Detection of unknown deletions in β‐globin gene cluster using relative quantitative PCR methods

β‐Thalassemia is mainly caused by mutations involving single base substitution and small deletions. However, a considerable number of carriers are suspected to have large deletions in β‐globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, however, some cases with indefinite breakpoints which usually cannot be detected by this method. We developed and optimized a quantitative real‐time PCR assay for copy number analysis of β‐globin gene cluster. The copy number of target fragments (i.e. β, δ or ^Gγ‐globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation‐dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown samples were successfully determined according to the ranges obtained from the ratios of normal and definite carrier samples. Interestingly, large deletions involving the entire β‐globin gene cluster were observed in six carrier individuals. This study showed that the MLPA as a preliminary screening test can be followed by SYBR Green real‐time PCR for analysis of copy number variations in β‐globin gene cluster. Combination of these relative quantitative PCR methods could be an appropriate approach for accurate diagnosis of unknown β‐thalassemia deletions in routine diagnosis of β‐thalassemia mutations.     

Effect of dietary behaviour modification on anthropometric indices and eating behaviour in obese adolescent girls

Title. Effect of dietary behaviour modification on anthropometric indices and eating behaviour in obese adolescent girls. Aim. This paper is a report of a study conducted to evaluate the effects of behaviour modification on anthropometric indices and to explore if behaviour modification could improve eating behaviour in adolescents. Background. Obesity is currently the most important nutritional disease of children and adolescents. To date, several attempts to achieve weight loss in children have been made, but little is known about their effects on improving eating behaviours. Method. Sixty obese adolescent girls participated in a behaviour modification program which was held for 16 weeks in 2007. The participants were randomly selected from two different schools and were assigned to an experimental and control group (30 participants each). Anthropometric indices and eating behaviours were assessed before and after the program. Eating behaviour was assessed using the Dutch Eating Behaviour Questionnaire. Result. There were statistically significant differences in changes in body weight (?2·75 kg vs. 0·62 kg), body mass index (?1·07 kg/m² vs. 0·24 kg/m²) and arm circumference (?2·31 cm vs. 0·5 cm) in the experimental group in contrast to controls (P < 0·001). There were also statistically significant differences in scores for eating behaviour, emotional eating (0·63, 0·17), external eating (0·99, 0·05) and restrained eating (0·72, 0·03) in the experimental vs. the control group respectively (P < 0·001). Conclusion. Nurses, more than other healthcare professionals, can address obesity in adolescents and they should not concentrate solely on weight reduction, but also encourage children to acquire a healthy lifestyle.     

Intra-retinal layer segmentation of 3D optical coherence tomography using coarse grained diffusion map

Optical coherence tomography (OCT) is a powerful and noninvasive method for retinal imaging. In this paper, we introduce a fast segmentation method based on a new variant of spectral graph theory named diffusion maps. The research is performed on spectral domain (SD) OCT images depicting macular and optic nerve head appearance. The presented approach does not require edge-based image information in localizing most of boundaries and relies on regional image texture. Consequently, the proposed method demonstrates robustness in situations of low image contrast or poor layer-to-layer image gradients. Diffusion mapping applied to 2D and 3D OCT datasets is composed of two steps, one for partitioning the data into important and less important sections, and another one for localization of internal layers. In the first step, the pixels/voxels are grouped in rectangular/cubic sets to form a graph node. The weights of the graph are calculated based on geometric distances between pixels/voxels and differences of their mean intensity. The first diffusion map clusters the data into three parts, the second of which is the area of interest. The other two sections are eliminated from the remaining calculations. In the second step, the remaining area is subjected to another diffusion map assessment and the internal layers are localized based on their textural similarities. The proposed method was tested on 23 datasets from two patient groups (glaucoma and normals). The mean unsigned border positioning errors (mean ± SD) was 8.52 ± 3.13 and 7.56 ± 2.95 μm for the 2D and 3D methods, respectively.     

Characterization of Shigella strains in Iran by plasmid profile analysis and PCR amplification of ipa genes

To characterize Shigella clinical strains, we studied 82 Shigella strains recovered from 719 stool samples of patients with bloody diarrhea in Shiraz, Iran, over the period from April to October 2003. Serological assay classified the Shigella isolates as follows: 61 (74.39%) Shigella sonnei isolates, 16 (19.51%) Shigella flexneri isolates, 3 (3.65%) Shigella boydii isolates, and 2 (2.43%) Shigella dysenteriae isolates. In an antibiogram test, all Shigella strains were susceptible to ceftazidime, ciprofloxacin, and ceftriaxone. They showed high degrees of sensitivity to nalidixic acid, gentamicin, cephalothin, and amikacin. Approximately 90.24% of the Shigella isolates were resistant to co-trimoxazole. The plasmid profile patterns of all strains were determined by a modified alkaline lysis method. The average number of plasmid bands for each strain was 9.5. By plasmid profile analysis we identified 56 genotypes among all isolates and 42, 14, 3, and 2 genotypes among the S. sonnei, S. flexneri, S. boydii, and S. dysenteriae strains, respectively. PCR assays showed that all isolates were positive for two virulence genes, ipaBCD and ipaH. In conclusion, these data mandate local monitoring of drug resistance and its consideration in the empirical therapy of Shigella infections. These results also demonstrate that plasmid profile analysis is more reliable than antibiotic susceptibility pattern analysis for the identification of Shigella epidemic strains isolated in Iran.     

Neutrophil gelatinase-associated lipocalin acts as a protective factor against H(2)O(2) toxicity

BACKGROUND: Lipocalin 2 (Lcn2, NGAL) is a member of the lipocalin superfamily for which a variety of functions have been reported. However, the precise biological roles of NGAL are not fully known. We have investigated the ability of NGAL to prevent H(2)O(2) toxicity, which is considered to be the classical inducer of oxidative stress caused by ROS generation in an in vitro model. METHODS: NGAL cDNA was isolated from HepG2 cell line and cloned to pcDNA3.1(+) vector. The construct was transfected to CHO cell line. Stable clones were generated, and the expression of NGAL was determined by RT-PCR, Western blot analysis and ELISA. NGAL gene in A549 cell line was downregulated with the siRNA. CHO and A549 cells were intoxicated with H(2)O(2) and cell proliferation was performed by MTT assay. Apoptotic cells were detected by flow cytometry. RESULTS: Cell proliferation was higher in CHO expressing NGAL in doses of 5 and 10 mM H(2)O(2) after 2h compared with the control. H(2)O(2) was also more toxic in the presence of NGAL siRNA compared with the control in A549 cell. Our results also revealed that NGAL protect cells from apoptosis. CONCLUSIONS: Overall, our results revealed for the first time a new function for NGAL/Lcn2: acting as a protective factor against H(2)O(2) toxicity. In the future, NGAL may have the potential application to ameliorate the toxicity induced by oxidative stress conditions.     

Peripheral distributions of IL-4-producing CD4 + T cells and CD4 + CD25 + FoxP3 + T cells (Tregs) in rheumatoid arthritis patients with poor response to therapy are associated with HLA shared epitope alleles and ACPA status

Specific profiling of CD4?+?T cell subsets in the circulation and inflamed joints of rheumatoid arthritis (RA) patients may have therapeutic implications. This study aimed to evaluate the peripheral distributions of Th2 and Treg cells in relation to HLA-shared epitope (SE) alleles and anti-cyclic citrullinated peptide antibody (ACPAs) status in patients with good response (GR) and poor response (PR) to treatment. The frequencies of IL-4-producing CD4?+?T cells (Th2) and CD4?+?CD25?+?Foxp3?+?T cells (Tregs) were determined by flow cytometry in 167 RA patients including 114 GR and 53 PR cases. CD4?+?T cell subsets were also analyzed based on HLA-SE and ACPAs statuses. One hundred nine of 167 patients were positives for HLA-SE, 63.4% for ACPAs, 43.7% for SE/ACPAs and 14.9% were negatives for SE/ACPAs. Higher frequencies of Th2 (P?=?0.001) and Treg cells (P?=?0.03) were found in the patients versus controls. Increased and decreased frequencies of Th2 and Tregs cells were observed in the PR versus GR patients respectively (P?=?0.003 and P?=?0.004). Higher proportions of Th2 cells were observed in the SE⁺RA versus SE^?RA (P?=?0.001), in ACPA⁺RA versus ACPA^?RA (P?=?0.005) and in the SE⁺ACPA⁺RA versus SE^?ACPA^?RA patients (P?=?0.002). Treg cells frequencies decreased in the SE⁺RA versus SE^?RA (P?=?0.03) and in SE⁺ACPA⁺RA versus SE^?ACPA^?RA (P?=?0.02). ACPA⁺GR and SE⁺PR patients showed higher proportions of Th2 cells than ACPA^?GR and SE^?PR patients respectively (P?=?0.02 and P?=?0.01). Analysis of the CD4?+?T cell subsets profiles in conjunction with genetic background and autoantibodies patterns can be useful for precise therapeutic response monitoring in the RA patients.

     

Drug Avoidance Self Efficacy Scale (DASES): A cultural adaptation and validation study

Background: Drug avoidance self efficacy has always been an important issue in substance abuse treatment and its important has emphasized in specific models of addiction. This study describes the process of cultural adaptation, validation and dimensionality of the Persian version of “Drug Avoidance Self Efficacy Scale”.

Methods: Cultural adaptation process followed the procedure outlined by Beaton et al. Psychometric properties and dimensionality of the scale were assessed using sample of Iranian addicts (n?=?317). The DASES was subject to independent exploratory (n?=?157, mean age 29.40?±?7.26) and confirmatory (n?=?160, mean age 27.99?±?5.70) factor analysis. Internal consistency of the questionnaire also was assessed using Cronbach’s alpha.

Results: There was no eliminated item in the cultural adaptation process. Results of exploratory factor analysis revealed a two-factor 14-item structure for the DASES. Secondary order, two-factor model provided a good statistical and conceptual fit for the data. The analysis of internal consistency of DASES was very satisfactory (α?=?0.809).

Conclusions: Although culturally adapted DASES is a potentially reliable and valid measure of substance users’ perceptions of avoidance self-efficacy, the findings should be viewed as preliminary in nature. Future research needs to determine to what extent substance users’ perceptions of avoidance self efficacy impact on treatment outcomes.     

The effects of two-stage carotid occlusion on spatial memory and pro-inflammatory markers in the hippocampus of rats

The purpose of this study was to evaluate the effects of cerebral hypoperfusion on cognitive ability, TNFα, IL1β and PGE2 levels in both hippocampi in a modified two-vessel occlusion model. Both common carotid arteries of adult male Wistar rats were permanently occluded with an interval of 1 week between occlusions. Learning and memory were significantly decreased after 1 month. This reduction was not significant after 2 months, which may be attributed to blood flow compensation. The TNFα level was significantly increased after 3 h and 1 day. IL1β was significantly increased after 1 day. After a week there was no significant difference in pro-inflammatory levels. Furthermore, there was no difference between right and left hippocampi. It is possible that TNFα and IL1β elevation initiates pathologic processes that contribute to memory impairment.     

Synthesis, characterization, and biocompatibility of novel injectable, biodegradable, and in situ crosslinkable polycarbonate-based macromers

A series of novel self-crosslinkable and biodegradable polymers, poly(hexamethylene carbonate-fumarate) and poly(hexamethylene carbonate) diacrylate, and their amphiphilic copolymers with polyethylene glycol, poly(ethylene glycol fumarate-co-hexamethylene carbonate-fumarate) (PEGF-co-PHMCF), were developed for tissue engineering using propylene oxide as an acid scavenger. The synthesized polymers are white, which makes them more suitable for self-crosslinking via photopolymerization. These novel polymers were fully characterized using nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, gel permeation chromatography, differential scanning calorimetry, and rheometry. The cytocompatibility of the photocrosslinked networks were evaluated by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. These polymers can be used as precursors to prepare polymer networks and scaffolds with controlled hydrophilicity, biodegradability, and mechanical characteristics. Results obtained suggest that these polymers are potentially useful as injectable and photocrosslinkable materials for cell delivery, tissue engineering, and drug delivery applications.