Expression of soluble, active, fluorescently tagged hephaestin in COS and CHO cell lines

Hephaestin (Hp) is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originally identified as the gene responsible for the phenotype of sex-linked anaemia in the sla mouse. The mutation in the sla protein causes ac-cumulation of dietary iron in duodenal cells, causing severe microcytic hypochromic anaemia. Although mucosal uptake of dietary iron is normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin (Cp), a member of the family of multi copper fer-roxidases (MCFs) and possesses ferroxidase activity that facilitates iron release from the duodenum and load onto the serum iron trans-port protein transferrin. In the present study, attempts were made to produce biologically active recombinant mouse hephaestin as a secretory form tagged with green fluorescent protein (GFP), Hpsec-GFP. Plasmid expressing Hpsec-GFP was constructed and transfected into COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression and provided a tag for purifying and analysing Hpsec-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-based assays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteins using complementary fluorescent tags on target proteins that would facilitate the fluorescence resonance energy transfer measurements, for example with transferrin or colocalisation studies, and help to discover more about hephaestin works at the molecular level.</jats:p

Effect of sodium hydroxide on mechanical characteristics of kenaf fibers reinforced concrete

This article shows the experimental analysis of sodium hydroxide (NaOH) effects on the mechanical properties upon the addition of kenaf fibres to the concrete mixture. The compression and flexural tests were carried out on seven different concrete mixtures for 28 days to evaluate the mechanical characteristics. Hence, the experimental trials comprise of seven different mixtures of concrete; the control (without the addition of kenaf fibre) was represented as the first mixture. The following six concrete mixtures contain different kenaf fibre volume fractions (1% and 2%); they were treated with 1%, 3% and 6% of sodium hydroxide concentrations in order to investigate their performances. The experimental results show that the compressive and flexural strengths of kenaf fibres reinforced concrete increase as sodium hydroxide concentration increases. Besides, it is worth noting that the addition of kenaf fibres has an insignificant effect on the compressive strength of the concrete owing to the reduction in concrete density. Therefore, the results of this study demonstrated that the concrete flexural strength increased to up to 12%) upon the addition of 1% kenaf fibre associated with 6% NaOH. Moreover, the failure state increasingly changed to ductile from brittle

Evaluation of the knowledge and practices of pregnant Yemeni Women regarding teratogens

Purpose: To investigate the knowledge and practice of pregnant women with regards to teratogens.Methods: A month-long cross-sectional study was carried out among 150 pregnant women selected from four Motherhood and Child Healthcare Centers (MCHCs) in Mukalla District of Yemen. Data collection was conducted during face-to-face interviews using a questionnaire. Descriptive and simple regression analyses were used.Results: Of the 150 pregnant women who participated in the study, 95.3 % of the pregnant women were &lt; 36 years old, 7.4 % had children with congenital malformations, 62 % indicated that they had heard about folic acid; however, only 16.6 % knew the significance of folic acid. Regarding toxoplasmosis, 94.7 % indicated that they had heard about toxoplasmosis, and 76 % knew about the serious consequences of the disease (congenital malformation and abortion) during pregnancy. Based on simple regression analysis, the results indicate that education and parity, irrespective of age or income level, were the major factors determining better knowledge and practices in pregnancy with regards to toxoplasmosis.Conclusion: Knowledge of folic acid deficiency among pregnant women in Mukalla District of Yemen is relatively low. Furthermore, preventive practices to avoid folic acid deficiency are minimal.Keywords: Knowledge, Practices, Teratogens, Pregnant Yemeni women, Folic acid deficienc

Acute bilateral simultaneous angle closure glaucoma after topiramate administration: a case report

<p>Abstract</p> <p>Introduction</p> <p>A case of severe acute bilateral angle closure glaucoma with complete visual loss after oral topiramate therapy.</p> <p>Case presentation</p> <p>A 34 year-old woman developed bilateral severe visual loss 2 days after doubling the dosage of topiramate. Her best-corrected visual acuity (BCVA) was counting fingers in both eyes (OU). Intraocular pressures were 49 mm and 51 mm of Hg in right and left eyes respectively, with conjunctival chemosis, corneal edema, shallow anterior chamber and closed angles on gonioscopy. B-scan ultrasound revealed annular peripheral choroidal effusions in both eyes.</p> <p>Conclusion</p> <p>Intraocular pressures and anterior chamber depth were normalized after discontinuation of topiramate and initiation of antiglaucoma therapy. Two weeks later, visual acuities improved to 20/25 in the right eye and 20/40 in the left eye. B-scan ultrasound showed resolution of choroidal effusion. Topiramate, an oral sulpha-derivative medication is known to cause ciliochoroidal effusions, which lead to forward rotation of the ciliary body and displacement of the lens-iris diaphragm, with resultant acute angle closure glaucoma and myopic shift.</p

Cingulate cortex hypoperfusion predicts Alzheimer's disease in mild cognitive impairment

BACKGROUND: Mild cognitive impairment (MCI) was recently described as a heterogeneous group with a variety of clinical outcomes and high risk to develop Alzheimer's disease (AD). Regional cerebral blood flow (rCBF) as measured by single photon emission computed tomography (SPECT) was used to study the heterogeneity of MCI and to look for predictors of future development of AD. METHODS: rCBF was investigated in 54 MCI subjects using Tc-99m hexamethylpropyleneamine oxime (HMPAO). An automated analysis software (BRASS) was applied to analyze the relative blood flow (cerebellar ratios) of 24 cortical regions. After the baseline examination, the subjects were followed clinically for an average of two years. 17 subjects progressed to Alzheimer's disease (PMCI) and 37 subjects remained stable (SMCI). The baseline SPECT ratio values were compared between PMCI and SMCI. Receiver operating characteristic (ROC) analysis was applied for the discrimination of the two subgroups at baseline. RESULTS: The conversion rate of MCI to AD was 13.7% per year. PMCI had a significantly decreased rCBF in the left posterior cingulate cortex, as compared to SMCI. Left posterior cingulate rCBF ratios were entered into a logistic regression model for ROC curve calculation. The area under the ROC curve was 74%–76%, which indicates an acceptable discrimination between PMCI and SMCI at baseline. CONCLUSION: A reduced relative blood flow of the posterior cingulate gyrus could be found at least two years before the patients met the clinical diagnostic criteria of AD

The predictive and prognostic value of tumour necrosis in muscle invasive bladder cancer patients receiving radiotherapy with or without chemotherapy in the BC2001 trial (CRUK/01/004)

Background: Severe chronic hypoxia is associated with tumour necrosis. In patients with muscle invasive bladder cancer (MIBC), necrosis is prognostic for survival following surgery or radiotherapy and predicts benefit from hypoxia modification of radiotherapy. Adding mitomycin C (MMC) and 5-fluorouracil (5-FU) chemotherapy to radiotherapy improved locoregional control (LRC) compared to radiotherapy alone in the BC2001 trial. We hypothesised that tumour necrosis would not predict benefit for the addition of MMC and 5-FU to radiotherapy, but would be prognostic. Methods: Diagnostic tumour samples were available from 230 BC2001 patients. Tumour necrosis was scored on whole-tissue sections as absent or present, and its predictive and prognostic significance explored using Cox proportional hazards models. Survival estimates were obtained by Kaplan–Meier methods. Results: Tumour necrosis was present in 88/230 (38%) samples. Two-year LRC estimates were 71% (95% CI 61–79%) for the MMC/5-FU chemoradiotherapy group and 49% (95% CI 38–59%) for the radiotherapy alone group. When analysed by tumour necrosis status, the adjusted hazard ratios (HR) for MMC/5-FU vs. no chemotherapy were 0.46 (95% CI: 0.12–0.99; P=0.05, necrosis present) and 0.55 (95% CI: 0.31–0.98; P=0.04, necrosis absent). Multivariable analysis of prognosis for LRC by the presence vs. absence of necrosis yielded a HR=0.89 (95% CI 0.55–1.44, P=0.65). There was no significant association for necrosis as a predictive or prognostic factor with respect to overall survival. Conclusions: Tumour necrosis was neither predictive nor prognostic, and therefore MMC/5-FU is an appropriate radiotherapy-sensitising treatment in MIBC independent of necrosis status

Directing cell therapy to anatomic target sites in vivo with magnetic resonance targeting

Cell-based therapy exploits modified human cells to treat diseases but its targeted application in specific tissues, particularly those lying deep in the body where direct injection is not possible, has been problematic. Here we use a magnetic resonance imaging (MRI) system to direct macrophages carrying an oncolytic virus, Seprehvir, into primary and metastatic tumour sites in mice. To achieve this, we magnetically label macrophages with super-paramagnetic iron oxide nanoparticles and apply pulsed magnetic field gradients in the direction of the tumour sites. Magnetic resonance targeting guides macrophages from the bloodstream into tumours, resulting in increased tumour macrophage infiltration and reduction in tumour burden and metastasis. Our study indicates that clinical MRI scanners can not only track the location of magnetically labelled cells but also have the potential to steer them into one or more target tissues

Characterization of an Enantioselective Odorant Receptor in the Yellow Fever Mosquito Aedes aegypti

Enantiomers differ only in the left or right handedness (chirality) of their orientations and exhibit identical chemical and physical properties. In chemical communication systems, enantiomers can be differentially active at the physiological and behavioral levels. Only recently were enantioselective odorant receptors demonstrated in mammals while their existence in insects has remained hypothetical. Using the two-microelectrode voltage clamp of Xenopus oocytes, we show that the yellow fever mosquito, Aedes aegypti, odorant receptor 8 (AaOR8) acts as a chiral selective receptor for the (R)-(—)-enantiomer of 1-octen-3-ol, which in the presence of other kairomones is an attractant used by blood-sucking insects to locate their hosts. In addition to steric constraints, chain length and degree of unsaturation play important roles in this recognition process. This is the first characterization of an enantioselective odorant receptor in insects and the results demonstrate that an OR alone, without helper proteins, can account for chiral specificity exhibited by olfactory sensory neurons (OSNs)