Cloning, sequence analysis and confirmation of derived gene sequences for three epitope-mapped monoclonal antibodies against human phagocyte flavocytochrome b

The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the NADPH oxidase complex, a multicomponent enzyme system that initiates a cascade of reactive oxygen species that play a critical role in innate immunity and vascular physiology. Epitope-mapped, monoclonal antibodies (mAb) that recognize the large (gp91phox) and small (p22phox) subunits of Cyt b provide valuable reagents that have been used to examine structural and mechanistic aspects of oxidase function. In the present study, the heavy and light chain variable region genes of the Cyt b-specific mAbs 44.1, NS5, and NL7 have been amplified by RT-PCR, cloned and subject to DNA sequence analysis. Since the 5' degenerate primer sets used for mAb gene amplification were observed to introduce extensive heterogeneity into the heavy and light chain FR1 regions, N-terminal protein sequence analysis was also conducted to obtain the correct amino acid sequence of this region. In order to confirm the identity of the cloned genes, intact mAbs were resolved by two-dimensional electrophoresis and subject to in-gel tryptic digestion for analysis by both MALDI and nanospray LC-MS/MS. Databases searches using the derived mAb sequences predicted residues comprising CDR loops, identified candidate germline genes, and showed the respective germline genes to accurately predict the N-terminal amino acid residues for each variable region. The above studies report the amino acid sequence of Cyt b-specific mAb variable region genes with high confidence and provide essential information for future efforts at Cyt b structure analysis by resonance energy transfer and X-ray crystallography.     

An exploration of parents’ knowledge,attitudes and practices towards the use of antibiotics in childhood upper respiratory tract infections in a tertiary Jordanian Hospital

Objectives

The present study aimed to evaluate the parents' knowledge, attitudes and practices (KAP) towards the use of antibiotics for childhood upper respiratory tract infections (URTIs), at the Jordanian University Hospital.

Low-energy laser therapy application on knee joints as an auxiliary treatment in patients with polyarticular juvenile idiopathic arthritis: a dual-arm randomized clinical trial

Lasers in Medical Science - Patients with juvenile idiopathic arthritis (JIA) always experience persistent pain and stiffness which induces muscle weakness, fatigue, and functional limitations....     

Evidence of Circulation of Several HAV Genetic Variants and Emergence of Potential Antigenic Variants in an Endemo-Epidemic Country before Vaccine Introduction

Similar to several other countries in the world, the epidemiology of hepatitis A virus changed from high to intermediate endemicity level in Tunisia, which led to the occurrence of outbreaks. This study aimed to determine the genetic and antigenic variability of HAV strains circulating in Tunisia during the last few years. Genotyping using complete VP1 gene and VP1-2A junction confirmed the predominance of genotype IA, with co-circulation of several genetic and antigenic variants. Phylogenetic analysis including Tunisian and strains from other regions of the world showed the presence of at least two IA-variants within IA subgenotype. Amino-acid analysis showed several mutations in or close to epitope regions in the VP1-region. This study provides a baseline on the genetic and antigenic variability of HAV circulating strains before the introduction of vaccination into the national immunization schedule.     

Sewage‐based epidemiology in monitoring the use of new psychoactive substances: Validation and application of an analytical method using LC‐MS/MS

Sewage‐based epidemiology (SBE) employs the analysis of sewage to detect and quantify drug use within a community. While SBE has been applied repeatedly for the estimation of classical illicit drugs, only few studies investigated new psychoactive substances (NPS). These compounds mimic effects of illicit drugs by introducing slight modifications to chemical structures of controlled illicit drugs. We describe the optimization, validation, and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine (MPA), 4‐methoxymethamphetamine (PMMA), and 4‐methoxyamphetamine (PMA). Sample preparation was performed using solid‐phase extraction (SPE) with Oasis MCX cartridges. The LC separation was done with a HILIC (150 x 3 mm, 5 µm) column which ensured good resolution of the analytes with a total run time of 19 min. The lower limit of quantification (LLOQ) was between 0.5 and 5 ng/L for all compounds. The method was validated by evaluating the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recoveries and matrix effects. The method was applied on sewage samples collected from sewage treatment plants in Belgium and Switzerland in which all investigated compounds were detected, except MPA and PMA. Furthermore, a consistent presence of MXE has been observed in most of the sewage samples at levels higher than LLOQ. Copyright © 2015 John Wiley & Sons, Ltd.     

Influence of Hydrofluoric and Nitric Acid Pre-Treatment and Type of Adhesive Cement on Retention of Zirconia Crowns

Background: The aim of this study was to test the impact of hot acids etching and two types of adhesive cement on the retention of zirconia crowns. Methods: Forty maxillary premolars were prepared, and zirconia crowns were designed and fabricated with proximal extensions, then divided into 4 groups (n = 10). Group AP; the crowns were air-abraded and cemented using Panavia SA Cement. Group AL; the crowns were air- abraded and cemented using GC LinkForce. Group AHP; the crowns were air-abraded, etched with the hot acids (48% hydrofluoric acid and 69% nitric acid), and cemented using Panavia SA Cement. Group AHL; the crowns were air-abraded, etched with the hot acids, and cemented using GC LinkForce. Each zirconia crown was pre-treated and bonded to its corresponding tooth. After thermocycling (5–55 °C/10,000), the retention test was performed and the load required to dislodge the crown was reported in Newton (N), and mode of failure was recorded. The retention strength (MPa) was calculated for each tested variable and statistically analyzed. Results: Group AHP showed the highest mean value of the retention strength, followed by group AP then group AHL. Group AL showed the lowest value. A statistically significant effect (p = 0.001) of the hot acids etching on the retention of zirconia crown was found. Also, there was a significant effect (p = 0.000) of the cement type. The interaction between surface treatment and the cement type has no significant impact (p = 0.882). The main mode of failure for Panavia SA Cement is mixed mode of failure, while for G-CEM LinkForce is adhesive failure. Conclusions: Hot acid etching pre-treatment improved the retention of zirconia crown. Usage of Panavia SA Cement with hot acids etching is effective can be used for adhesive cementation of zirconia crown.     

Biliary stent migration causing incarceration and perforation of the ileum in a previously uncomplicated inguinal hernia: Report of a case and review of literature

Endoscopic biliary stent insertion is a well-established minimally invasive procedure used in the management of hepatic, biliary or pancreatic disorders. Dislocation and migration of endoscopically inserted biliary stents is a late complication; it has a reported incidence of about 7%–10% of cases. Most of the migrated stents pass naturally without complications. Very occasionally, (less than 1%), the stent becomes stuck and produces gut perforation. Very few case reports in the literature describe plastic biliary stents perforating a bowel in a hernia. Herein, we report the case of a migrated plastic biliary stent that perforated an ileal loop inside a sac of a previously uncomplicated inguinal hernia.