[Association of sleep duration and risk of frailty among the elderly over 80 years old in China: a prospective cohort study].

Objective: To explore the association between sleep duration and the risk of frailty among the elderly over 80 years old in China. Methods: Using the data from five surveys of the China Elderly Health Influencing Factors Follow-up Survey (CLHLS) (2005, 2008-2009, 2011-2012, 2014, and 2017-2018), 7 024 elderly people aged 80 years and above were selected as the study subjects. Questionnaires and physical examinations were used to collect information on sleep time, general demographic characteristics, functional status, physical signs, and illness. The frailty state was evaluated based on a frailty index that included 39 variables. The Cox proportional risk regression model was used to analyze the correlation between sleep time and the risk of frailty occurrence. A restricted cubic spline function was used to analyze the dose-response relationship between sleep time and the risk of frailty occurrence. The likelihood ratio test was used to analyze the interaction between age, gender, sleep quality, cognitive impairment, and sleep duration. Results: The age M (Q1, Q3) of 7 024 subjects was 87 (82, 92) years old, with a total of 3 435 (48.9%) patients experiencing frailty. The results of restricted cubic spline function analysis showed that there was an approximate U-shaped relationship between sleep time and the risk of frailty. When sleep time was 6.5-8.5 hours, the elderly had the lowest risk of frailty; Multivariate Cox proportional risk regression model analysis showed that compared to 6.5-8.5 hours of sleep, long sleep duration (>8.5 hours) increased the risk of frailty by 13% (HR: 1.13; 95%CI: 1.04-1.22). Conclusion: There is a nonlinear association between sleep time and the risk of frailty in the elderly.

[The application of Hoffmann method in the establishment of children's reference intervals of erythrocyte count].

OBJECTIVE: To verify the reliability and feasibility of Hoffmann method in establishing pediatric reference intervals (RI) of erythrocyte count. METHODS: Three hundreds and ninty-two thousands of hospital-based data for erythrocyte count of children aged in 1 to 17, measured by the Sysmex Xs-800i, was collected from Beijing Children's Hospital during January to December 2014. Outliers were removed using the Dixon method, then Hoffmann method was conducted to establish the gender and age stratified pediatric RIs of erythrocyte count. The erythrocyte count of 2 217 healthy children, recruited from Beijing Children's Hospital and Liaocheng Children's Hospital in Shandong province, was conducted as normal reference to verify the reliability of Hoffmann method in establishing RIs and to compare with existing RIs. RESULTS: In 4 subgroups as following, male aging 1 to 12 years, male aging 13 to 17 years, female aging 1 to 12 years, female aging 13 to 17 years, the RIs of erythrocyte count established using Hoffmann method were (4.1-5.4)×10(12)/L, (4.4-5.7)×10(12)/L, (4.0-5.3)×10(12)/L, (4.0-5.3)×10(12)/L, respectively. The verification results in 2 217 healthy children showed that the proportions of out of range in four subgroups were 6.17%, 8.81%, 6.22%, 7.78%, respectively. CONCLUSION: Hoffmann method produce reliable RIs according with the actual situation in healthy children, which is also convenient and is worth popularizing in clinical practice.

Influence of adenosine on preservation of porcine pancreas in islet transplantation.

Ischemia time during transplantation has greatly restricted the quality and utilization of grafts. To improve the quality of islet transplantation, adenosine was added into the University of Wisconsin (UW) pancreas perfusate to assess its effect on islet yield and function in porcine pancreas. Ten pancreata from donation after cardiac death pigs were obtained and randomly divided into two groups: control group (N = 5) with UW perfusion solution, and experimental group (N = 5) with adenosine-enriched UW perfusion solution. The yield and purity of the islet cells were counted after they were collected, purified, and stained with dithizone. Acridine orange/propidium iodide staining was applied to determine islet cell viability. Islet function was assessed by glucose stimulated insulin secretion assays, and released insulin was quantified by enzyme-linked immunosorbent assay. The metabolic substrates of the pancreas were analyzed by trace dialysis technology. We found that the addition of adenosine in UW perfusion solution significantly increased the yield, purity and viability of islet cells, as well as enhanced their insulin release. In addition, the levels of metabolic substrates, pyruvate and lactate, were significantly reduced. The addition of adenosine could effectively increase islet cell viability during mechanical perfusions, which may improve islet transplantation. This perfusion protocol may be clinically feasible, and should be considered in the clinical setting.

Identification of long non-coding RNA involved in osteogenic differentiation from mesenchymal stem cells using RNA-Seq data.

The aim of this study was to identify long non-coding RNA (lncRNA) associated with osteogenic differentiation from mesenchymal stem cells (MSCs) using high-throughput RNA sequencing (RNA-Seq) data. RNA-Seq dataset was obtained from the European Bioinformatics Institute (accession No. PRJEB4496), including two replicates each for immortalized mesenchymal stem cells iMSC#3 cultured in growth medium (GM) and differentiation medium (DM) for 28 days. The clean reads were aligned to a hg19 reference genome by Tophat and assembled by Cufflinks to identify the known and novel transcripts. RPKM values were calculated to screen for differentially expressed RNA. Novel lncRNA were screened based on various filter criteria. Subsequently, the underlying function of novel lncRNAs were predicted by functional annotation by ERPIN, a co-expression network was constructed by WGCNA and the KEGG pathway enriched by KOBAS. A total of 3171 RNA differentially expressed between the DM and GM groups (2597 mRNA and 574 lncRNA) were identified. Among the 574 differentially expressed lncRNA, 357 were known and 217 were novel lncRNA. Furthermore, 32 novel lncRNA were found to be miRNA precursors (including miR-689, miR-640, miR-601, and miR-544). A total of 14,275 co-expression relationships and 217 co-expression networks were obtained between novel lncRNA and mRNA. The differentially expressed lncRNA and mRNA were enriched into 6 significant pathways, including those for cancer, ECM-receptor interaction, and focal adhesion. Therefore, novel lncRNAwere identified and their underlying function predicted, which may provide the basis for future analyses of the role of lncRNA in osteoblastic differentiation.

[Association of lifestyle and apolipoprotein E gene with risk for cognitive frailty in elderly population in China].

Objective: To investigate the impact of lifestyle, apolipoprotein E (ApoE) gene, and their interaction on the risk for cognitive frailty in the elderly population in China. Methods: The study participants were from the Chinese Longitudinal Healthy Longevity Survey. The information about their lifestyles were collected by questionnaire survey, and a weighted lifestyle score was constructed based on ß coefficients associated with specific lifestyles to assess the combined lifestyle. ApoE genotypes were assessed by rs429358 and rs7412 single nucleotide polymorphisms. Cognitive frailty was assessed based on cognitive function and physical frailty. Cox proportional hazards regression model was used to analyze the association of lifestyle and ApoE gene with the risk for cognitive frailty and evaluate the multiplicative and additive interactions between lifestyle and ApoE gene. Results: A total of 5 676 elderly persons, with median age [M (Q1, Q3)] of 76 (68, 85) years, were included, in whom 615 had cognitive frailty. The analysis by Cox proportional hazards regression model indicated that moderate and high levels of dietary diversity could reduce the risk for cognitive frailty by 18% [hazard ratio (HR)=0.82, 95%CI: 0.68-1.00] and 28% (HR=0.72, 95%CI: 0.57-0.91), respectively; moderate and high levels of physical activity could reduce the risk by 31% (HR=0.69, 95%CI: 0.56-0.85) and 23% (HR=0.77, 95%CI: 0.64-0.93), respectively. Healthy lifestyle was associated with a 40% reduced risk for cognitive frailty (HR=0.60, 95%CI: 0.46-0.78). ApoE ε4 allele was associated with a 26% increased risk for cognitive frailty (HR=1.26, 95%CI: 1.02-1.56). No multiplicative or additive interactions were found between lifestyle and ApoE gene. Conclusions: Dietary diversity and regular physical activity have protective effects against cognitive frailty in elderly population. Healthy lifestyle can reduce the risk for cognitive frailty in elderly population regardless of ApoE ε4 allele carriage status.

The gelsolin level in patients with primary Sjogren's syndrome.

OBJECTIVE: Gelsolin (GSN) is a multifunctional protein that can regulate cell proliferation, apoptosis, inflammation and infection. GSN has been reported to be involved in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and many other diseases. The role of GSN in primary Sjogren's syndrome (pSS) remains still unclear. The aim of this study is to investigate the changes of GSN level in serum and whole blood cells of pSS patients and evaluate the relationship between GSN and fatigue or other clinical indicators. PATIENTS AND METHODS: The cross-sectional study included 47 pSS patients (1 male and 46 females, average age: 52.83±12.63 years) and 51 healthy controls (all females, average age: 50.61±9.86 years). The patients were collected from the Second Affiliated Hospital of Harbin Medical University, China, without the age and sex differences. The levels of GSN in serum of pSS patients and the healthy controls were measured by Western blotting. The sequencing gene expression omnibus (GEO) data from National Center for Biotechnology Information (NCBI) about GSN levels in the whole blood cells of pSS patients and the healthy controls were analyzed by R language. RESULTS: Compared with healthy controls, the level of GSN was significantly decreased in the serum of pSS patients (98.89 ± 28.94 vs. 131.6 ± 37.1 µg/ml, p<0.001). The expression of GSN in the whole blood cells of pSS patients was significantly lower than that in the healthy controls (6.4 ± 0.19 vs. 6.6 ± 0.17, p<0.01). Compared to non-fatigued pSS patients, the level of GSN was down-regulated in serum (85.69 ± 27.08 vs. 111.52 ± 24.71 µg/ml, p<0.01) and whole blood cells (6.43 ± 0.18 vs. 6.58 ± 0.21, p<0.001) in fatigue pSS patients. However, there was no significant correlation between the level of GSN and EULAR Sjogrens syndrome disease activity index (ESSDAI) in pSS patients (p=0.73). CONCLUSIONS: GSN is decreased in serum and whole blood cells of pSS patients, and it is much lower in fatigue patients than that in non-fatigue patients. The correlation between the level of GSN and ESSDAI was not significant in pSS patients.

Detection of fetal sex in the peripheral blood of pregnant women.

OBJECTIVE: To choose the best method to examine fetal sex in maternal blood as early as possible and evaluate the quantitative change of fetal-free DNA in maternal plasma. METHOD: One hundred and fifty pregnant women were studied at 5-9 completed weeks of gestation. Fetal cells were isolated using lymphocyte separation liquid and 3% gelatin. Furthermore, fluorescence in situ hybridization was used to examine the terminal of the Y chromosome. Nested polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (FQ-PCR) were used to amplify the SRY gene of the plasma DNA extracted from the same 150 samples of maternal blood. Sequential analysis was performed using FQ-PCR during the whole pregnancy on 32 pregnant women carrying male fetuses. RESULTS: Using fluorescence in situ hybridization, we can find male fetal cells in maternal blood as early as the 49th day. We can also find free fetal DNA in maternal plasma as early as the 49th and the 42nd day of pregnancy using nested PCR and FQ-PCR. The amount of fetal DNA was increasing with the gestation week. The standard value of every gestation week was obtained by FQ-PCR. CONCLUSION: FQ-PCR was the best method to detect fetal sex in early pregnancy. There is a principle of quantitative change of free fetal DNA in maternal plasma.

Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection.

A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification, characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR). The PCR products were labeled as probes with DIG-11-dUTP using the random priming method. Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4. A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed. Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences. The size of inserts generated by PCR ranged from 140bp to 500bp. This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.

[Phylogenetic study of Artemia from China using RAPD and AFLP markers].

We have applied the techniques of RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism) to the analysis of the relationships among Artemia species and strains. RAPD markers were successfully employed to detect diversity and genetic differentiation among four species of brine shrimp: A. franciscana, A. urmiana, A. sinica, and A. parthenogenetica. Seventy, ten-base synthetic oligonucleotides were used to amplify a total of 458 distinct fragments. DNA polymorphisms were found in all the species examined; The highest percentage of polymorphic bands found in A. parthenogenetica, was 28.8 per cent. There are significant differences between bisexual sibling species and parthenogenetic populations. A. parthenogenetica provided 94 specific molecular markers, while bisexual sibling species gave 27 specific molecular markers. A. sinica is a species distinct from the other Old World bisexual species. AFLP were used to analyze 15 Artemia species and strains for genetic diversity. They are extremely sensitive to even a small sequence variation and more polymorphism than RAPD. Using only 10 pairs of primer combinations, we detected 580 AFLP bands of which were polymerphic. The RAPD and AFLP techniques are powerful DNA fingerprinting methods for classification of Artemia species and strains.

The cotton ATP synthase δ1 subunit is required to maintain a higher ATP/ADP ratio that facilitates rapid fibre cell elongation.

The δ subunit of mitochondrial ATP synthase serves as a linker between the F(0) and F(1) sectors. Here, through microarray and quantitative RT-PCR, we found that the δ1 subunit was significantly up-regulated during cotton fibre cell elongation. Both the relative level and duration of GhATPδ1 transcripts correlated positively with the final length of different cotton germplasms. Elongating fibre cells had a significantly elevated ATP/ADP ratio, suggesting that a higher energy input is probably required for primary fibre cell wall formation and elongation. We obtained a putative full-length GhATPδ1 cDNA that shows 37% sequence identity to the Saccharomyces cerevisiae ATP16 at the deduced amino acid level. An almost wild-type growth rate was restored in atp16Δ cells that expressed GhATPδ1, with a resultant ATP/ADP ratio similar to that found in wild-type cells, indicating that the cotton gene was functional in yeast. Mitochondria prepared from 10 dpa wild-type fibre cells showed significantly higher ATP synthase activity in comparison to ovule samples from wild type and leaf samples. Exogenous application of piceatannol (PA) or oligomycin (OM), inhibitors of ATP synthase F(1) or F(0) subunits, respectively, in ovule culture media resulted in much shorter fibre cells and a significantly lower ATP/ADP ratio. Our data suggest that GhATPδ1 is important for activity of mitochondrial ATP synthase and is probably related to cotton fibre elongation.

Prognosis of colorectal cancer with liver metastasis: value of a prognostic index.

The objective of the present study was to explore the factors related to the prognosis of colorectal cancer (CRC) and to establish a prognostic model for the selection of patients who might benefit from hepatic resection for metastatic CRC. A total of 293 patients undergoing liver resection for metastatic CRC (172 males and 80 females ranging in age from 26 to 80 years) were selected and clinical, pathological and outcome data were examined in this retrospective study. The prognostic index (PI) of the patients was calculated on the basis of results of multivariate analysis. Patients were stratified into different groups, with survival curves projected according to PI. The 1-, 3-, and 5-year overall survival rates were 58.3, 26.4, and 11.3%, respectively. Univariate analysis indicated that degree of primary tumor differentiation, resection margin, preoperative carcinoembryonic antigen (CEA) level, number of liver metastases, and resection of liver metastases were associated with prognosis (P < 0.05). In multivariate analysis, the last three factors were found to be independent prognostic factors. The resection of liver metastases was a favorable factor. Patients were classified into three groups according to PI, which differed significantly in survival rate (P < 0.05). The individual survival rate was evaluated based on PI. Resection of hepatic colorectal metastases may produce long-term survival and cure. The proposed PI was easy to use, was highly predictive of patient outcome, and permitted categorization of patients into treatment groups.

Methylation status of the fragile histidine triad and E-cadherin genes in plasma of cervical cancer patients.

Recent evidence suggests that tumor cells may release DNA into the serum and plasma of afflicted cancer patients. However, no report existed regarding the methylation status of the fragile histidine triad (FHIT) and E-cadherin genes in plasma samples of cervical cancer patients. Methylation-specific PCR (MSP) was employed to examine CpG island methylation of the FHIT and E-cadherin genes in 151 pretreatment plasma samples and 30 tumor tissue samples from cervical cancer patients. MSP products were cloned and sequenced. CpG island methylation of the FHIT and E-cadherin genes was detected in 30.46% and 39.74% of plasma samples, respectively, and in 53.33% and 60.0% of tissue samples, respectively. The total concordance rate of methylation between plasma samples and tissue samples in FHIT gene was 80.00% and that in E-cadherin gene was 76.66%. At least one of the two methylated genes was detected in 56.29% of plasma samples and 76.7% of tissue samples. The presence of both methylated genes was detected in 13.9% of plasma samples and 36.67% of tissue samples. We found that the higher the clinical stage and histologic grade, the higher the rate of methylation in both genes in plasma samples. CpG island methylation of the FHIT and E-cadherin genes is present in plasma of cervical cancer patients. Using the two genes as markers simultaneously may allow clinicians to diagnose and evaluate the effect of treatment earlier and using fewer invasive procedures.

Prognosis of colorectal cancer with liver metastasis: value of a prognostic index

The objective of the present study was to explore the factors related to the prognosis of colorectal cancer (CRC) and to establish a prognostic model for the selection of patients who might benefit from hepatic resection for metastatic CRC. A total of 293 patients undergoing liver resection for metastatic CRC (172 males and 80 females ranging in age from 26 to 80 years) were selected and clinical, pathological and outcome data were examined in this retrospective study. The prognostic index (PI) of the patients was calculated on the basis of results of multivariate analysis. Patients were stratified into different groups, with survival curves projected according to PI. The 1-, 3-, and 5-year overall survival rates were 58.3, 26.4, and 11.3 percent, respectively. Univariate analysis indicated that degree of primary tumor differentiation, resection margin, preoperative carcinoembryonic antigen (CEA) level, number of liver metastases, and resection of liver metastases were associated with prognosis (P < 0.05). In multivariate analysis, the last three factors were found to be independent prognostic factors. The resection of liver metastases was a favorable factor. Patients were classified into three groups according to PI, which differed significantly in survival rate (P < 0.05). The individual survival rate was evaluated based on PI. Resection of hepatic colorectal metastases may produce long-term survival and cure. The proposed PI was easy to use, was highly predictive of patient outcome, and permitted categorization of patients into treatment groups.

Quantitative evaluation of ethanol effects on diffusion and metabolism of beta-estradiol in hairless mouse skin.

The influence of low levels of ethanol on the simultaneous diffusion and metabolism of beta-estradiol (E2 beta) in hairless mouse skin was quantitatively evaluated. A wide range of diffusion/metabolism experiments was conducted with full-thickness skin, stripped skin, and dermis at the various ethanol levels. The experiments were carried out in a two-chamber diffusion-cell system where ethanol was present in both the donor and the receiver chambers at equal concentrations. Analysis of the experimental data with several enzyme distribution models further showed that the best model was that for which the enzyme activity resided totally in the epidermis and near the basal layer of the epidermis. The ethanol effects were separated and quantified in terms of the diffusion and metabolism parameters. Aqueous ethanol, even at low concentrations (greater than or equal to 25%), was found to have two important effects on E2 beta transport: ethanol functions as an inhibitor of the enzymatic conversion of E2 beta to estrone (E1) in the viable epidermis, and ethanol is able to enhance the transport of permeants across the lipoidal pathway of the stratum corneum.

Estimation of skin target site acyclovir concentrations following controlled (trans)dermal drug delivery in topical and systemic treatment of cutaneous HSV-1 infections in hairless mice.

The use of controlled transdermal delivery of acyclovir (ACV) in the treatment of cutaneous herpes simplex virus type 1 infections in hairless mice was investigated. Using an in vivo animal model (A. Gonsho, et al. Int. J. Pharm. 65:183-194 (1990)) made it possible to quantify both, the topical and the systemic antiviral efficacy of ACV transdermal patches as a function of the drug delivery rate of the patches. Drug delivery rates required to attain systemic efficacy were found to be higher than the rates required to attain the same magnitude of topical efficacy. The ACV concentrations in the basal cell layer of the epidermis for 50% topical efficacy and 50% systemic efficacy were estimated. The basal epidermis layer was considered to be the site of antiviral drug activity (skin target site). Systemic plasma levels were obtained from pharmacokinetic studies and were used to estimate the ACV concentration achieved systemically in the basal epidermis layer. A computational model for drug permeation across skin was employed to estimate the ACV concentration achieved topically in the basal epidermis layer. Equal topical and systemic efficacies were found to correspond to equal drug concentrations at the site of antiviral activity. The length of the effective diffusion pathway of drug molecules in the dermis prior to entering the blood circulation was assumed to be approximately equal to 1/20 of the anatomical dermis thickness because of dermis vascularization.