Human bone marrow mesenchymal stem cell behaviors on PCL/Gelatin nanofibrous scaffolds modified with a collagen IV-derived RGD-containing peptide

We introduce an RGD [Arg-Gly-Asp]-containing peptide of collagen 4 origin that possesses potent cell adhesion and proliferation properties. In this experimental study, the peptide was immobilized on an electrospun nanofibrous polycaprolactone/gelatin [PCL/Gel] hybrid scaffold by a chemical bonding approach to improve cell adhesion properties of the scaffold. An iodine- modified phenylalanine was introduced in the peptide to track the immobilization process. Native and modified scaffolds were characterized with scanning electron microscopy [SEM] and fourier transform infrared spectroscopy [FTIR]. We studied the osteogenic and adipogenic differentiation potential of human bone marrow-derived mesenchymal stem cells [hBMSCs]. In addition, cell adhesion and proliferation behaviors of hBMSCs on native and peptide modified scaffolds were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay and 4',6-diamidino-2-phenylindole [DAPI] staining, and the results compared with tissue culture plate, as the control. FTIR results showed that the peptide successfully immobilized on the scaffold. MTT assay and DAPI staining results indicated that peptide immobilization had a dramatic effect on cell adhesion and proliferation. This peptide modified nanofibrous scaffold can be a promising biomaterial for tissue engineering and regenerative medicine with the use of hBMSCs

[Study of hBMSC adhesion and proliferation on RGD-modified polycaprolactone/gelatin nanofibrous scaffold]

In this study we introduced an RGD-containing peptide of collagen IV origin that possesses potent cell adhesion and proliferation properties. This peptide was immobilized on a nanofibrous polycaprolactone/gelatin scaffold after which we analyzed human bone marrow-derived mesenchymal stem cells [hBMSCs] adhesion and proliferation on this peptide-modified scaffold. Nanofibrous scaffold was prepared by electrospinning. The peptide was synthesized by solid-phase peptide synthesis and immobilized on electrospun nanofibrous a polycaprolactone/gelatin scaffold by chemical bonding. Native and modified scaffolds were characterized with Scanning Electron Microscope [SEM] and Fourier-Transform Infra-red Spectroscopy [FTIR]. Adhesion and proliferation of hBMSCs on native and modified scaffolds were analyzed by the Methylthiazol Tetrazolium [MTT] assay. SEM images showed that electrospun scaffolds had homogenous morphology and were 312 +/- 89 nm in diameter. There was no significant difference in scaffold morphology before and after peptide immobilization. FTIR results showed that the peptide was successfully immobilized on the scaffold. Based on MTT assay, cell adhesion studies indicated that peptide immobilization improved cell adhesion on RGD-modified scaffolds at all corresponding time points [p<0.05]. RGD immobilization led to increased cell proliferation potential of the scaffold compared with tissue culture plate and native scaffold [P<0.05]. This novel peptide and modified nanofibrous scaffold, having improved cell adhesion and proliferation properties, can be used for tissue engineering and regenerative medicine by using hBMSCs.

Expression and characterization of bacterial organophosphorus hydrolase in pichia pastoris with the intent to degrade organophosphate neurotoxins

Organophosphorus hydrolase [OPH] is a homodimeric enzyme that can hydrolyze phosphoester bonds and reduce the toxicity of organophosphorus compounds. This makes OPH a suitable element for the biodegradation of these compounds. We successfully cloned the OPH gene from Pseudomonas diminuta, after optimization for Pichia pastoris, into a yeast expression vector [pPICZlphaB]. After transformation and induction of recombinant yeasts, the expressed enzyme was investigated for its biochemical and kinetical parameters. The enzyme was purified 7.49-fold to a specific activity of 0.421×10[3] U/mg protein from the supernatant with a yield of 33%. The purified enzyme was able to degrade organophosphates. It had an optimal activity and stability up to 50°C, and a pH range of 7.0-10.0. The enzyme had a Km of 45.96 µM and a Vmax of 11.23 microM/min [421 microM/min/mg] for paraoxon as a substrate. This enzyme was sensitive to divalent cations and inactivated by denaturing compounds such as SDS. The molecular mass of the purified enzyme as estimated by SDS-PAGE analysis was approximately 40 kDa. In this study, the purified enzyme effectively hydrolyzed paraoxon, an organophosphorus compound. The activity and stability of this enzyme at high temperatures and pH, and low Km in comparision with bacterial isolates could make it an attractive biocatalyst for applied bioremediation and biosensing

Factors associated with secondhand smoke exposure in infants

Determining the factors associated with secondhand smoke [SHS] exposure in children provides valuable information for smoking control strategies. This study aimed to assess factors related to SHS exposure in infants based on urinary cotinine measures. A cross-sectional analysis of the data that were collected as part of the randomized controlled trial was conducted. Participants were 130 smoking households with children under the age of 1 year attending a health care center in southern Tehran. Eligible parents consented to participate in this study and completed a questionnaire including demographic data, questions regarding smoking at home, smoking status and Fagerstrom test through face-to-face interview. The Infants' urinary cotinine level was measured using gas chromatography, adjusted with urinary creatinine level and reported as cotinine [ng]/ creatinine [mg]. Factors related to infants' SHS exposure were assessed using the multivariate logistic regression model based on standard cut-point [30 ng of urinary cotinine/mg creatinine]. The final multivariate logistic regression model showed that social status [p=0.002], home smoking restriction [p=0.05] and the infant's age [p=0.01] were associated with the infants' SHS exposure determined based on urinary cotinine levels. These results support the influence of social status, home smoking restriction and infant's age on the exposure of infants to SHS

Estimation of atlatoxin-albumin adduct in serum sample by optimized HPLC-fluorescence

With consideration of lethal effects of aflatoxins specially Bl on human health. Estimation of aflatoxin-albumin adduct, as an important marker of aflatoxin exposure, seems essential. The aim of this study is optimization of HPLC-fluorescence method for measurement of this important marker in blood serum. In this study, blood serum of three groups of rats as A] positive controls [treated with AFB1], B] negative controls [without treatment] and standard rats [treated with radiolabeled AFBl] were used. After albumin isolation using ammunium sulphate and acetic acid, purity of albumin was tested by SDS-PAGE electrophoresis and albumin concentration was quantified by bradford method. Then albumin was hydrolysed by pronase and aflatoxin bound to albumin was released as aflatoxin-lysine. Pronase was precipitated and albumin was digested by aceton in cold, the volume of supernatant was reduced by freeze-drier and injected into HPLC system. Aflatoxin was quantified in comparison to standard rats samples. The purity of this isolated albumin was confirmed by SDS-PAGE electrophoresis. Albumin concentration in positive, negative and standard samples were 10, 13 and 12.5 mg/ml, respectively. Detection limit [20 pg/mg Alb] for measurement of aflatoxin was determined by HPLC method, specificity and sensitivity of method were 92% and 100% respectively. The mean concentration of AF-Alb adducts in serum of positive control rats was 10 ng/mg Alb and the reproducibility of the method after several repeat was very good. In this study, for AF-Alb adduct quantification by HPLC method, mobile phase, I percentage of solvents and run time were changed and the affinity chromatography before HPLC, was deleted. Therefor HPLC- fluorescence which is a precise and specific method, and since it is fast, highly reproducible and cost effective, also with improvement made, could easily be used for the quantification of this important marker in serum