[A conceptual framework for dynamics of cccDNA in hepatitis B virus].

The resolution of the hepatitis C issue has raised expectations for a chronic hepatitis B cure, driving the industry to expand investment in research and development efforts to strengthen functional cure strategies. These strategies have a wide variety of types, and the published research findings are heterogeneous. The theoretical analysis of these strategies is of great significance for determining prioritized research orientations as well as sensibly allocating research and development resources. However, due to a paucity of necessary conceptual models, current theoretical analysis has not been able to unify various therapeutic strategies into a proper theoretical framework. In view of the fact that the decrease in the quantity of cccDNA is an inevitable core event accompanied by the process of functional cure, this paper intends to analyze several chronic hepatitis B cure strategies using cccDNA dynamics as a framework. Furthermore, there are currently few studies on the dynamics of the cccDNA field, hoping that this article can promote recognition and research in this field.

[Clinical cure strategies for hepatitis B: direct-acting antiviral drugs].

Direct-acting antivirals (DAAs) play a critical role for the therapy of chronical hepatitis B. DAAs can decrease the production of viral progeny of hepatitis B virus (HBV), breaking the viral dynamic equilibrium between: (1) virion production from hepatocytes and clearance from circulation; (2) replenishment and decay of covalently closed circular (ccc)DNA pool inside infected hepatocytes. Nucleos(t)ide analogues can potently shift the first balance to undetectable viremia in the blood, but have limited or no effect on the second one, thus making it imperative to develop new agents targeting additional step(s) of HBV life cycle. We herein briefly introduce the DAAs currently in development by classifying them as agents affecting the replenishment or the decay of cccDNA pool.

Regulatory roles of miR-593 in the proliferation and invasion of human hepatic carcinoma.

OBJECTIVE: The aim of this study was to explore the expression manuals of microRNA-593 (miR-593) in hepatocellular carcinoma (HCC), and to investigate its role in mediating the proliferation, migration and invasion of HCC cells. PATIENTS AND METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect miR-593 expression in HCC tissues and cells. HCC cells were, then, transfected with miR-593 mimic to exogenously overexpress miR-593. Cell counting kit 8 (CCK8) assay was exploited to detect the proliferation ability of HCC cells. In addition, wound healing assay and transwell assay were adopted to determine the migration and invasion abilities of HCC cells, respectively. RESULTS: MiR-593 was abnormally down-expressed in HCC tissues and cells. Up-regulation of miR-593 significantly inhibited the proliferation, migration and invasion of HCC cells. CONCLUSIONS: MiR-593 acted as a tumor suppressor gene in the development of HCC. Hence, we proposed that miR-593 might be a new potential therapeutic target marker for HCC treatment.

LEF-AS1 participates in occurrence of colorectal cancer through adsorbing miR-505 and promoting KIF3B expression.

OBJECTIVE: To explore the possible role and mechanism of long non-coding ribonucleic acid LEF-AS1 (lncRNA LEF-AS1) in the pathogenesis of colorectal cancer (CRC). PATIENTS AND METHODS: The expression levels of LEF-AS1 in 54 CRC tissue samples and adjacent normal ones were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). In the meantime, CRC cell lines were screened for subsequent in vitro experiments. LEF-AS1 siRNA was transfected into CRC cells using the liposome method. Then, cell counting kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assays were conducted to detect cell proliferation. Thereafter, transwell assay was performed to evaluate the cell migration capacity, as well as invasiveness, Caspase-3 activity was examined to estimate cell apoptosis, and flow cytometry was applied for cell cycle detection. Subsequently, bioinformatics analysis was carried out to predict the target genes of micro RNA (miR)-505 that were predicted to be able to bind to LEF-AS1, while the relative activity of luciferase between miR-505 and KIF3B or LEF-AS1 was examined via luciferase gene reporter assay. In addition, the interaction between KIF3B and miR-505, as well as LEF-AS1, was further verified by RNA knockdown assay and cell reversal experiments. RESULTS: The expression of LEF-AS1 in CRC tissue specimens was found to be markedly higher than that in normal colon tissues. After transfection with LEF-AS1 siRNA, the cell viability, as well as cell migration and invasion capacities, were both attenuated. However, the cell apoptosis rate was conversely elevated. Dual-Luciferase reporter assay revealed that LEF-AS1 could combine with miR-505, which was capable of targeted binding to KIF3B. In addition, LEF-AS1 siRNA transfection attenuated cell proliferation, migration, and invasion capacities, which could be partially reversed by the overexpression of KIF3B. CONCLUSIONS: In this research, LEF-AS1 is highly expressed in CRC tissues and cell lines. However, the down-regulation of LEF-AS1 reduces the proliferation rate and suppresses the invasiveness and metastasis of CRC cells through the LEF-AS1/miR-505/KIF3B axis.

[Mode of median nerve irritation under different wrist forcing postures observed by ultrasonography and foot pressure measurement system].

Objective: To analyze the influence of simulation mouse use motion under different wrist forcing postures on median nerve, tendons and ligaments in the carpal tunnel. Methods: From June to November in 2017, a total of 49 healthy volunteers [aged from 18 to 27 years, 24 males (48 cases of hands) and 25 females (50 cases of hands)] were selected in the Institute of Digitized Medicine and First Affiliated Hospital of Wenzhou Medical University.Three hand postures of the volunteers were simultaneously and continuously measured by using LOGIQ E9 ultrasonic diagnostic apparatus and Zebris foot pressure distribution measurement system.Seventeen parameters of nerves, tendons and ligaments in carpal tunnel were observed under natural (0 N), and two forced (25 and 50 N) states.Double factor variance analysis was performed with generalized estimating equation (GEE). Results: With increasing pressure (0, 25 and 50 N) of hand postures, the distance between median nerve and transverse carpal ligament were all less than 0.2 cm.The differences in both the distance between median nerve and flexor pollicis longus under the hand pressure changes or under the hand posture changes and the top angle of a triangle composed of median nerve, flexor pollicis longus and flexor digitorum superficialis group under the hand pressure changes or under the hand posture changes were all significant under the GEE analysis (all P<0.01). There were no significant changes in all other structural parameters in the carpal tunnel with the increasing of hand pressure (all P>0.05). Conclusions: The influence of the transverse carpal ligament to the median nerve belongs to the mechanism of pressure-induced irritation damage.The influence of flexor pollicis longus to median nerve belongs to the mechanism of tension-induced irritation damage.The influence of flexor digitorum superficialis to median nerve belongs to the mechanism of mixed shear irritation damage.

[Study on HIV prevention related knowledge-motivation-psychological model in men who have sex with men, based on a structural equation model].

OBJECTIVE: Knowledge-motivation-psychological model was set up and tested through structural equation model to provide evidence on HIV prevention related strategy in Men who have Sex with Men (MSM). METHODS: Snowball sampling method was used to recruit a total of 550 MSM volunteers from two MSM Non-Governmental Organizations in Urumqi, Xinjiang province. HIV prevention related information on MSM was collected through a questionnaire survey. A total of 477 volunteers showed with complete information. HIV prevention related Knowledge-motivation-psychological model was built under related experience and literature. Relations between knowledge, motivation and psychological was studied, using a ' structural equation model' with data from the fitting questionnaires and modification of the model. RESULTS: Structural equation model presented good fitting results. After revising the fitting index: RMSEA was 0.035, NFI was 0.965 and RFI was 0.920. Thereafter the exogenous latent variables would include knowledge, motivation and psychological effects. The endogenous latent variable appeared as prevention related behaviors. The standardized total effects of motivation, knowledge, psychological on prevention behavior were 0.44, 0.41 and 0.17 respectively. Correlation coefficient of motivation and psychological effects was 0.16. Correlation coefficient on knowledge and psychological effects was -0.17 (P<0.05). Correlation coefficient of knowledge and motivation did not show statistical significance. CONCLUSION: Knowledge of HIV and motivation of HIV prevention did not show any accordance in MSM population. It was necessary to increase the awareness and to improve the motivation of HIV prevention in MSM population.

Sirtuin 3 inhibits hepatocellular carcinoma growth through the glycogen synthase kinase-3ß/BCL2-associated X protein-dependent apoptotic pathway.

SIRT3 is a class III histone deacetylase that has been implicated in a variety of cancers. The role of SIRT3 in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that SIRT3 expression was frequently repressed in HCC and its downregulation was closely associated with tumor grade and size. Ectopic expression of SIRT3 inhibited cell growth and induced apoptosis in HCC cells, whereas depletion of SIRT3 in immortalized hepatocyte promoted cell growth and decreased epirubicin-induced apoptosis. Mechanistic studies revealed that SIRT3 deacetylated and activated glycogen synthase kinase-3ß (GSK-3ß), which subsequently induced expression and mitochondrial translocation of the pro-apoptotic protein BCL2-associated X protein (Bax) to promote apoptosis. GSK-3ß inhibitor or gene silencing of BAX reversed SIRT3-induced growth inhibition and apoptosis. Furthermore, SIRT3 overexpression also suppressed tumor growth in vivo. Together, this study reveals a role of SIRT3/GSK-3ß/Bax signaling pathway in the suppression of HCC growth, and also suggests that targeting this pathway may represent a potential therapeutic approach for HCC treatment.

Construction and functional identification of a hepatitis B virus S protein small hairpin RNA recombinant adenovirus.

Hepatitis B virus S protein (HBs) plays an important role in hepatocellular carcinoma progression. However, to date, no direct and effective methods exist to research the function of HBs. Here, we combined the technology of RNA interference with recombinant adenovirus, constructed a recombinant adenovirus-expressing small hairpin RNA of HBs, and infected HepG2.2.15 cells. Then, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, enzyme-linked immunosorbent assay, and Western blot analysis were performed to verify the interference effects. As a result, a recombinant adenovirus was successfully constructed and effectively packaged in AD293 cells, and it significantly inhibited HBs mRNA and protein expression in vitro. Our study may provide a novel tool to study HBs function.

An improved reverse dot hybridization for simple and rapid detection of adefovir dipivoxil-resistant hepatitis B virus.

Early detection of adefovir dipivoxil-resistant mutants during long-term treatment of chronic hepatitis B virus (HBV) infection with this drug is of great clinical importance. We developed an improved reverse dot hybridization test for simple and rapid detection of the rtA181V/T and rtN236T mutations associated with adefovir dipivoxil resistance in chronic hepatitis B patients. Probes were designed for genotypes B, C, and D of this resistance characteristic; a total of 70 clinical samples were analyzed with this improved reverse dot hybridization assay. Its usefulness was validated by comparing with sequencing data. Discordant results were confirmed by subclone sequencing. This reverse dot hybridization assay was sufficiently sensitive to detect 10(3) copies/mL; it also detected adefovir dipivoxil-resistant mutant strains when they comprised more than 5% of a mixed virus population. This reverse dot hybridization array correctly identified adefovir dipivoxil-resistant mutants; it had high concordance (98.5%) with direct sequencing data. There was no clear relationship between the HBV genotype and the development of adefovir dipivoxil-resistant mutants. This reverse dot hybridization assay proved to be simple and rapid for detection of rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil.

Molecular, cellular and functional characterizations of a novel ICAM-like molecule of the immunoglobulin superfamily from Leishmania mexicana amazonensis.

A molecule with two immunoglobulin (Ig) domains cloned from Leishmania mexicana amazonensis was characterized to have a sequence homology to the Ig domains of an ICAM-like molecule telencephalin, cloned from the brain of mammals, as well as to the variable domains of human immunoglobulin lambda light chain. The molecule therefore appears to be an ICAM-like molecule as well as a member of the immunoglobulin superfamily. We thus named it ICAM-L for Leishmania ICAM. The gene was coamplified with the ribonucleotide reductase M(2) subunit gene responsible for hydroxyurea resistance from hydroxyurea (Hu)-resistant Leishmania variants. As expected, an increase of the ICAM-L protein as well as an increase of the specific ICAM-L transcript of 2.1 kb was detected in the Hu-resistant variants with increasing doses of the drug used for resistance selection. Structurally, ICAM-L is more similar to the secretory adhesive molecules, such as 1Bgp and the link protein of the immunoglobulin superfamily, in that it lacks a transmembrane region and a GPI anchor sequence. Although ICAM-L was mainly localized in the nucleus of the parasite by confocal microscopy, however, detailed studies by electron microscopy and FACS analysis indicated that the protein was also localized on the surface of the parasite. The surface localization of the protein was furthered strengthened by the observations that anti-ICAM-L or ICAM-L itself can significantly block the binding of the parasite to macrophages. The blocking of the attachment of parasite to macrophages may indicate that ICAM-L functions as an intercellular adhesive molecule.

Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates.

Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. As the first step in our pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesise environmental-friendly bioplastics, we investigated the usage of soya wastes from a soya milk dairy, and malt wastes from a beer brewery plant as the carbon sources for the production of polyhydroxyalkanoates (PHA) by selected strain of microorganism. Bench experiments showed that Alcaligenes latus DSM 1124 used the nutrients from malt and soya wastes to biosynthesise PHAs. The final dried cell mass and specific polymer production of A. latus DSM 1124 were 32g/L and 70% polymer/cells (g/g), 18.42 g/L and 32.57% polymer/cell (g/g), and 28 g/L and 36% polymer/cells (g/g), from malt waste, soya waste, and from sucrose, respectively. These results suggest that many types of food wastes might be used as the carbon source for the production of PHA.

Plasma lipoprotein cholesterol levels in rats fed a diet enriched in cholesterol and cholic acid.

To investigate the effects of dietary cholesterol and cholic acid on plasma cholesterol levels, rats fed a cholesterol-free diet or a diet enriched in cholesterol (0.5% or 1%) with or without cholic acid supplementation were studied for 4 weeks. Although 0.5% cholesterol supplementation showed no effect on plasma total cholesterol and LDL-cholesterol levels in rats fed a diet without cholic acid treatment, the addition of dietary cholic acid caused an increase in plasma total cholesterol, LDL-cholesterol and VLDL-cholesterol levels in rats fed a cholesterol-rich diet. There was no significant change in HDL-cholesterol levels among the dietary groups. Rats fed a diet enriched in cholesterol have increased liver total lipids and total cholesterol contents. In addition, lower liver lipid peroxide concentration was found in rats fed a cholesterol-rich diet when compared with those fed the control diet. It is interesting that cholic acid supplementation led to an increase in hepatic cholesterol content and a decrease in liver lipid peroxide concentration in rats fed a cholesterol-rich diet. Results from this study suggest that dietary cholesterol and cholic acid might play an important role in regulation of lipid metabolism in rats.

Colocalization of iron and ceroid in human atherosclerotic lesions.

The presence of ceroid, a complex of protein associated with oxidized lipids, is commonly observed in human atherosclerotic lesions. When the human aortic walls were examined by Perls' staining, it was found that the iron deposits were evident in aortas with atherosclerosis. The extent of iron deposition was associated with the severity of the lesion. Furthermore, the iron deposits appeared to be colocalized with ceroids either extracellularly or intracellularly in foam cell-like macrophages or smooth muscle cells. Electron microscopy and X-ray microanalysis revealed that some of the extracellular iron aggregates were present within the ceroids. Likewise, some of the subcellular iron aggregates were found to be located near the lipid droplets or within the ceroids of foam cells. Collectively, these observations support the theory that the lipid oxidation occurring in lipid-laden cells of aortic lesions is facilitated by iron-overload in these cells.

Conversion of food industrial wastes into bioplastics.

The usage of plastics in packaging and disposable products, and the generation of plastic waste, have been increasing drastically. Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. In the authors' laboratories, various carbohydrates in the growth media, including sucrose, lactic acid, butyric acid, valeric acid, and various combinations of butyric and valeric acids, were utilized as the carbon (c) sources for the production of bioplastics by Alcaligenes eutrophus. As the first step in pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesize bioplastics, the authors investigated the usage of malt wastes from a beer brewery plant as the C sources for the production of bioplastics by microorganisms. Specific polymer production yield by A. Latus DSM 1124 increased to 70% polymer/cell (g/g) and 32 g/L cell dry wt, using malt wastes as the C source. The results of these experiments indicated that, with the use of different types of food wastes as the C source, different polyhydroxyalkanoate copolymers could be produced with distinct polymer properties.

Pulmonary edema induced by upper airway obstruction--report of 3 cases.

Pulmonary edema is a well-recognized complication of upper airway obstruction, and has been reported sporadically both in children and adults since 1977. Although the pathogenesis of pulmonary edema associated with upper airway obstruction is multifactorial, attention is primarily focused on excessive negative intrapleural and transpulmonary pressure produced by forceful inspiration against a closed glottis that results in transudation of fluid from the pulmonary capillary into the interstitial and alveolar spaces. We report 3 cases of pulmonary edema induced by upper airway obstruction after extubation following general anesthesia.

Role of intercellular junctions in the passage of horseradish peroxidase across aortic endothelium.

BACKGROUND: The manner in which molecules are transported across the arterial endothelial layer has been a subject open to much interpretation and controversy. Further elucidation and clarification of these mechanisms are of primary interest. EXPERIMENTAL DESIGN: To investigate the ultrastructural features of arterial endothelial junctions and to evaluate their functional roles as a transendothelial pathway for macromolecular transport, experiments were performed on the thoracic aortae of adult male Sprague-Dawley rats by using the ultrathin serial sectioning technique and horseradish peroxidase (HRP). The aorta was perfusion-fixed with or without prior intravenous injection of HRP. RESULTS: The intercellular clefts exhibited a great deal of variety in shape, being linear, winding, interdigitated, irregular and/or dumbbell-shaped in appearance. Besides the typical 20-nm width encountered at the uniform region of intercellular clefts, local widenings (up to several hundred nm) were quite common. The arterial endothelial junctions were highly organized. Junctional elements, including tight junctions and gap junctions, were frequently present in the same intercellular cleft, even on the same plane of sectioning. Sometimes, gap junctions were found without tight junctions, but the intercellular clefts were rarely obliterated by tight junctions alone. Some intercellular clefts were not obliterated by either gap or tight junctions, and HRP was found to reach the subendothelial space by passing through these junctionless clefts. Densitometric determination of the HRP concentration profile in such junctionless clefts showed a decreasing gradient from the luminal to the abluminal front. The serial sections provided evidence that the apparently free vesicles were actually plasmalemmal membrane invaginations open to the luminal or abluminal front in the arterial endothelium. CONCLUSIONS: The present study showed that the junctionless normal endothelial clefts, in addition to the transiently open junctions surrounding mitotic cells, might provide a significant pathway in the transendothelial transport of macromolecules with the size of HRP.