Overexpression of miR-214-3p in esophageal squamous cancer cells enhances sensitivity to cisplatin by targeting survivin directly and indirectly through CUG-BP1.

Based on its marked overexpression in multiple malignancies and its roles in promoting cell survival and proliferation, survivin is an attractive candidate for targeted therapy. Toward this end, a detailed understanding of the mechanisms regulating survivin expression in different cancer cells will be critical. We have previously shown that the RNA-binding protein (RBP) CUG-BP1 is overexpressed in esophageal cancer cells and post-transcriptionally regulates survivin in these cells. The objective of this study was to investigate the role of microRNAs (miRs) in regulating survivin expression in esophageal cancer cells. Using miR expression profiling analysis, we found that miR-214-3p is one of the most markedly downregulated miRs in two esophageal squamous cancer cell lines compared with esophageal epithelial cells. Interestingly, using miR target prediction programs, both survivin and CUG-BP1 mRNA were found to contain potential binding sites for miR-214-3p. Forced expression of miR-214-3p in esophageal cancer cells leads to a decrease in the mRNA and protein levels of both survivin and CUG-BP1. This effect is due to decreased mRNA stability of both targets. By contrast, silencing miR-214-3p in esophageal epithelial cells leads to an increase in both survivin and CUG-BP1 mRNA and protein. To determine whether the observed effect of miR-214-3p on survivin expression was direct, mediated through CUG-BP1, or both, binding studies utilizing biotin pull-down assays and heterologous luciferase reporter constructs were performed. These demonstrated that the mRNA of survivin and CUG-BP1 each contain two functional miR-214-3p-binding sites as confirmed by mutational analysis. Finally, forced expression of miR-214-3p enhances the sensitivity of esophageal cancer cells to cisplatin-induced apoptosis. This effect is abrogated with rescue expression of survivin or CUG-BP1. These findings suggest that miR-214-3p acts as a tumor suppressor and that its downregulation contributes to chemoresistance in esophageal cancer cells by targeting both survivin and CUG-BP1.

Characterization of the expression and inflammatory activity of NADPH oxidase after spinal cord injury.

Reactive oxygen species (ROS) and the NADPH oxidase (NOX) enzyme are both up-regulated after spinal cord injury (SCI) and play significant roles in promoting post-injury inflammation. However, the cellular and temporal expression profile of NOX isotypes, including NOX2, 3, and 4, after SCI is currently unclear. The purpose of this study was to resolve this expression profile and examine the effect of inhibition of NOX on inflammation after SCI. Briefly, adult male rats were subjected to moderate contusion SCI. Double immunofluorescence for NOX isotypes and CNS cellular types was performed at 24 h, 7 days, and 28 days post-injury. NOX isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on injury status. NOX2 and 4 were found in all cell types assessed, while NOX3 was positively identified in neurons only. NOX2 was the most responsive to injury, increasing in both microglia and astrocytes. The biggest increases in expression were observed at 7 days post-injury and increased expression was maintained through 28 days. NOX2 inhibition by systemic administration of gp91ds-tat at 15 min, 6 h or 7 days after injury reduced both pro-inflammatory cytokine expression and evidence of oxidative stress in the injured spinal cord. This study therefore illustrates the regional and temporal influence on NOX isotype expression and the importance of NOX activation in SCI. This information will be useful in future studies of understanding ROS production after injury and therapeutic potentials.

Genetic association of DLG5 R30Q with familial and sporadic inflammatory bowel disease in men.

BACKGROUND: The association of DLG5 R30Q with IBD has been replicated in several populations, but is not statistically significant in others. We studied the incidence of DLG5 alleles in a population of IBD patients from Pennsylvania. METHODS: DLG5 R30Q (rs1248696) and G1066G (rs1248634) were analyzed with PCR-based RFLP methods in a total of 521 subjects, that included 105 individuals with IBD and 139 without IBD from a familial IBD registry, 107 with sporadic IBD, and 170 unrelated healthy controls. R30Q was further analyzed with SNPlex Genotyping System in 473 samples. RESULTS: RFLP genotyping data showed that, DLG5 R30Q was significantly associated with IBD overall (p=0.006), and separately with CD (p=0.009) and UC (p=0.024). The association of R30Q with IBD was entirely due to a male-associated effect (male vs female p=0.015 vs 0.241 (IBD), p=0.024 vs 0.190 (CD), and p=0.019 vs 0.575 (UC)). The frequency of the A allele carriage was elevated in both affected and unaffected members in the familial IBD cohort compared to healthy controls (p=0.037). In the family pedigrees, we observed differences in the expression of IBD in individuals carrying the A allele between families. CONCLUSIONS: In the studied population, DLG5 R30Q was associated with all forms of IBD. An elevated presence of the R30Q variant was observed in all members of a familial IBD registry. This association of the R30Q variant with IBD was male-specific.

Comparative Evaluation of the Q3 and Hybrid III 3-Year-Old Dummies in Biofidelity and Static Out-of-Position Airbag Tests.

A comparison of the Q3 and Hybrid III 3-year-old crash test dummies is presented in this paper. The performance of the dummies were compared in sixty biofidelity tests, seventy-seven static out-of-position airbag tests and sixty-three calibration tests. Various time histories and other data pertaining to accelerations, deflections, forces and moments are compared. In addition, the ease of positioning, handling, and the durability of the dummies in various out-of-position test configurations was assessed. Both the Q3 and Hybrid III 3-year-old dummies were calibrated to their respective specifications. The Hybrid III 3-year-old met its calibration requirements, while the Q3 did not always meet its own calibration requirements. The calibration specifications of the Q3 dummy need to be re-examined and possibly refined. The biofidelity of the Q3 and Hybrid III 3-year-old dummies were evaluated in both frontal and lateral test modes. Each dummy was evaluated against its own and the other's specified requirements, when possible. In the frontal test mode, the Hybrid III 3-year-old acceptably met all of its requirements. The Q3 dummy did not meet all of its own frontal biofidelity requirements. Based on these results, the Hybrid III 3-year-old is more biofidelic for primarily frontal loading conditions. With respect to the lateral biofidelity specifications, neither the Hybrid III 3-year-old nor the Q3 dummy met the requirements for the thorax and pelvis tests performed. Both dummies met the head drop requirements. Neither dummy is recommended for lateral loading conditions. For lateral testing where only the head is impacted, the Hybrid III 3-year-old could be used. In general, the responses of both dummies were repeatable in both the frontal and lateral biofidelity tests performed. The Hybrid III 3-year-old and the Q3 dummies were evaluated in static out-of-position airbag tests with three different side airbag systems (two seat-mounted and one door-mounted system), and one frontal passenger airbag system. Throughout this testing, the Q3 resultant head accelerations exhibited an excessive amount of high-frequency noise causing this dummy to be unacceptable for static out-of-position airbag testing. No significant issues were found with the Hybrid III 3-yearold. It was also determined that the Q3 dummy was more difficult to position repeatedly than the Hybrid III 3-yearold. This was due to the dummy's construction and its lack of rigid landmarks.

Spontaneous locomotor activity and pharmacokinetics of intravenous methamphetamine and its metabolite amphetamine in the rat.

The purpose of these studies was to better understand the behavioral effects and pharmacokinetics of an i.v. bolus dose of (+)-methamphetamine [(+)-METH] in a rat model of (+)-METH abuse. We characterized the behavioral effects after increasing (+)-METH doses (0.1, 0.3, and 1.0 mg/kg) and the pharmacokinetics of (+)-METH (and its metabolite (+)-amphetamine [(+)-AMP)]) at the lowest and highest of these doses in adult male Sprague-Dawley rats. The doses and route of administration were selected to mimic aspects of human use on a dose/body weight basis. Although the 0.1 mg/kg dose did not cause statistically significant increases in locomotor activity compared with saline controls, the higher doses (0.3 and 1.0 mg/kg) caused statistically significant increases in locomotor activity (p <.05), which lasted for up to 3 h at the highest dose. After the 1.0 mg/kg dose, the volume of distribution at steady state was 9.0 liters/kg, the total clearance was 126 ml/min/kg, and the average distribution and elimination half-lives were 9.2 and 63.0 min, respectively. Because the pharmacokinetic values after the 0.1 mg/kg dose were not different from those after the 1.0 mg/kg dose, the pharmacokinetics of (+)-METH were considered to be independent of the dose over this 10-fold range. (+)-AMP serum concentrations after the 1.0 mg/kg dose peaked from 10 to 30 min, and exhibited a T(1/2lambdaz) of 98.5 min. The statistically longer T(1/2lambdaz) of (+)-AMP (p <.05) suggested that the (+)-AMP terminal elimination rate and not the (+)-AMP metabolic formation rate is the rate-limiting step in (+)-AMP elimination following i.v. (+)-METH dosing.

Identification and characterization of a novel beta subunit of soluble guanylyl cyclase that is active in the absence of a second subunit and is relatively insensitive to nitric oxide.

Previously characterized soluble guanylyl cyclases form alpha-beta heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named alpha1, alpha2, beta1, and beta2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase beta3 (MsGC-beta3). It is most closely related to the mammalian beta subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-beta3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat beta1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-beta3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-beta3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-beta3 is primarily expressed in the nervous system of Manduca.

Central line associated bacteremia in the pediatric patient.

PURPOSE: To define the per-day risk of central line associated bacteremia in an infant-toddler population and to describe risk factors associated with the development of central line bacteremia. METHOD: The Central Line Data Tool collected information on 102 central venous catheters from 73 patients ranging in age from 1 day to 29 months. Each line was in place for 3 days or longer. FINDINGS: There were 17 documented catheter-related infections during the 1-year study period (7.7 infections per 1,000 catheter days). Factors significantly associated with central line bactermia included: PAS infusion, catheter type and site, medication administration, blood withdrawal, and accidental line disruption. CONCLUSIONS: Use of central lines for multiple purposes should be minimized.

Conducting the pediatric health history: a guide.

Although obtaining thorough and accurate pediatric health histories can be a challenging task, they are critically important to the health and well-being of children. A thorough history is a crucial component to both acute and episodic visits. Components of the health history are reviewed.

A competitive enzyme immunoassay for the direct determination of mometasone furoate (SCH 32088) in human plasma.

Mometasone furoate (SCH 32088) is a synthetic corticosteroid which has a topical anti-inflammatory activity with a minimal potential for suppressing hypothalamic-pituitary-adrenocortical (HPA) axis. A sensitive competitive enzyme immunoassay (EIA) for measuring SCH 32088 in unextracted human plasma has been developed. The 3-(O-carboxymethyl)oxime (CMO) of SCH 32088 was synthesized and conjugated with bovine thyroglobulin, and the product was used for the production of antibodies in rabbits. SCH 32088-3-CMO was also conjugated with horseradish peroxidase, which was used as the tracer. The EIA thus developed can detect 1 pg SCH 32088 per assay or 25 pg per ml of human plasma. It can reliably quantitate SCH 32088 in human plasma from 50 pg ml-1 to 2.5 ng ml-1 with good linearity, accuracy and precision. The assay can be extended to measure SCH 32088 in plasma of laboratory animals. The availability of this sensitive assay makes it possible to evaluate the pharmacokinetics and toxicokinetics of SCH 32088 in laboratory animals and man.

Seroepidemiology of arboviruses among seabirds and island residents of the Great Barrier Reef and Coral Sea.

Duplicate neutralization tests were done on 401 avian and 101 human sera from island residents collected in the Coral Sea and on Australia's Great Barrier Reef against 19 known arboviruses. Antibodies to a potentially harmful flavivirus, Gadget's Gully virus, were equally present (4%) in both avian and human sera. Antibodies to another flavivirus, Murray Valley Encephalitis, and an ungrouped isolate, CSIRO 1499, were also present in both populations with non-significantly different incidences. Antibodies to Upolu, Johnston Atoll, Lake Clarendon, Taggert, Saumarez Reef and CSIRO 264 viruses were restricted to seabirds. Island residents with antibodies to Ross River and Barmah Forest viruses are thought to have been exposed to these viruses on the mainland as antibody to both viruses was absent among seabirds. These results indicate that consideration should be given to tick-associated arboviruses as potential public health hazards on islands where both seabird and human activities interact.