RNF125­mediated ubiquitination of MCM6 regulates the proliferation of human liver hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated mortality worldwide. Minichromosome maintenance proteins (MCMs), particularly MCM2-7, are upregulated in various cancers, including HCC. The aim of the present study was to investigate the role of MCM2-7 in human liver HCC (LIHC) and the regulation of the protein homeostasis of MCM6 by a specific E3 ligase. Bioinformatics analyses demonstrated that MCM2-7 were highly expressed in LIHC compared with corresponding normal tissues at the mRNA and protein levels, and patients with LIHC and high mRNA expression levels of MCM2, MCM3, MCM6 and MCM7 had poor overall survival rates. Cell Counting Kit-8 and colony formation assays revealed that the knockdown of MCM2, MCM3, MCM6 or MCM7 in Huh7 and Hep3B HCC cells inhibited cell proliferation and colony formation. In addition, pull-down, co-immunoprecipitation and ubiquitination assays demonstrated that RNF125 interacts with MCM6 and mediates its ubiquitination. Furthermore, co-transfection experiments indicated that RNF125 promoted the proliferation of HCC cells mainly through MCM6. In summary, the present study suggests that the RNF125-MCM6 axis plays an important role in the regulation of HCC cell proliferation and is a promising therapeutic target for the treatment of LIHC.

Zolpidem and zolpidem phenyl-4-carboxylic acid pharmacokinetics in oral fluid after a single dose.

BACKGROUND: Oral fluid zolpidem detection in the settings of drug-facilitated crime and roadside drug testing indicates recent zolpidem intake. Zolpidem pharmacokinetics in classical biological matrices such as blood and urine have been described; however, reports of such data based on oral fluids are limited. OBJECTIVE: The aim of this study is to describe the pharmacokinetics of zolpidem and its major metabolite zolpidem phenyl-4-carboxylic acid (ZPCA) in oral fluids after intake. METHODS: Ten milligrams of zolpidem tartrate tablets were orally administered to 14 volunteers, and oral fluid samples were collected at various times up to 72 hours and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) with post-column reagent addition. RESULTS: Both zolpidem and ZPCA could be detected in oral fluid after 1 hour and were rapidly eliminated, with half-lives of 2.77 ± 0.71 hours and 5.11 ± 0.67 hours, respectively. Maximum zolpidem concentrations (36.73 ± 10.89 ng/mL) occurred at 2 ± 0.52 hours, and maximum ZPCA concentrations (0.28 ± 0.16 ng/mL) occurred at 2 ± 0.37 hours. Zolpidem/ZPCA ratios decreased after zolpidem intake, an observation that might be helpful for determining the time of drug use. CONCLUSION: The results showed that the measurement of zolpidem in oral fluid can be used for the non-invasive monitoring of zolpidem consumption and misuse in drug-facilitated crime and roadside drug testing settings.

Promoting early neovascularization of SIS-repaired abdominal wall by controlled release of bioactive VEGF.

Insufficient early neovascularization post-operation is thought to be the main reason of surgical recurrence of porcine small intestinal submucosa (SIS)-repaired abdominal wall defects. The controlled release of exogenous angiogenic growth factors (GFs) from biocompatible carriers is a possible way to solve this problem. In the present study, dextran nanoparticles (DNPs) loaded with vascular endothelial growth factor 165 (VEGF165) were pre-formulated by dual-aqueous phase separation method and then electrospun into the poly(lactic-co-glycolic acid) (PLGA) polymer fibers. The aim of this material is to release VEGF in a sustained manner with the degradation of PLGA and maintain its bioactivity concurrently. The prepared VEGF/DNPs-PLGA membrane was sandwiched by dual-layer SIS to construct a SIS-DNPs/VEGF-PLGA-SIS (SVDPS) composite scaffold. The in vitro study showed that the VEGF/DNPs-PLGA obtained higher VEGF encapsulation efficiency as well as better release property and bioactivity than the emulsion electrospun VEGF-PLGA and PLGA fibrous membranes by ELISA and HUVEC proliferation. The in vivo study showed that the SVDPS composite scaffold promoted significantly higher early therapeutic neovascularization within 2 weeks post-surgery than SIS-VEGF-PLGA-SIS (SVPS) and SIS-PLGA-SIS (SPS) by immunohistochemical and immunoblotting examination.

LC-MS-MS with Post-Column Reagent Addition for the Determination of Zolpidem and its Metabolite Zolpidem Phenyl-4-carboxylic Acid in Oral Fluid after a Single Dose.

A rapid, selective and sensitive LC-MS-MS method with a post-column addition of acetonitrile was developed and fully validated for the quantitative determination of zolpidem and its major metabolite, zolpidem phenyl-4-carboxylic acid (ZPCA), in oral fluid. Preliminary sample treatment was limited to a simple dilution of 1 mL oral fluid specimen aliquots with methanol. Chromatography was performed on a Capcell Pak C18 MGII column (250 × 2.0 mm, 5 µm i.d., Shiseido, Tokyo, Japan) with isocratic elution using a water-acetonitrile mobile phase with 0.1% formic acid, 5% acetonitrile and 20 mM ammonium acetate in an aqueous phase at a flow rate of 0.4 mL/min. Acetonitrile was added post-column at a flow rate of 0.4 mL/min to enhance ionization of the analytes in the MS source. Detection was carried out on a QTrapTM 6500 mass spectrometer in positive ionization mode. Good linearities were generated over the range of 0.05-200 ng/mL for zolpidem and 0.1-200 ng/mL for ZPCA. Limit of detection for zolpidem and ZPCA were 0.01 ng/mL and 0.05 ng/mL, respectively, whereas LLOQs were 0.05 ng/mL and 0.1 ng/mL, respectively. This method meets the required criteria for bioanalytical analyses to be used for clinical and forensic purposes. Application of this method was demonstrated by testing authentic samples collected after a single oral dose to obtain insights into the general detectability and detection windows of zolpidem and ZPCA in oral fluid.

Carbon nanotubes as VEGF carriers to improve the early vascularization of porcine small intestinal submucosa in abdominal wall defect repair.

Insufficient early vascularization in biological meshes, resulting in limited host tissue incorporation, is thought to be the primary cause for the failure of abdominal wall defect repair after implantation. The sustained release of exogenous angiogenic factors from a biocompatible nanomaterial might be a way to overcome this limitation. In the study reported here, multiwalled carbon nanotubes (MWNT) were functionalized by plasma polymerization to deliver vascular endothelial growth factor165 (VEGF165). The novel VEGF165-controlled released system was incorporated into porcine small intestinal submucosa (PSIS) to construct a composite scaffold. Scaffolds incorporating varying amounts of VEGF165-loaded functionalized MWNT were characterized in vitro. At 5 weight percent MWNT, the scaffolds exhibited optimal properties and were implanted in rats to repair abdominal wall defects. PSIS scaffolds incorporating VEGF165-loaded MWNT (VEGF-MWNT-PSIS) contributed to early vascularization from 2-12 weeks postimplantation and obtained more effective collagen deposition and exhibited improved tensile strength at 24 weeks postimplantation compared to PSIS or PSIS scaffolds, incorporating MWNT without VEGF165 loading (MWNT-PSIS).

Comparison of open and laparoscopic preperitoneal repair of groin hernia.

BACKGROUND: Compared with laparoscopic groin herniorrhaphy, the open procedure used in most former studies was Lichtenstein repair. However, unlike the totally extraperitoneal (TEP) or transabdominal preperitoneal (TAPP) laparoscopic techniques, Lichtenstein procedure is a premuscular but not preperitoneal repair. This retrospective study compared the outcomes between laparoscopic preperitoneal and open preperitoneal procedure-modified Kugel (MK) herniorrhaphy. METHODS: Groin hernia patients older than 18 years who underwent open MK or laparoscopic preperitoneal herniorrhaphy in our hospitals between January 2008 and December 2010 were enrolled. Baseline characteristics, recurrence, and intraoperative, short-term, and long-term postoperative complications were recorded. RESULTS: Among the 1,760 included patients (530 open and 1,230 laparoscopic), 96.08% completed the follow-up (24-60 months). The patients in the open group were older than laparoscopic group (p < 0.001). More bilateral (91.45%) and recurrent (82.12%) hernia patients underwent laparoscopic procedures (p < 0.001 and p = 0.004, respectively). The overall recurrence rate was 0.71%, with no significant difference between the two approaches (p = 0.227). The overall complication rate was lower for the laparoscopic than the open approach (14.47 vs. 19.25%, p = 0.012), whereas the rates of life-threatening complications were similar (1.51 vs. 0.98%, p = 0.332). The laparoscopic group had significantly lower incidence rates of wound infection and chronic pain (p = 0.016 and p < 0.001, respectively), shorter operative time, lower visual analogue scale scores, and faster recovery than the open group (p < 0.001). CONCLUSIONS: As preperitoneal herniorrhaphy, both MK and laparoscopic (TEP/TAPP) procedures are safe and effective, with low incidence rates of life-threatening complications and recurrence. The laparoscopic approach is superior in terms of lower incidence rates of infection and chronic pain, shorter operative time, and faster recovery; however, careful surgical procedure selection and implementation of technical details are required.