[Roles and mechanisms of m6A modification regulating RP11-426A6.5 in laryngeal squamous cell carcinoma].

Objective: To investigate the roles of N6-methyladenosine (m6A) modification in regulating RP11-426A6.5 in the development of laryngeal squamous cell carcinoma (LSCC). Methods: The methylation and expression levels of lncRNAs were identified and important lncRNAs were screened utilizing long non-coding RNA (lncRNA) m6A methylation microarray. Cancer and para cancer tissue samples were taken from 48 LSCC patients hospitalized to the Department of Otolaryngology-Head and Neck Surgery of the Second Affiliated Hospital of Harbin Medical University between January and September 2017. Expression profiling microarray was performed in 3 of 48 LSCC samples, and methylated RNA immunoprecipitation-quantitative PCR (MeRIP-qPCR) and quantitative real-time fluorescent PCR (qRT-PCR) were performed in the remaining 45 LSCC samples to verify the m6A modification and expression levels of RP11-426A6.5. Correlations between RP11-426A6.5 and clinical factors were anlysed. Laryngeal cancer cell line with low expression of RP11-426A6.5 was created in vitro using RNA interference (RNAi) technology. The 5-Ethynyl-2'-deoxyuridine (EdU) cell proliferation experiment, wound healing experiment, and transwell invasion experiment were used respectively to measure the proliferation, migration, and invasion of LSCC cells. The effect of RP11-426A6.5 down-regulation on the growth of transplanted tumors in vivo was verified by nude mice tumorigenesis assay. The Cancer Genome Atlas (TCGA) database and sequence-based RNA adenosine methylation site predictor (SRAMP) website were used to predict the enzymes and corresponding methylation sites. MazF digestion was chosen to validate the binding sites. RNAi technology was used to observe the changes in cell function after interfering with the expression of the corresponding genes of the modified enzymes. MeRIP-qPCR was used to detect the level of RP11-426A6.5 m6A cell line treated with actinomycin D was used to observe the stability of RP11-426A6.5. Results: RP11-426A6.5 methylation and expression levels were significantly higher in LSCC tissues than those in paracancerous tissues (methylation levels: 23.828±4.975 vs 20.280±3.607; expression levels: 1.197±0.314 vs 1.015±0.170, all P values<0.05). RP11-426A6.5 expression levels were closely correlated with T stage (T1-2: 1.081±0.298 vs T3-4: 1.306±0.292, χ2=5.35, P<0.05). The postoperative survival of patients with high RP11-426A6.5 expressions was significantly lower than that of patients with low RP11-426A6.5 expression (P=0.046). Assays in vitro and in vivo showed that the downregulation of RP11-426A6.5 significantly decreased the proliferation, migration, and invasion abilities of LSCC cells and the growth of transplanted tumors. The binding of methyltransferase-like 3 (METTL3), an m6A-modified enzyme, to the corresponding methylation site of RP11-426A6.5 enhanced its stability and mediated its regulation of malignant behaviors of LSCC cells. Conclusions: RP11-426A6.5 can regulate the malignant behaviors of LSCC cells, which is mediated by the m6A modification process involving in the methyltransferase METTL3.

The complete mitochondrial genome and molecular phylogeny of Lueyang black-bone chicken.

1. The objectives of the current study were to investigate the mitochondrial genome and molecular phylogeny of Lueyang black-bone chicken, and  provide  molecule base to preserve and explore the specific chicken strain. 2. Based on sequencing and clustering, the complete mitochondrial DNA map and sequences of Lueyang black-bone chicken were revealed, and two phylogenetic trees of Lueyang black-bone chickens based on D-loop sequences and the mitochondrial genome were constructed. 3. The results showed that the complete mitochondrial genome of Lueyang black-bone chickens is 16,784bp in size, consisting of 22 transfer RNA genes, two ribosomal RNA genes, 13 protein-coding genes, and one non-coding control region. The base composition of the complete mtDNA sequence is 30.28% for A, 23.78% for T, 32.42% for C, 13.52% for G. Additionally, 10 haplotypes of D-loop sequences in 32 Lueyang black-bone chickens were detected, which were distributed into 4 clades (A, B, C and E). 4. It was concluded that genetic diversity is wide in Lueyang black-bone chickens, and this strain has multiple maternal origins from different regions in China and neighbouring regions.

[HBsAg clearance rate in patients with HBeAg-positive chronic hepatitis B: a comparative effect analysis based on the real world].

OBJECTIVE: To investigate HBsAg clearance rate in previously untreated patients with HBeAg-positive chronic hepatitis B (CHB) treated with nucleos(t)ides and interferons and its influencing factors based on the clinical diagnosis and treatment dat. METHODS: A retrospective cohort study was conducted in 1 767 previously untreated HBeAg-positive CHB patients who visited Beijing You'an Hospital from February 14, 2008 to December 31, 2012. HBsAg clearance rates were calculated for patients with different characteristics, and the Cox regression model was used to investigate the influencing factors for HBsAg clearance. RESULTS: The overall annual HBsAg clearance rate was 0.46% in 1767 patients, and in the patients treated with adefovir, entecavir, telbivudine, and common interferon, the annual HBsAg clearance rate was 0.52%, 0.47%, 0.45%, and 1.18%, respectively. No patients in the lamivudine and pegylated interferon-α groups experienced HBsAg clearance, which might be due to the small sample size. The univariate analysis showed that HBsAg clearance rate was associated with the patient's age when he/she visited the hospital and baseline HBsAg titer level. After adjustment for other factors, the patients treated with common interferon had a significantly higher possibility of HBsAg clearance than those treated with entecavir (HR = 8.33, 95% CI: 1.19-58.50, P = 0.0329), but the possibility of HBsAg clearance showed no significant difference between patients treated with other nucleos(t)ides and entecavir. The patients aged≥50 years had a probability of HBsAg clearance 4.92 times that of those aged < 50 years (HR = 4.92, 95% CI: 1.38-17.50, P = 0.0139) and the patients with baseline HBsAg titer level < 3 log10 IU/ml had a probability of HBsAg clearance 22.77 times higher than that of those with baseline HBsAg titer level≥3 log10 IU/ml (HR = 23.77, 95% CI: 6.17-91.51, P < 0.0001). CONCLUSION: The previously untreated CHB patients achieve a low annual HBsAg clearance rate under current antiviral therapeutic regimens, especially nucleos(t)ides. Baseline HBsAg titer level is closely associated with HBsAg clearance.

Correlations between folate, B12, homocysteine levels, and radiological markers of neuropathology in elderly post-stroke patients.

OBJECTIVE: To investigate serum levels of folate, B12, and total homocysteine (tHcy) in elderly post-stroke patients, and the possible correlations with radiological markers of neuropathology. DESIGN: Cross-sectional study. SETTING: Department of Neurology, Cardinal Tien Hospital. SUBJECTS: Eighty-nine elderly post-stroke patients were enrolled for dietary assessment and blood tests. Neuroradiological assessment was done in 62 of these patients. MAIN OUTCOME MEASURES: Dietary folate and vitamin B12 intakes were evaluated by a 24-h recall system using a semi-quantitative questionnaire. Circulating levels of folate, B12, and tHcy were measured. Magnetic resonance imaging (MRI) or computed tomography (CT) was used for evaluation of brain lesions including infarction and atrophy. RESULTS: Mean folate and B12 intakes of these post-stroke patients were 69% and 261% of the recommended dietary allowances (RDA), respectively. Inadequate folate levels, defined as serum folate < 6 ng/mL, was noted in 68% of these patients. Hyperhomocysteinemia levels (tHcy >or=15 micromol/L) were observed in 48%. According to tertiles of serum tHcy and folate levels, the rate of brain atrophy, but not brain infarctions, are significantly associated with elevated tHcy (P = 0.0126) and decreased folate levels (P = 0.0273). After adjustments for age, sex, disease status, brain infarctions and carotid stenosis, the odds ratio of brain atrophy was 9.8 (95% CI: 1.7-56.4, P = 0.0101) in the hyperhomocysteinemia group and 9.6 (95% CI: 1.1-81.3, P = 0.0377) in the low folate group (serum folate < 3.0 ng/mL) compared with the group with normal tHcy and folate levels. No significant association was noted between vitamin B12 levels and brain lesions. CONCLUSIONS: Our data shows that folate deficiency and hyperhomocysteinemia are prevalent in elderly post-stroke patients. These two conditions are strongly and independently associated with the development of brain atrophy.

Quantifying individual fruit fly consumption with Anastrepha suspensa (Diptera: Tephritidae).

We needed a technique to compare the consumption of baits by individual Carribbean fruit fly, Anastrepha suspensa (Loew). By improving consumption and determining individual dose, we could lower pesticide concentration while retaining bait/pesticide efficacy and potentially reduce the environmental impact of fruit fly bait/pesticide eradication methods. We report here a precise dye-based technique for the quantification of consumption by individual adult A. suspensa fruit flies. Fluorescein, measured at 491 nm, and cresol red, measured at 573 nm, were efficiently extracted with 0.1 M NaOH and quantified with a spectrophotometer. The lower limit for this method with 0.1% dye concentration is 300 nl consumed by an individual fly. Dye movement to the hindgut and possible defecation occurred in approximately 4 h; maximum ingestion occurred in approximately 1 h. Maximum experimental time is limited to 4 h. Flies preferred feeding upside down compared with right side up when given a choice; consumption was equal when flies were given no choice of feeding position. Thus, maximum bait/pesticide efficacy might be achieved with an upside-down presentation. Regurgitation led to a 100% overestimation of actual consumption with the J-tube presentation of food. Our individual fly consumption technique will be useful in comparing consumption in phagostimulant studies, estimating dose in oral toxicity tests, differentiating behavioral and physiological resistance in toxicant studies, ultimately leading to improved bait/pesticide methods and reduced environmental impact of area wide fruit fly eradication programs. This technique could be applied to studies of tephritid consumption, to the consumption of other insects, and to regurgitation studies.

Ardisiaquinones from Ardisia teysmanniana.

Bioassay-guided fractionation of a CHCl(3) extract from the leaves of Ardisia teysmanniana Scheff. (Myrsinaceae) has led to the isolation of three new alkyldibenzoquinone derivatives that showed inhibitory activity in an in vitro assay for UDP-MurNac synthesis. The structures of ardisiaquinone G, H and I were established using MS and NMR spectroscopic methods.

Sodium tetraborate effects on mortality and reproduction of Anastrepha suspensa (Diptera: Tephritidae).

When flies were treated with 0- 0.5% sodium tetraborate by feeding for 24 h, mortality in treatments was not different from controls. Fecundity and fertility were reduced by 0.5% sodium tetraborate. When flies were fed for 48 h, mortality of both males and females increased in the 0.5% sodium tetraborate treatment; oviposition was eliminated for 20 d after treatment. When treatment was extended to 168 h, 0.1% sodium tetraborate caused increased mortality and decreased fecundity and fertility. Fed for 168 h, 0.2 and 0.5% sodium tetraborate killed almost all flies within the 7-d treatment. Oviposition of survivors in 0.1 and 0.2% sodium tetraborate treatments was arrested for 20 d after treatment.

Serum and egg yolk IgG antibody titers from laying chickens vaccinated with Pasteurella multocida.

Through determining the serum and egg yolk antibody titers in immunized laying hens to Pasteurella multocida regularly, the growth-decline trend of the egg yolk antibody levels was found to be similar to that of the serum antibody levels (r = 0.94), but the growth and decline of the egg yolk antibody seemed to be delayed 3-6 days compared with that of the serum antibody, and the egg yolk antibody titers were generally lower than those of the serum antibody (P < 0.01). Serum and egg yolk antibody levels declined 3 and 6 days, respectively, after booster immunizations. The higher the antibody levels were before booster immunization, the more they declined.

Pathological observations of hepatic tumours in ducks.

During August to October, 1983, 73 of 113 ducks submitted to our laboratory from a duck farm for pathological examination were found to have a variety of hepatic tumours. Grossly, the lesions could be divided into three groups: nodular, macro-nodular and diffuse forms; the nodular form was the most frequently seen (54/73). Histopathologically, the tumours could be classified into six categories: liver cell adenoma, cholangioadenoma, hepatocellular carcinoma, cholangiocarcinoma, mixed hepatic carcinomas and metastatic hepatic carcinoma, and the hepatocellular carcinoma was the most common (68/73).

Recombinant human prorenin from CHO cells: expression and purification.

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from approximately 1-5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr approximately 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence and pH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P'3 sequence of human angiotensinogen.