall thickness (LVPWD) were measured by echocardiography. Results: Compared with the normal control group (1.95±0.79), the relative expression of microRNA-29b in the patients both in the HBP-NLVH group (2.67±0.92) and the HBP-LVH group (5.12±1.23) was up-regulated, and the difference between normal control and patients was statistically significant (P<0.05). In patients, the microRNA-29b level in the HBP-LVH group was significantly higher than that in the HBP-NLVH group (P<0.05). The expression level of microRNA-29b was positively correlated with IVSD (r=0.71, P<0.05) and LVPWD (r=0.74, P<0.05), respectively. The sensitivity and specificity of serum microRNA-29b levels in the diagnosis of left ventricular hypertrophy in hypertension patients were 96.8% and 91.3%, respectively. Conclusion: Serum microRNA-29b level is elevated in hypertensive patients with left ventricular hypertrophy, and is positively correlated with left ventricular hypertrophy. The circulation microRNA-29b might be a useful biomarker with prognostic value in left ventricular hypertrophy in hypertension patients.

[The effect of estrogen level on Budd Chiari syndrome related hepatocellular carcinoma].

Objective: To investigate the effect of estrogen level on Budd Chiari syndrome related hepatocellular carcinoma. Methods: Immunohistochemical method was used to detect estrogen receptor-α and estrogen receptor-ß expression in 38 cases of Budd Chiari syndrome related hepatocellular carcinoma and 50 cases of HBV related hepatocellular carcinoma.Hepatoma cells of Budd Chiari syndrome related hepatocellular carcinoma were exposed to different concentrations of Estrogen for 48 hours. Tetrazolium bromide (MTT) colorimetry was used to analyze cell proliferation activities; cell cycle was analyzed by flow cytometry (FCM); cell apoptosis was analyzed by flow cytometry (FCM) and Casepase-3 activity was measured after induced by adriamycin(ADM). Results: The positive rate of estrogen receptor-α expression in the tissues of Budd Chiari syndrome related hepatocellular carcinoma was 71.05%, which was higher than that (32%)in HBV related hepatocellular carcinoma tissue evidently (P<0.01). The positive rate of estrogen receptor-ß expression in the tissues of Budd Chiari syndrome related hepatocellular carcinoma was 68.4%, which was higher than that (26%)in HBV related hepatocellular carcinoma tissue evidently (P<0.01). With the concentrations of estrogen increasing, MTT Assays showed that estrogen level increased the cell proliferation activities of Budd Chiari syndrome related hepatocellular carcinoma. The number of cells at stage S and G2/M were significantly increased and cells at stage G0/G1 were reduced with the increasing concentrations of estrogen. After being incubated under the different concentrations of estrogen for 48 h, the apoptosis rates decreased gradually and the Casepase-3 activity was significantly reduced with the increasing concentration of estrogen. Conclusions: Estrogenreceptor expression may have an important influence on hepatocellular carcinoma cell biology difference between Budd Chiari syndrome related hepatocellular carcinoma and HBV related hepatocellular carcinoma. Estrogen level can promote cell proliferation and cell cycle, and inhibit the apoptosis of hepatoma cells of Budd Chiari syndrome related hepatocellular carcinoma in vitro, and these effects were increased with the increasing of estrogen level.

[Value of RNA oxidation product 8-oxo-Gsn in evaluating renal function in patients with chronic kidney disease].

Objective: To investigate the relationship of DNA oxidative product 8-oxo-dGsn and RNA oxidative product 8-oxo-Gsn with chronic kidney disease (CKD). Methods: Between January 2015 and December 2016, 146 cases of CKD (30, 30, 31, 30 and 25 cases of CKD stage 1-5, respectively) were collected in the Department of Nephrology in Beijing Hospital. Among them, 70 cases were male, accounting for 47.95%. The age distribution ranged from 21 to 88 years, with an average age of (56.43±16.79) years. Their fasting blood and morning urine were collected. The levels of 8-oxo-dGsn and 8-oxo-Gsn in plasma and urine were quantified by isotope-diluted liquid chromatography mass spectrometry (MS)/MS (ID-LC-MS/MS). Results: The urine 8-oxo-Gsn/Cr in patients with CKD stage 1-5 was (3.07±1.07) µmol/mol, (3.42±1.34) µmol/mol, (3.72±1.47) µmol/mol, (3.90±1.93) µmol/mol and (3.75±2.26) µmol/mol, respectively. The urinary 8-oxo-Gsn content in CKD stage 4 patients was significantly higher than those of other 4 stages (P<0.05). The serum/urine ratio of 8-oxo-Gsn was 0.02±0.02, 0.03±0.02, 0.06±0.04, 0.10±0.05 and 0.34±0.03, respectively, and in CKD stage 4 and 5 patients, it increased significantly, especially in CKD stage 5 cases (P<0.05). Expression of 8-oxo-Gsn had a good correlation with renal function[the Spearman 's correlation coefficient: serum 8-oxo-Gsn and serum creatinine was 0.629 (P<0.001); urine/serum 8-oxo-Gsn and eGFR was 0.799 (P<0.001); serum/urine 8-oxo-Gsn and serum/urine creatinine was 0.888 (P<0.001)]. With age increasing, CKD patients showed increased RNA oxidation, and 8-oxo-Gsn increased significantly in patients over 60 years (P<0.05). After multiple linear regression analysis, 8-oxo-Gsn was only associated with serum creatinine (ß=0.656, t=8.275, P<0.001). Conclusions: Our finding indicates that the RNA oxidation occurs in patients with renal disease, and its oxidation increased as the disease progressing. The significant increase in the ratio of plasma and urinary 8-oxo-Gsn is of great importance on evaluating renal function.

Identification of a new non-synonymous mutation in HLA-B gene, HLA-B*15:320, in a Chinese individual by sequence-based typing.

The novel allele HLA-B*15:320 differs from HLA-B*15:01:01:01 at position 709 in exon 4 (A>T, Ile>Phe).

In vitro assessment of 39 CYP2C9 variants found in the Chinese population on the metabolism of the model substrate fluoxetine and a summary of their effects on other substrates.

WHAT IS KNOWN AND OBJECTIVE: Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme that is responsible for clearing approximately 15% of clinically important drugs. The objective of this study was to assess the catalytic characteristics of 39 CYP2C9 isoforms found in the Chinese population and their effects on the metabolism of the model substrate fluoxetine in vitro. METHODS: Baculovirus-mediated expressing system was used to highly express wild-type and the 38 CYP2C9 allelic variants in insect cell microsomes. Then, the enzymatic characteristics of each variant were evaluated using fluoxetine as the substrate. Reactions were performed at 37 °C with the insect microsomes and 10-200 µm fluoxetine for 60 min. After termination, the products were precipitated and used for signal collection by UPLC-MS/MS. RESULTS AND DISCUSSION: Of the 39 tested CYP2C9 isoforms, only four variants (CYP2C9*3, CYP2C9*27, CYP2C9*34 and CYP2C9*37) exhibited similar relative clearance values to that of the wild-type CYP2C9*1. Moreover, five variants (CYP2C9*14, CYP2C9*36, CYP2C9*45, CYP2C9*48 and CYP2C9*55) showed a higher intrinsic clearance value than the wild-type protein, whereas the remaining 29 CYP2C9 isoforms exhibited significantly decreased clearance values (from 6·23% to 87·74%) compared to CYP2C9*1. In addition, 28 CYP2C9 isoforms including CYP2C9*3 exhibited a trend towards substrate inhibition for fluoxetine. WHAT IS NEW AND CONCLUSION: This study provides the most comprehensive data on the enzymatic activities associated with all reported CYP2C9 variants in the Chinese population with regard to the widely used antidepressant drug, fluoxetine. Our data indicate that more attention should be paid to subjects carrying the corresponding infrequent CYP2C9 alleles when administering fluoxetine in the clinic.

293FT is a highly suitable mammalian cell line for the in vitro enzymatic activity analysis of typical P450 proteins.

Mammalian cells have been widely used for the in vitro evaluation of the functional effect of allelic variants of cytochrome P450 (CYP). The aim of this study was to determine the most suitable mammalian cell line for the in vitro drug metabolism analysis of CYP variants. Three reported cell lines (COS-7, HepG2, 293T) and one fast-growing variant of the 293 cell line 293FT were transfected with vectors expressing green fluorescent protein or typical variants of CYP2C9, CYP2C19 or CYP2D6 to investigate the protein expression levels and the catalytic activity of expressed CYP allelic variants. The transfected 293FT cells had the highest protein expression level and exhibited the highest enzymatic activity, while HepG2 cells showed the lowest activity among the four tested cell lines. Simultaneously, 293FT cells still maintained the similar relative enzymatic ratio among three typical CYP2C9 variants to that of the commonly used COS-7 cells. In addition, 293FT cells could also be used for the in vitro functional evaluation of two other typical P450 proteins, CYP2C19 and CYP2D6. Therefore, the 293FT cell line is more suitable for the in vitro enzymatic activity analysis of typical P450 proteins than any other reported mammalian cell lines.

A new HLA-A*24 allele, HLA-A*24:02:87, identified by sequencing-based typing in a Chinese volunteer bone marrow donor.

Novel allele HLA-A*24:02:87 has one nucleotide change with A*24:02:01:01 in exon 3 at position 594 C>T.

CYP2C9 polymorphism analysis in Han Chinese populations: building the largest allele frequency database.

Genetic polymorphisms of CYP2C9 significantly influence the pharmacokinetics and pharmacodynamics of some drugs, which might result in adverse drug effects and therapeutic failure. Several studies have been performed on CYP2C9 genetic polymorphisms in Han Chinese populations. However, these studies only focused on two commonly investigated alleles, *2 and *3, in relatively small sample sizes. To scale up the gene-scanning region and determine relatively precise data on the genetic distribution pattern in Chinese populations, unrelated healthy Han Chinese volunteers from Zhejiang Province (n=1127) and Hebei (n=1000) Province were recruited as subjects for the direct sequencing of all exons of CYP2C9. As a result, 14 previously reported alleles were detected in this work, and 8 of these alleles (*14, *16, *19, *23, *27, *29, *33 and *34) were described for the first time in Chinese populations. In addition, 37 novel mutations were also detected, of which 22 variants were non-synonymous, and 21 new alleles, *36-*56, were designated by the Human CYP Allele Nomenclature Committee. In vitro functional analysis of these 22 novel CYP2C9 variants revealed that 17 mutations had a significant influence on the protein's catalytic activity. Our study provides the most accurate data on CYP2C9 polymorphisms in Han Chinese populations and detects the largest number of novel allelic variants existing to date. These new alleles will greatly enrich the current knowledge of naturally occurring CYP2C9 variants in Chinese populations.

HOTAIR: a cancer-related long non-coding RNA.

Long non-coding RNA was dismissed as merely transcriptional "noise" in the past decades. Numerous researches have shown that lncRNAs regulated gene expression at the epigenetic level. Moreover, lncRNAs played important roles in proliferation, apoptosis and invasiveness of tumor cells, and participated in metastatic capacity of cancers. Recent studies revealed HOX transcript antisense RNA, a lncRNA with regulatory functions of transcription, could bind PRC2 and LSD1/CoREST/REST complexes and direct to the specific gene sites, resulted in H3K27 methylation and H3K4 demethylation and ultimately gene silencing. Aberrant HOTAIR expression was associated with various sites of cancers such as breast, hepatocellular, gastric, colorectal, pancreatic et al; and affected survival and prognosis of cancer patients. In this review, we introduce an overall view of HOTAIR by describing the known molecular mechanisms and potential functions of HOTAIR and summarizing the latest progresses on the research of HOTAIR in various human cancers.

Identification of a new HLA-B*46 allele, B*46:37, in a Chinese individual.

A novel allele B*46:37 was identified in a Chinese individual.

Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models.

Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection-galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis.

Causal roles of daytime sleepiness in cardiometabolic diseases and osteoporosis.

OBJECTIVE: Daytime sleepiness has some association with cardiometabolic diseases and osteoporosis, but it is unknown whether their relationship is causal. This two-sample Mendelian randomization (MR) study aims to explore their causal relationship. MATERIALS AND METHODS: We included the largest genome-wide association studies (GWASs) associated with daytime sleepiness, cardiometabolic diseases and osteoporosis. 34 single nucleotide polymorphisms (SNPs) were used as the instrumental variables of daytime sleepiness. RESULTS: Genetic predisposition to excessive daytime sleepiness was strongly associated with increased risk of coronary artery disease (beta-estimate: 0.610, 95% confidence interval [CI]: 0.128 to 1.093, standard error [SE]: 0.246, p-value=0.013) and may increase the incidence of type 2 diabetes (beta-estimate: 0.614, 95% CI: 0.009 to 1.219, SE: 0.309, p-value=0.047). We found no causal influence of daytime sleepiness on heart failure, atrial fibrillation, cerebral ischemia, intracerebral hemorrhage, forearm bone mineral density (FA-BMD), femoral neck BMD (FN-BMD), and lumbar spine BMD (LS-BMD). CONCLUSIONS: This study suggested that excessive daytime sleepiness was causally associated with increased risk of coronary artery disease, which may benefit to prevent this disease.

Identification of a novel allele HLA-A*2489 by sequence-based typing in a Chinese individual.

A novel human leukocyte antigen-A allele, A*2489, was identified in a Chinese registered donor. The A*2489 allele differs from the closest matching allele A*2428 by two nucleotide substitutions, 257(A-->G) and 265(G-->C), in exon 2.

Analysis of high-resolution HLA-A, -B, -Cw, -DRB1, and -DQB1 alleles and haplotypes in 718 Chinese marrow donors based on donor-recipient confirmatory typings.

High-resolution human leucocyte antigen (HLA)-A, -B, -Cw, -DRB1, and -DQB1 alleles and haplotype frequencies were analysed from 718 Chinese healthy donors selected from the Chinese Marrow Donor Program registry based on HLA donor-recipient confirmatory typings. A total of 28 HLA-A, 61 HLA-B, 30 HLA-Cw, 40 HLA-DRB1 and 18 HLA-DQB1 alleles were identified, and HLA-A*1101, A*2402, A*0201, B*4001, Cw*0702, Cw*0102, Cw*0304, DRB1*0901, DRB1*1501, DQB1*0301, DQB1*0303 and DQB1*0601 were found with frequencies higher than 10% in this study population. Multiple-locus haplotype analysis by the maximum-likelihood method revealed 45 A-B, 38 Cw-B, 47 B-DRB1, 29 DRB1-DQB1, 24 A-B-DRB1, 38 A-Cw-B, 23 A-Cw-B-DRB1, 33 Cw-B-DRB1-DQB1 and 22 A-Cw-B-DRB1-DQB1 haplotypes with frequencies >0.5%. The most common two-, three-, four- and five-locus haplotypes in this population were: A*0207-B*4601 (7.34%), Cw*0102-B*4601 (8.71%), B*1302-DRB1*0701 (6.19%), DRB1*0901-DQB1*0303 (14.27%), A*3001-B*1302-DRB1*0701 (5.36%), A*0207-Cw*0102-B*4601 (7.06%), A*3001-Cw*0602-B*1302-DRB1*0701 (5.36%), Cw*0602-B*1302-DRB1*0701-DQB1*0202 (6.12%) and A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0202 (5.29%). Presentation of the high-resolution alleles and haplotypes data at HLA-A, -B, -Cw, -DRB1 and -DQB1 loci will be useful for HLA matching in transplantation as well as for other medical and anthropological applications in the Chinese population.

Characterization of the novel HLA-C*03:02:17 allele by sequencing-based typing.

HLA-C*03:02:17 differs from HLA-C*03:02:02:01 by one nucleotide substitution at position 393.

Characterization of the novel HLA-B*40:01:51 allele by sequencing-based typing.

HLA-B*40:01:51 differs from HLA-B*40:01:01 by 2 nucleotide substitutions at position 72 and 126.