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The aim of this study was to examine the impact of intake of a lactic acid bacterium, Lactobacillus plantarum 299v (Lp299v) on aflatoxin-induced hepatotoxicity in broilers. For this, broilers were ...

Growing broiler chicks on a diet contaminated with aflatoxins (200 or 2000 ppb) for entire growth period resulted in increased oxidative stress and liver damage markers. The toxicity was subsided in broilers received a specific aflatoxin-binding probiotic i.e., Lactobacillus plantarum 299v (Lp299v). There was a substantial (30–90%) increase in antioxidant activity of plasma, which was suppressed due to dietary aflatoxins. Probiotic also reduced serum lactate dehydrogenase and alanine amino transferase together with lipid peroxidation products in liver, which were elevated due to aflatoxin. Because of Lp299v consumption, there was ∼20–55% recovery in body weight gain in broilers intoxicated with aflatoxins. Comparison of the Lp299v effects with that of a commercial aflatoxin binder revealed that, improved antioxidant activity of the chicks was associated with growth performance. These data suggest that aflatoxin-binding probiotics are beneficial with multi-functional effects and can efficiently help reducing aflatoxins in food chain associated with poultry industry.

BackgroundIdentification of molecular markers, such as miRNAs is promising for the diagnosis of asthma and its clinical phenotypes. The aim of this study was to examine the changes in the expression of selected microRNAs in plasma exosomal fractions of severe asthma patients. The expression of miRNAs was determined in relation to the changes in inflammatory markers.MethodSevere asthma patients (n = 30) and healthy subjects (n = 30) were selected among the individuals referred to asthma and allergy clinic. Blood was collected from each participant to determine the serum high-sensitive C-reactive protein (hs-CRP) and total IgE. The exosomal fraction of plasma was isolated and processed for quantitation of miR-124, miR-125b, miR-133b, miR-130a and miR-125b-1-3p expression using quantitative real time-PCR (qRT-PCR).ResultsSerum hs-CRP and total IgE were significantly higher in asthma patients compared to controls. Expression of miR-124, miR-133b, and miR-130a was down-regulated in asthma patients as compared to controls (p ConclusionOverexpression of miR-125b in severe asthma which was associated with serum IgE and hs-CRP may suggest that this molecule is linked to inflammatory reactions. Up-regulation of miR-125b together with decreased expression of miR-124, miR-133b, and miR-130a may suggest that this miRNA profile is useful for diagnosis and discrimination of clinical phenotypes of asthma.

Background: Oral Squamous Cell Carcinoma (OSCC) is among the ten most common cancers worldwide. Hypermethylation of CpG sites in the promoter region and subse-quent down-regulation of a tumor suppressor gene, TGM-3 has been proposed to be linked to different types of human cancers including OSCC. In this study, methylation sta-tus of CpG sites in the promoter region of TGM-3 has been evaluated in a cohort of pa-tients with OSCC compared to normal controls. Methods: Forty fresh tissue samples were obtained from newly diagnosed OSCC patients and normal individuals referred to dentistry clinic for tooth extraction. DNA was extract-ed, bisulfite conversion was performed and it was subjected to PCR using bisulfite-sequencing PCR (BSP) primers. Prepared samples were sequenced on a DNA analyzer with both forward and reverse primers of the region of interest. The peak height values of cytosine and thymine were calculated and methylation levels for each CpG site within the DNA sequence was quantified. Results: Quantitative DNA methylation analyses in CpG islands revealed that it was sig-nificantly higher in OSCC patients compared to controls. DNA methylation at CpG1/CpG3/CpG5 (p=0.004-0.01) and CpG1/CpG3 (p=0.001-0.019) sites was associated with tumor stage and grade, respectively. Male OSCC patients had higher methylation rate at CpG3 (p=0.032), while smoker patients showed higher methylation rate at CpG6 (p=0.045). Conclusion: These results manifested the contribution of DNA methylation of TGM-3 in OSCC and its potential association with clinico-pathologic parameters in OSCC.

AIM: Due to lipases’ regio-selectivity and ability to catalyze different reactions such as hydrolysis, esterification, and transesterification, the enzyme is attractive in biotransformation technology. Besides, another technology, namely enzyme immobilization, has attracted scientists/technologists’ attention to employ immobilized lipase in such a field. Thus lipase of Candida rugosa was immobilized onto silica nanoparticles through adsorption. Furthermore, the immobilized biocatalyst was characterized and used to esterify ibuprofen enantioselectively. METHODS: To characterize immobilized lipase onto silica nanoparticles scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used. RESULTS: The catalytic properties of both immobilized and free lipases such as optima pH and temperature were not different. According to the results, the immobilized lipase on silica nanoparticles showed 45% and 96% conversion (C) and enantioselectivity (ee(s)), respectively. In comparison to free lipase, the immobilized enzyme came with better catalytic activity. CONCLUSION: Silica nanoparticles as one of the most promising materials for the immobilization of lipase in enantioselective esterification of ibuprofen, were introduced in this work. GRAPHICAL ABSTRACT: [Image: see text]

This study was carried out to examine the role of intracellular albumin in the modulation of oxidative damage induced by glutathione modifiers in HepG2 cells. Also, the relationship of albumin synthesis with oxidative stress factors including antioxidants was studied. HepG2 cell culture was supplemented with glutathione modifiers; L-Buthionine-sulfoximine (BSO; 0.1 and 1.0 mM) or N-acetyl cysteine (NAC; 1 and 10 mM) and the cell viability and changes in reduced glutathione (GSH), oxidized glutathione (GSSG), reactive oxygen species (ROS), catalase, and superoxide dismutase were measured. Besides, albumin expression at protein and mRNA levels was determined in cells pretreated with BSO or NAC. Kinetic studies showed that albumin expression in HepG2 cells is correlated with GSH and GSSG levels. Changes in albumin expression at protein and mRNA levels reached their maximum (19% and 55%, respectively) in the cells 6 H after NAC treatments. A substantial decrease in intracellular albumin due to BSO (27%) was associated with a significant increase in the generation of cellular ROS (17%). In contrast, increased albumin synthesis (intracellular and secretory) was associated with inhibition in cellular ROS. Overall data may suggest that albumin expression in coordination with the glutathione redox system is part of the antioxidant defense mechanism in liver cells.

Background A number of complex and rare mutations in epidermal growth factor receptor (EGFR) gene have been identified and the clinical implication of serum EGFR ligands has also been reported. However, the prognostic significance of these mutations and also the serum EGFR and its ligands in Non-Small Cell Lung Carcinoma (NSCLC) has remained a challenging issue. This study is aimed at finding the prognostic importance of EGFR rare mutations and serum EGFR, amphiregulin (AR), and TGF-α (Transforming Growth Factor-alpha) in NSCLC. Materials and method NSCLC patients ( n = 98) with mean age of 59 ± 10.5 were enrolled (M/F: 75/23). DNA was extracted from formalin fixed paraffin embedded tissues. Exons 19 and 21 were amplified using polymerase chain reaction followed by direct sequencing for identification of mutations. Serum EGFR, AR, and TGF-α were measured by ELISA. Results EGFR mutation rate in patients was 37% (exon 19 deletions: 72.2%, exon 21 substitutions: 27.8%). The E872K in exon 21 mutation-positive cases was the most frequent rare mutation detected (90%; 9/10 samples). A significant relationship was found between EGFR exon 21mutations and serum EGFR and TGF-α ( P 3 pg/ml) and TGF-α (> 10.5 pg/ml) were associated with shorter overall survival ( P Conclusions The data clearly show that elevation of serum TGF-α and AR are associated with poor prognosis of NSCLC. In addition to the close relationship between EGFR mutations and serum EGFR, serum TGF-α changes was associated with the gene mutations. These findings could be implicated in clinical decision making related to EGFR-TKIs.

micro (mi)-RNAs are vital regulators of multiple processes including insulin signaling pathways and glucose metabolism. Pancreatic β-cells function is dependent on some miRNAs and their target mRNA, which together form a complex regulative network. Several miRNAs are known to be directly involved in β-cells functions such as insulin expression and secretion. These small RNAs may also play significant roles in the fate of β-cells such as proliferation, differentiation, survival and apoptosis. Among the miRNAs, miR-7, miR-9, miR-375, miR-130 and miR-124 are of particular interest due to being highly expressed in these cells. Under diabetic conditions, although no specific miRNA profile has been noticed, the expression of some miRNAs and their target mRNAs are altered by posttranscriptional mechanisms, exerting diverse signs in the pathobiology of various diabetic complications. The aim of this review article is to discuss miRNAs involved in the process of stem cells differentiation into β-cells, resulting in enhanced β-cell functions with respect to diabetic disorders. This paper will also look into the impact of miRNA expression patterns on in vitro proliferation and differentiation of β-cells. The efficacy of the computational genomics and biochemical analysis to link the changes in miRNA expression profiles of stem cell-derived β-cells to therapeutically relevant outputs will be discussed as well.

Despite the current guidelines for the management of type 2 diabetes mellitus (T2DM), patients still struggle with the hyperglycemia consequences. Imbalance in zinc homeostasis, in particular, renders diabetic patients more susceptible to the damages of oxidative stress. This study aimed to evaluate the effects of zinc supplementation on the superoxide dismutase gene expression and enzyme activity in overweight individuals with T2DM. Additionally, biochemical parameters, such as fasting blood glucose (FBG), insulin, glycated hemoglobin (HbA1c), homeostasis model of assessment-insulin resistance (HOMA-IR), serum levels of zinc and lipid profile, were assessed.In this randomized, double-blind, placebo-controlled trial, 70 overweight (BMI 25) T2DM patients were selected based on the inclusion criteria. They were divided into two groups for supplementation of daily 50 mg zinc gluconate or placebo for 8 weeks. Blood samples were collected from all the individuals in the zinc group and controls for analysis.The results showed that, in comparison with the control group, zinc supplementation increased both gene expression and enzyme activity of SOD (p 0.01) as well as the levels of insulin (p = 0.02) among the patients in the zinc group. Moreover, there was a meaningful reduction in the levels of FBG, HbA1c and HOMA-IR value (p 0.001), triglycerides and total cholesterol (p 0.05) after the zinc treatment.Taken together, the current study suggests that daily supplementation with 50 mg zinc gluconate could be a useful approach for the management of overweight T2DM.IRCT2015083102.

The aim of this study was to investigate the relationship between the H-ras and Cox-2 gene expression in tumors from Iranian Oral Squamous Cell Carcinoma (OSCC) patients. Fresh tumor biopsies removed from oral cavity were collected from 67 new cases. Total RNA was extracted from biopsies and processed for quantification of H-ras and Cox-2 specific RNA expression using real-time PCR (QPCR). In addition, 59 gingival biopsies from apparently normal individuals were processed for QPCR assays. The results showed that Cox-2 expression at mRNA levels was at minimal levels in normal gingival biopsies. However, there was a surge in Cox-2 expression in tumor tissues (11.5 fold, p

Differentiation of mesenchymal stem cells (MSCs) to hepatocytes-like cells is associated with alteration in the level of reactive oxygen species (ROS) and antioxidant defense system. Here, we report the role of glutathione in the functions of hepatocytes derived from MSCs. The stem cells undergoing differentiation were treated with glutathione modifiers [buthionine sulfoxide (BSO) or N-acetyl cysteine (NAC)], and hepatocytes were collected on day 14 of differentiation and analysed for their biological and metabolic functions. Differentiation process has been performed in presence of glutathione modifiers viz. BSO and NAC. Depending on the level of cellular glutathione, the proliferation rate of MSCs was affected. Glutathione depletion by BSO resulted in increased levels of albumin and ROS in hepatocytes. Whereas, albumin and ROS were inhibited in cells treated with glutathione precursor (NAC). The metabolic function of hepatocytes was elevated in BSO-treated cells as judged by increased urea, transferrin, albumin, alanine transaminase and aspartate transaminase secretions in the media. However, the metabolic activity of the hepatocytes was inhibited when glutathione was increased by NAC. We conclude that the efficiency of metabolic function of hepatocytes is inversely related to the levels of cellular glutathione. These data may suggest a novel role of glutathione in regulation of metabolic function of hepatocytes. Copyright © 2013 John Wiley & Sons, Ltd.

Background Fibroblasts are the most prominent cells of the skin. Consequently, skin aging depends predominantly on these cells. Applying herbs in anti-aging, antioxidant, anti-pollutant, anti-bacterial, and anti-inflammatory formulas are now growing. On the basis of the fact that Iranian traditional medicine has utilized herbs for several centuries in order to improve various kinds of health issues, Rosemarinus officinalis leaf extract and Altheae officinalis root extract have been selected for evaluating their effects on viability and proliferation on HDF in comparison to modern cosmeceutical approaches including growth factor application as anti-aging ingredients. This survey has been performed for assessing the effect of herbs on proliferation, viability, and differentiation of cells. Methods Human dermal fibroblasts (HDF) were isolated from the human foreskin. The methods used for viability measurement of fibroblasts were Tripan blue and Bromodeoxyuridine (Brdu) incorporation assays, which were used to study the cell proliferation effect of herbal extracts in HDF. Results HDF were isolated by tissue culture. Herbal extracts and bFGF were found to induce significant proliferation and viability of HDFs at concentrations ranging from 10 to 20 µL/mL and 15 to 25 µL/mL and were reduced in presence of 1 and 5 μM of LBSO as the positive control to assess the oxidant potential of this substance. Conclusions These results suggest that herbal extract may have inhibitory effects on senescence of dermal fibroblasts and suggesting that herbal extracts may be a good candidate to utilize in anti-aging, wound, and burn healing formulations and products.

Stem cell therapy for degenerative diseases has been established; however there are controversies over the treatment of solid tumors with stem cell transplantation. In the present study, the anti-tumor action of mesenchymal stem cells (MSCs) has been examined in a mouse model of breast cancer with emphasize on tumor growth, angiogenesis and c-Myc expression in breast tumors. For this purpose, MSCs were isolated from bone marrow of Balb/c mice and characterized. A Balb/c mouse model of breast cancer was developed and subjected to cell therapy intra venous (I.V) or intra tumor (I.T) with MSCs. Tumor growth was measured using a digital caliber for until the end of experiment (30days). Then the mice were sacrificed and their tumors were removed and processed for histopathological examination, immunohistochemical assay of CD31 and measuring of c-Myc expression using quantitative PCR. Detection of the labeled-MSCs in tumors following injection of the cells (I.V or I.T) clearly showed the homing of MSCs into tumors. Tumor growth in case of MSC-treated mice by I.V and I.T routes was inhibited by approximately 28% and 34% respectively compared to controls. The suppression of angiogenesis was reflected in Micro Vessel Density (MVD) following I.V or I.T delivery of the MSCs. c-Myc gene expression in tumor tissues of mice treated I.V or IT with MSCs was down-regulated to 28.0% and 16.0% respectively compare to control groups. In conclusion, growth inhibition of breast tumors in mice due to MSC therapy is associated with modulation of c-Myc activation and angiogenesis markers.

Incorporation of endothelial cells or their progenitor cells into newly sprouting blood vessels can contribute to tissue vascularization after ischemic injury. However, the interaction of the stem cells-derived endothelial cells with angiogenesis within tumors is not well understood. The aim of this study was to examine the efficiency of endothelial-like cells derived from MSCs in controlling breast tumor growth associated with abnormal angiogenesis. For this purpose, Balb/c mouse model of breast carcinoma was developed and subjected to intra tumor (I.T)/intra venous (I.V) therapy with undifferentiated MSCs or endothelial cells derived from them. The homing of the stem cells was approved by measuring different markers as well as tracing green fluorescence protein (GFP)-labeled MSCs in the tumors. Tumor growth was measured following cell therapy using a digital caliper. At the end of treatment period (30 days) the angiogenesis markers; VEGFR2 expression as well as micro-vessel density (MVD) using CD31 were estimated in tumor tissues. Stem cell transplantation to mice bearing breast tumors resulted in tumor growth suppression in all experimental groups. The endothelial markers; CD31 and VEGFR2 were down regulated following I.T delivery of the endothelial cells. Accordingly, angiogenesis was suppressed following I.T administration of endothelial cells which was associated with increased focal necrosis in the tumors. In conclusion, data show that endothelial cells directly injected into tumors is more efficient compared to undifferentiated MSCs in controlling tumor-associated angiogenesis and tumor growth.

Two experiments were performed to screen bacilli isolated from quails for their aflatoxin removal potential and to assess the efficiency of their amelioration of experimental aflatoxicosis. Nonhemolytic bacilli were selected for in vitro aflatoxin B1 (AFB1) removal and conventional probiotic tests. The isolate with the highest scores was selected for assessment in field experiments and was identified as Berevibacillus laterosporus (Bl). In the second experiment, 125 male Japanese quails (21 d old) were divided into 5 groups with 5 replications to compare the toxin removal efficiency of Bl with that of a commercial toxin binder, improved Millbond-TX (IMTX). The experimental groups were as follows: Control (without any feed additive or AFB1); AFB1 (2.5 mg/kg); AFB1+Bl (2.5 mg/kg+10(8) cfu/mL); AFB1+IMTX (2.5 mg/kg+2.5 g/kg); and Bl (10(8) cfu/mL). The greatest BW gain and slaughter and carcass weights were found in the Bl group and the lowest values were observed in the AFB1 group (P

The aim of this study was to investigate the contribution of acute alcohol in sepsis-related liver damages using a Cecal Ligation and Puncture (CLP) model. Rats were divided into 7 groups (5 rats/group): control (saline-injected), sham-operated, CLP, ethanol (1.0 and 2.0 g/kg b.w) and CLP+ethanol. The CLP+ethanol group received a single dose of ethanol following sepsis induction. Sepsis induction caused early changes in lipid peroxidation products in liver, whereas ethanol alone (2.0 g/kg b.w) resulted in a significant increase (~21%) in lipid peroxidation, which was further increased (~57%) in CLP rats treated with alcohol. CLP operation and alcohol treatment exhibited additive effects on plasma catalase, liver glutathione and glutathione S-transferase (GST), which were primarily suppressed due to ethanol. Hepatic cytochrome P4501A1, which was elevated in CLP rats, was reversed in the CLP+ethanol group. Plasma tumor necrosis factor-α was markedly elevated (~85%) in septic rats, but was unaffected in septic rats having received ethanol. Histopathological observations revealed that inflammatory reactions in liver in response to CLP operation are not intensified by ethanol administration. On the basis of biochemical and histopathological results, it can be concluded that acute ethanol treatment is responsible for early changes in oxidative stress, which may lead to polymicrobial sepsis-related organ damage.

Telomerase has been proposed as a novel and potentially selective target in cancer therapy. Silymarin, which is a standardized mixture of flavonolignans from the medical plant Silybum marianum, has potent effects against various types of cancer cells, but its effect on telomerase activity in the human leukemia cell line K562 has not been investigated. The aim of this study was to examine the mechanism of silymarin-induced apoptosis in K562 cells, with particular emphasis on its effect on telomerase activity. The antiproliferation effect of silymarin on K562 cells was evaluated by the MTT assay. To measure apoptosis, Hoechst 33342 staining and flow cytometry were used. The telomerase activity was determined using the telomeric repeat amplification protocol (TRAP)-ELISA assay. The treatment of the K562 cells with silymarin resulted in a significant inhibition of cell growth and telomerase activity. Also, a positive correlation was found between telomerase inhibition and induction of apoptosis in silymarin-treated K562 cells. These results suggest a novel mechanism in the anticancer activity of silymarin in human leukemia K562 cells and may provide a basis for future development of anti-telomerase therapies.

We have recently reported that the inhibition of colonic premalignant lesions induced by 1,2-dimethylhydrazine (DMH) is mediated by the interference of caraway oil components in the activities of the main hepatic xenobiotic metabolizing enzymes. The present study was carried out to examine the effect of dietary caraway oils on the progression of cancer, with emphasis on β-catenin expression in the colon during DMH-induced colonic carcinogenesis. For this purpose, colon cancer was induced by DMH in rats (20 mg/kg body weight for 5 weeks) and groups of animals were given dietary caraway essential oils at two levels (0.01 and 0.1%) for 16 weeks. After 16 weeks and at the end of the experimental period the colon tissue biopsies were processed for histopathological examination and the expression of β-catenin at mRNA and protein levels was estimated by polymerase chain reaction and enzyme-linked immunosorbent assay. The formation of premalignant lesions based on aberrant crypt foci (ACF) in DMH-treated rats was greatly inhibited (72-87%) in rats given dietary essential oils when compared to respective controls. There was a correlation between the number of colonic ACF formation and the expression levels of β-catenin measured at protein and mRNA levels. These results indicate that the Wnt/β-catenin signaling pathway is activated during colon cancer promotion and that the expression of colonic β-catenin is altered in long-term caraway oil feeding, leading to suppression of DMH-induced premalignant lesions in rat colon.

In the present study, genetic diversity and mycotoxin profiles of Aspergillus flavus isolated from air (indoors and outdoors), levels (surfaces), and soils of five hospitals in Southwest Iran were examined. From a total of 146 Aspergillus colonies, 63 isolates were finally identified as A. flavus by a combination of colony morphology, microscopic criteria, and mycotoxin profiles. No Aspergillus parasiticus was isolated from examined samples. Chromatographic analyses of A. flavus isolates cultured on yeast extract-sucrose broth by tip culture method showed that approximately 10% and 45% of the isolates were able to produce aflatoxin B(1) (AFB(1)) and cyclopiazonic acid (CPA), respectively. Around 40% of the isolates produced sclerotia on Czapek-Dox agar. The isolates were classified into four chemotypes based on the ability to produce AF and CPA that majority of them (55.5%) belonged to chemotype IV comprising non-mycotoxigenic isolates. Random amplified polymorphic DNA (RAPD) profiles generated by a combination of four selected primers were used to assess genetic relatedness of 16 selected toxigenic and non-toxigenic isolates. The resulting dendrogram demonstrated the formation of two separate clusters for the A. flavus comprised both mycotoxigenic and non-toxigenic isolates in a random distribution. The obtained results in this study showed that RAPD profiling is a promising and efficient tool to determine intra-specific genetic variation among A. flavus populations from hospital environments. A. flavus isolates, either toxigenic or non-toxigenic, should be considered as potential threats for hospitalized patients due to their obvious role in the etiology of nosocomial aspergillosis.

This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34 + stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC 50 values of AFB1 for hepatocytes differentiated from CD34 + and MSCs were within the same range (44.7–46.8 μM). The IC 50 calculated for non-differentiated MSCs and CD34 + cells was slightly lower (42.0–43.4 μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20 μM) for 24 h were ∼15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34 + cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34 + cells, then UCB-MSCs and finally hepatocyte-CD34 + cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants.

In this study, a new effective, pH and thermally stable glucose oxidase (GOX)–silver nanoparticles (AgNPs) bioconjugate was designed. AgNPs were synthesized based on the reduction of silver nitrate (AgNO 3 ) by sodium borohydride (NaBH 4 ) using two simple procedures. Periodic acid was used for oxidation of the GOX and emission of Lucifer yellow (LyCH) was monitored by spectrofluorometer for evaluation of the oxidation properties of the GOX. The oxidized GOX (Ox-GOX) was immobilized on AgNPs by its sugar moieties via 6-aminohexanoic acid (6AHA) as linker. A sample of the synthesized bioconjugate was loaded on 7.5% non-denaturing polyacrylamide gel electrophoresis (PAGE) to confirm its structural and physical stability. The results from enzymatic activity assay showed that the bioconjugate, GOX and Ox-GOX were similar in stability and activity in acidic and basic pH (optimum pH = 7.0–8.0). Based on the results from thermal stability assay, it was found that the activity of the bioconjugate was found to be higher at lower temperatures. The V max of the bioconjugate, GOX, and Ox-GOX was estimated as 28.6, 6.2, and 6 IU μg −1 enzyme and the K m was calculated as 2.7, 9, and 9.5 mM, respectively. It was found that the immobilization method improves the activity and stability of the GOX in different pH and temperatures. As a conclusion, the proposed method opens up the way to the development of a new bioconjugate with potential use in sensing, and many find potential applications in clinical diagnostics, medicine, and industries.

BACKGROUND: The aim of this study was to investigate the effect of sodium selenite (SS) on reactive oxygen species (ROS) production, total antioxidant capacity (TAC) and glutathione peroxide (GPx) activity of cultured pre-antral follicles derived from vitrified and non-vitrified ovarian tissue. METHODS: Immature mouse ovaries were vitrified, and mechanically isolated pre-antral follicles from vitrified and non-vitrified samples were cultured in TCM 199 medium supplemented with different concentrations (0, 5 and 10 ng/ml) of SS. Follicular, oocyte and embryo development was assessed. In parallel, ROS, TAC and GPx levels were analyzed after 0, 12, 24, 48, 72 and 96 h of culture. RESULTS: Development rates of follicles, oocytes and embryos were significantly higher in SS-supplemented groups (P < 0.005). ROS production was increased, and TAC levels and GPx activities were decreased after 24 h of culture of pre-antral follicles in vitrified and non-vitrified groups, whereas in the presence of SS, ROS production was decreased and TAC levels and selenium-dependent GPx-specific activities were increased after 96 h of culture. Vitrified and non-vitrified samples responded in a similar manner. CONCLUSION: SS caused an increase in follicular TAC level and GPx activity and a decrease in ROS level, thus improving the in vitro development of follicles.

The aim of this study was to find out the profile of cellular glutathione (GSH) and GSH S-transferase (GST) in hepatocytes differentiated from adult mesenchymal stem cells (MSC). For this purpose, we have derived functionally active hepatocyte-like cells from normal human multipotent adult MSC. Then the differentiated cells were characterized by specific hepatic markers. The cellular GSH and GST catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined in hepatocyte-like cells differentiated from MSC compared with undifferentiated MSC. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting techniques were used to study GST-P1-1 and GST-A1-1 expression in differentiated and undifferentiated cells. The results showed that there is more than threefold increase in GST catalytic activity in hepatocytes recovered by day 14 of differentiation. GST-P1-1 mRNA expression was detected in both differentiated hepatocyte-like cells and their undifferentiated progenitors. Under similar conditions, only differentiated hepatocyte-like cells expressed GST-A1-1 mRNA. These results were further confirmed by showing that the undifferentiated cells expressed both GST-A and GST-P proteins. Unlike GST, the level of cellular GSH was declined (approximately 20%) in hepatocytes derived from MSC as compared to that of undifferentiated cells. These data may suggest that hepatogenic differentiation of human bone marrow MSC is accompanied with the regulation of factors participating in GSH conjugation pathway.

In this study characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells (hBMCs) was investigated in relation to their capillary network formation potential. Differentiation was performed in presence of vascular endothelial growth factor (VEGF) and insulin like growth factor-1 (IGF-1). A panel of cellular and molecular markers was used for characterization of the endothelial cells. The cells were strongly positive for von Willebrand factor (vWF) and vascular endothelial growth factor receptor 2 (VEGFR2) when measured at protein and mRNA levels. Development of endothelial cells was found to be associated with formation of typical organelles such as Weibel Palade (WP) bodies, Cavealae and pinocytic vesicles. Early vessel growth was also evidenced by showing specific junctions between the cells. The migratory and angiogenic properties of the cells were confirmed by showing capillary network formation in vitro. These results indicate that the capacity of endothelial cells differentiated from hBMSCs in formation of vascular system is consistent with molecular and structural development.

Aim To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold. Methods After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers. Results Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and α-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (pConclusion Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Natural products may regulate the cellular effects of aflatoxins and evidence suggests that aromatic organic compounds of spices can control the production of aflatoxins. With a view to controlling aflatoxin production, the essential oils from Rosmarinus officinalis and Trachyspermum copticum L. were obtained by hydrodistillation. Antifungal activities of the oils were studied with special reference to the inhibition of Aspergillus parasiticus growth and aflatoxin production. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oils were determined. T. copticum L. oil showed a stronger inhibitory effect than R. officinalis on the growth of A. parasiticus. Aflatoxin production was inhibited at 450 ppm of both oils with that of R. officinalis being stronger inhibitor. The oils were analyzed by GC and GC/MS. The major components of R. officinalis and T. copticum L. oils were Piperitone (23.65%), alpha-pinene (14.94%), Limonene (14.89%), 1,8-Cineole (7.43%) and Thymol (37.2%), P-Cymene (32.3%), gamma-Terpinene (27.3%) respectively. It is concluded that the essential oils could be safely used as preservative materials on some kinds of foods to protect them from toxigenic fungal infections.

Experimental autoimmune encephalomyelitis (EAE) was induced in a mouse model (C57/BL6) to investigate the antioxidant status of animals at various clinical stages of the disease. For this purpose, blood, brain and spinal cord samples from EAE mice were collected and examined at different scores following post-immunization with myelin oligodendrocyte glycoprotein (MOG). The clinical sign of mobility of animals on different days was associated with gradual increase in lipid peroxidation products (malondialdehyde, i.e. MDA) in brain and spinal cord. Changes in lipid peroxidation during EAE progression was inversely related to superoxide dismutase (SOD) activity in erythrocyte preparation. However, suppression of catalase in erythrocytes, tissue glutathione (GSH) and plasma total antioxidant capacity (FRAP assay) were the early events in EAE, occurred during scores 1 and 2. Biochemical alterations were corroborated with histopathological observations showing demyelination and inflammatory foci in central nervous system (CNS) of animals suffering from partial hind limb paralysis (score 3). These data suggest that generation of MDA in CNS is a continuous process during EAE induction and suppression of antioxidant factors are early events of the disease, but crucial in increasing the vulnerability of CNS to demyelinating lesions.

Resistance to oxidative damages in undifferentiated mesenchymal stem cells (MSCs) in comparison with the undifferentiated progenitor cells may differ depending on several factors. This study was carried out to examine the impact of hepatogenic differentiation process of MSCs on oxidative DNA damage markers. Hepatic differentiation of MSCs was carried out using a two-step conventional protocol and the cells were processed for characterization using morphological and biochemical markers. During the course of differentiation cellular levels of reactive oxygen species (ROS), 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and expression of ataxia-telangiectasia mutated (ATM) protein were estimated at time intervals (10, 20 and 30 days). The results showed a decrease in cellular ROS (13%, P

Aflatoxin contamination of grains and agro-products is a serious food safety issue and a significant economic concern worldwide. In the present study, the effects of eugenol on Aspergillus parasiticus growth and aflatoxin production were studied in relation to the expression of some essential genes involved in aflatoxin biosynthetic pathway. The fungus was cultured in presence of serial two-fold concentrations of eugenol (15.62-500 μg mL(-1)) for 3 days at 28 °C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography. The expression of aflatoxin biosynthetic genes including ver-1, nor-1, pksA, omtA and aflR were evaluated by real-time PCR. Eugenol strongly inhibited A. parasiticus growth in the range of 19.16-95.83 % in a dose-dependent manner. Aflatoxin B1 production was also inhibited by the compound in the range of 15.07-98.0 %. The expressions of ver-1, nor-1, pksA, omtA and aflR genes were significantly suppressed by eugenol at concentrations of 62.5 and 125 μg mL(-1). These results indicate that eugenol may be considered as a good candidate to control toxigenic fungal growth and the subsequent contamination of food, feed and agricultural commodities by carcinogenic aflatoxins.

The effects of Akacid(plus), a novel member of guanidine-based polymeric compounds recently introduced as a potent inhibitor of fungal growth and aflatoxin biosynthesis were studied on Aspergillus parasiticus by transmission electron microscopy (TEM). The toxigenic fungus was cultured on yeast extract-sucrose broth in presence of serial two-fold concentrations of Akacid(plus) (1.5-96 microL/50 mL medium) for 96 h at 28 degrees C with shaking. Mycelial samples exposed to fungistatic concentrations of compound (1.5-48 microL) were processed for TEM. Corresponding to the growth inhibition, TEM observations revealed morphological anomalies in fungal compartments. The results demonstrated that Akacid(plus) targets the plasma membrane of the hyphae by its breaking down at variable intervals with the formation of small membrane-bound vesicles inside the cytoplasm, while no obvious damage was observed on the cell wall. A marked depletion of cytoplasmic contents of hyphae accompanied with lysis and disruption of membranes of major organelles such as nuclei, mitochondria and endoplasmic reticulum indicates that in high fungistatic concentrations, Akacid(plus) passes not only through the cell wall but also through the plasma membrane and then interact with membranous structures of the cytoplasmic organelles. Ultrastructural changes of fungal compartments exposed to Akacid(plus) in relation to the fungal growth and aflatoxin biosynthesis are discussed.

Summary Objectives Listeria monocytogenes has gained increasing attention as a pathogen of public health importance owing to large numbers of food-borne outbreaks of listeriosis. Because of negative consumer perception of chemical preservatives, attention is shifting towards natural alternatives. Particular interest has been focused on the potential application of plant essential oils. The objective of the present study was to determine ultrastructural changes brought about by essential oils from two types of thyme, Thymus eriocalyx and Thymus x-porlock , on Listeria monocytogenes . Materials and methods Minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations and bactericidal kinetics of the oils were determined. Listeria monocytogenes were treated with essential oils from two thyme species and observed under a transmission electron microscope. Results The oils from the above plants were found to be strongly antimicrobial. Analysis of the oils by gas chromatography and gas chromatography/mass spectrometry lead to the identification of 18 and 19 components in T. eriocalyx and T. x-porlock oils, respectively. Listeria monocytogenes treated with essential oils from the two thyme species exhibited a thickened or disrupted cell wall with increased roughness and lack of cytoplasm. Conclusion The antilisterial effects of thyme oil are stronger than the action of electric shocks in combination with nisin reported in the literature. It is concluded that essential oils such as thyme oil, which inhibited the growth of L. monocytogenes at low concentrations, could be considered as preservative materials for some kinds of foods; they could find an application as additives to foodstuffs in storage to protect them from listerial contamination.

The antifungal effects of essential oils from Thymus eriocalyx and Thymus x-porlock were studied with special reference to the mechanism of inhibition of Aspergillus niger growth at ultrastructural level. Minimal inhibitory (MIC), minimal fungicidal (MFC) concentrations, and fungicidal kinetics of the oils were determined. Transmission electron microscopy (TEM) of A. niger exposed to MIC levels of the oils showed irreversible damage to cell wall, cell membrane and cellular organelles. The oils analyzed by GC and GC/MS led to identification of 18 and 19 components in T. eriocalyx and T. x-porlock oils respectively. The results are compared and discussed with the data in literature. It is concluded that the essential oils could be safely used as preservatives.

The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.

Aflatoxicosis is one of the major causes of mortality and low productivity in broiler chickens, which is induced by aflatoxin contaminated grains. Elimination of aflatoxins by treatment with ammonia is one of the approaches to reduce aflatoxicosis. This study was conducted to assess the production parameters with respect to biochemical and histopathological changes in chicks fed ammonia treated maize. Detoxification of aflatoxin contaminated maize grains was done in a pilot plant with aqueous ammonia (1%, v/w). One-day-old broiler chicks (Ross 308) were fed on different dietary treatments designed dividing 480 chicks into six groups. Various dietary treatments included: group A (control), basal diet containing uncontaminated maize; group B, basal diet containing ammonia treated uncontaminated maize; group C, basal diet containing aflatoxin B1 (AFB, 1 ppm); group D, basal diet having ammonia treated aflatoxin contaminated (1 ppm) maize; group E, basal diet containing AFB (2 ppm) contaminated maize; group F, basal diet containing ammonia treated AFB (2 ppm) contaminated maize. Chickens were monitored daily and then body weight and feed consumption were recorded. At the end of rearing period (6 weeks), aflatoxin treated groups resulted in significant ( P

Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

To develop an indirect enzyme-linked immunosorbent assay (ELISA) for measuring class-pi glutathione S-transferase (GST) in plasma, and tissue biopsies obtained from upper gastrointestinal cancer (UGI Ca) patients.GST activity and GST-pi concentration were detected in normal human squamous esophageal epithelium, normal gastric cardia and their corresponding malignant tumor biopsies.Plasma GST was significantly higher (p0.05) in UGI Ca patients as compared to those obtained from normal individuals. Plasma GST-pi concentration in normal subjects was 6.6 +/- 1.9 ng/mg protein, whereas it was higher in UGI Ca patients (esophageal, 10.0 +/- 1.8; gastric, 10.7 +/- 1.7 ng/mL, por= 0.01). GST and GST-pi levels were higher in surgically resected tumor biopsies as compared to normal tissues adjacent to a tumor (p0.05). GST-pi was also elevated in malignant esophagus and gastric biopsies taken at endoscopy as compared to normal and normal-appearing esophageal tissues (p0.05). Total GST was also increased significantly in gastric malignant tissues although its activity was within the same range in normal and normal-appearing tissues. A significant correlation between plasma GST-pi with that in malignant tissues was observed. Total GST and GST-pi were also correlated in different malignant tissues.Biopsies obtained at endoscopy can be used satisfactorily to measure tumor markers.

Previous studies from this laboratory indicated that glutathione (GSH) conjugate formation with acetaminophen (APAP) is remarkably induced in liver of weanling rats in response to a single overdose of the drug administered intraperitoneally (ip). Increased APAP-GSH conjugation has been attributed to inducible glutathione S-transferases (GSTs) in dividing hepatocytes. In order to verify this finding, an in vitro reconstitution assay containing liver microsomes (source of cytochrome P-450) and cytosolic fractions (source of GST) from livers and kidneys of adult and weanling rats has been established. In vitro incubation of the reaction mixture was followed by solvent extraction, enzymatic digestion and HPLC analysis of the conjugate. Under controlled conditions, in vitro, the rate of APAP-GSH conjugation reflected the GST activity of cytosolic sample added to incubation system. The activity of cytosolic GST in catalyzing this reaction was measured using cytosols prepared from various tissue sources, particularly from animals pretreated with dietary butylated hydroxylanisole (BHA). The extent of APAP-GSH conjugate formation mediated by cytosols varied in this order: BHA-treated adult liver>BHA-treated weanling liver>control adult liver>control weanling liver>BHA-adult kidney>control adult kidney>BHA weanling kidney>control weanling kidney. In contrast to findings obtained from in vivo experiments, the rate of GST-dependent APAP conjugate formation with GSH in vitro is not induced in the presence of exogenous drug.

The relationship between the activities of 3 cytosolic enzymes with aflatoxin biosynthesis in Aspergillus parasiticus cultured under different conditions has been investigated in order to find out the role of each enzyme in aflatoxin biosynthesis. Basically the activity of isocitrate dehydrogenase (IDH) was higher in non-toxigenic strains as compared to its counterpart toxigenic fungi (p 90% inhibition) when the concentration of neem extract was adjusted to 50% (v/v). No significant changes in FAS and IDH activities were observed when aflatoxin synthesis was under restraints by neem (Azadirachta indica) leaf extract. During a certain period of time of culture growth, when aflatoxin production reached to its maximum level, the activity of FAS was slightly induced in the toxigenic strains fed with a low concentration (1.56% v/v) of the neem leaf extract. At the time (96 h) when aflatoxin concentration reached to its maximum levels, the activity of GST in the toxigenic fungi was significantly higher (i.e., 7–11 folds) than that of non-toxigenic strains. The difference was highest in mycelial samples collected after 120 h. However unlike FAS and IDH, GST was readily inhibited (∼67%) in mycelia fed with 1.56% v/v of the neem extract. The inhibition reached to maximum of 80% in samples exposed to 6.25–12.5% of the extract. These results further substantiate previous finding that there is a positive correlation between GST activity and aflatoxin production in fungi.

Differentiation of mesenchymal stem cells (MSCs) to hepatocyte-like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator-activated receptors-α (PPAR-α) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte-like cells on selective culture media. The morphological and biochemical changes, PPAR-α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation-dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR-α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs.

International audience; Soil isolates of Aspergillus section Flavi from Mazandaran and Semnan provinces with totally different climatic conditions in Iran were examined for aflatoxins (AFs; B and G types), cyclopiazonic acid (CPA) and sclerotia production. A total of 66 Aspergillus flavus group strains were identified from three species viz. Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius in both locations. A. flavus (87.9%) was found to be the prominent species followed by A. nomius (9.1%) and A. parasiticus (3.0%). Only 27.5% of A. flavus isolates were aflatoxigenic (B(1) or B(1) and B(2)), out of which approximately 75% were capable to producing CPA. All the A. parasiticus and A. nomius isolates produced AFs of both B (B(1) and B(2)) and G (G(1) and G(2)) types, but did not produce CPA. Sclerotia production was observed in only 4 isolates of A. flavus among all 66 isolates from three identified species. A. flavus isolates were classified into various chemotypes based on the ability to produce aflatoxins and CPA. In this study, a new naturally occurring toxigenic A. flavus chemotype comprising of two strains capable of producing more AFB(2) than AFB(1) has been identified. A relatively larger proportion of aflatoxigenic A. flavus strains were isolated from corn field soils of Mazandaran province which indicate a possible relationship between high levels of relative humidity and the incidence of aflatoxin-producing fungi. The importance of incidence of Aspergillus section Flavi in corn field soils regard to their mycotoxin production profiles and crop contamination with special reference to climatic conditions is discussed.

The rate of glutathione (GSH) conjugate formation to acetaminophen (APAP) in livers of weanling and adult rats treated with a single i.p. dose of APAP was compared. HPLC analysis of cytosolic fractions revealed that rate of conjugation in weanling rat is 24-times greater than that of adults. Increased rate of GSH conjugation was independent of of the age-related difference observed in liver GSH content. The normal level of liver GSH in weanling rat was 57% of adult level. APAP treatment depleted GSH more significantly in weanling rats as compared to that in adults. N-acetylcystein (NAC) alone had little influence on liver GSH levels. However it was successful in reducing GSH depletion in tissues of growing rats. A 32% repletion in hepatic GSH level in NAC-treated weanling rats was associated with a further 13-fold increase in the rate of GSH conjugate formation. These data together with histopathological results, clearly showed that the inducible GSH system in weanling rat liver act as a safe guard against APAP toxicity. A surge in the rate of APAP-GSH conjugation in growing liver may function in compensation of other detoxification pathways which are saturated more readily at this age.

Background: Nosocomial fungal infections could arise from independent exposure to airborne spores of filamentous fungi existing in the hospital environment. Objectives: The present study aimed to determine the mycoflora of indoor and outdoor environments of five major hospitals in Khorramabad, Iran. Materials and Methods: Sampling of air was done from indoor and outdoor environments of wards, surroundings and green space of hospitals by settle plate method. To obtain the sample from surfaces, pre-moistened swabs with cotton-tipped sticks were applied on different surfaces (floor, the walls, windows, beds, trolleys, laryngoscope and angiography devices). Culture plates of air and surfaces on Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) were incubated in the dark at 28 C and examined daily for fungal colonies for two to three weeks. Fungal isolates were identified by a combination of their macroscopic and microscopic criteria after purification on isolation culture media. Results: A total of 707 fungal colonies including, Penicillium (29.14%), Cladosporium (24.04%), Aspergillus (20.65%), Fusarium (9.05%), Alternaria (3.96%), Rhodotorula (1.69%), Cryptococcus neoformans (0.7%) and other fungi (10.77%) were isolated. All the examined high-risk parts of the hospitals were found to be contaminated by various fungi. Conclusions: Aspergillus was the most prominent genus in Intensive Care Unit (ICU) and surgery, Cladosporium in Critical Care Unit (CCU), emergency and thalassemia, and Penicillium in orthopedic, emergency and neonatal sections. Among pathogenic yeasts, C. neoformans was isolated from ICU, surgery and orthopedic sections. The dimorphic fungal pathogen, Sporothrix schenckii, was reported from CCU. The isolated fungi specially the genera Aspergillus and Penicillium are potential threats for immunocompromised patients in the hospitals.

In this study, the ability of neonatal rat liver to metabolise [3H]aflatoxin B1 (AFB1) was compared to that of the adult animal. In order to make this comparison, neonatal and young adult rats were killed 2, 6, 12 and 24 h after injection with a single i.p. dose of AFB1. The rate of AFB1 adduct formation to nuclear DNA and protein was measured in hepatic and pulmonary tissues. The results demonstrated that AFB1 was epoxidized more rapidly by the adult's liver and lungs 2 h after the toxin administration, compared with those of the neonatal's (adult 30 pmol and neonatal 12 pmol AFB1 bound/mg DNA). However, these differences were more pronounced in hepatic than in pulmonary tissues. The same differences between AFB1-DNA adducts were also observed at different time points. These changes are certainly related to the level of hepatic cytochrome P-450. The delayed cytochrome P-450-dependent AFB1 activation in neonatal's liver provides time enough for de-epoxidation of slowly generated epoxide. The rate of AFB1-epoxide formation at this age was consistent with the activity of phase II metabolism of AFB1 (glutathione conjugation). In addition, the hydrolysis of AFB1-DNA adducts at a relatively higher rate by neonatal's liver may also contribute to the quick removal of the adducts. In spite of the aforementioned evidence which shows the capability of neonatal liver to handle AFB1, the fate of large amounts of free (non-metabolised) AFB1 deposited in neonatal's liver is not well understood.

Differentiation of human mesenchymal stem cells (MSCs) to metabolically active hepatocytes depends on different regulatory factors. Trans-differentiation of stem cells into specific cell lineage in the presence of specific stimuli is associated with the molecular and cellular damage. The aim of the present study was to examine the role of P53 in the regulation of cyclooxygenase-2 (COX-2) expression and the generation of protein and lipid oxidation during trans-differentiation of MSCs into hepatocyte-like cells. During the 3-week differentiation process of MSCs to hepatocyte-like cells we found that expression liver-specific markers was associated with increased levels of lipid peroxidation and protein carbonyl formation. Expression of P53 and COX-2 at mRNA and protein levels were evaluated in MSCs before and after differentiation on days 7, 14 and 21. We showed that the up-regulation of COX-2 was associated with augmentation of the rate of cell proliferation, morphological and biochemical changes of hepatocytes-like cells. However, in parallel the P53 at the mRNA level was down-regulated, and at protein levels accumulation in the nuclei was reduced during the hepatogenic differentiation time. Our results may suggest a P53-COX-2 pathway in the regulation of hepatogenic differentiation of stem cells, which is linked to differentiation-dependent molecular oxidative damage.

Aflatoxins are primarily produced by the fungi Aspergillus flavus and Aspergillus parasiticus, which contaminate a wide variety of food and feed commodities including maize, oilseeds, spices, groundnuts, tree nuts, milk and dried fruits [Strosnider et al., 2006]. Presence of aflatoxins in food chain is associated with decrease in quality and quantity of food and feed materials. In addition, consumption of aflatoxin-contaminated products can pose a risk of development of various diseases in human and animals. Aflatoxins are produced in toxigenic fungi after undergoing biosynthesis pathway involving several enzymes and reactions. Upon consumption of aflatoxin contaminated products by human and animals, the toxin undergoes metabolism via cytochrome P450 enzymes in the liver. Aflatoxin metabolism in mammalian organs is a committed process and different metabolites are produced which can exert adverse effects of toxic metabolites. Aflatoxin epoxide (8,9-epoxide) is the major toxic metabolite which can bind to DNA and induce hepatocellular carcinoms. The extent of aflatoxin toxicity and carcinogenicity in human and animals depends on several factors including the metabolic capacity of the organism. Aflatoxin contamination of food products is associated with health and socioeconomic costs which is difficult to valuate in the developing countries. Moreover, the current regulations do little to help reduce aflatoxin and related health effects. Therefore the focus should be on promoting the adaptation of strategies that can control aflatoxin and its associated health risks. According to Wu and Khlangwiset (2010), interventions to reduce aflatoxin-induced illness can be grouped into three categories; agricultural, dietary and clinical. Agricultural interventions are methods that can be applied either in the field (preharvest) or in drying, storage and transportation (postharvest) to reduce aflatoxin levels in food. The dietary and clinical interventioans are considered as secondary interventions by which the aflatoxinrelated illness can be reduced. These two types interventions are associated with advantages and disadvantages. Due to concern for the potential effects of aflatoxins on human health, most countries have legislation that restricts marketing of aflatoxin-contaminated grains [Van Egmond, 1989]. The United States Food and Drug Administration has set an aflatoxin limit of 20 μg/kg for

There are several issues to be considered to reduce the risk of rejection and minimize side effects associated with liver cell transplantation in chronic liver diseases. The source and the condition of stem cell proliferation and differentiation ex vivo and the transplantation protocols are important safety considerations for cell based therapy. The biochemical and molecular markers are important tools for safety evaluation of different processes of cell expansion and transplantation. Studies show that hepatocytes differentiated from adult and embryonic stem cells exhibit biochemical and metabolic properties resembling mature hepatocytes. Therefore these assays can help to assess the biological and metabolic performance of hepatocytes and progenitor stem cells. The assays also help in testing the contribution of transplanted hepatocytes in improving the repair and function of damaged liver in the recipient. Here we review the biochemical and metabolic markers, which are implicated in evaluation of safety issues of stem cells used for therapeutic purposes in chronic liver diseases and regeneration of damaged liver. We also highlight application of biochemical tests for assessment of liver cell transplantation.

Silymarin, a mixture of flavonolignans, is extracted from milk thistle (Silybum marianum) and has a strong antioxidant activity and exhibits anticarcinogenic, anti-inflammatory, and cytoprotective effects. In this study we attempted to determine whether silymarin and the glutathione modifiers, buthionine sulfoxamine (BSO) and N-acetylcysteine (NAC), are involved in regulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in proliferating mesenchymal stem cells (MSCs). Cellular glutathione was manipulated during a 14-day culture using BSO, NAC and silymarin. At intervals of 2, 7 and 14 days, cells were collected and COX-2 and iNOS levels were measured. In parallel, generation of cellular H(2)O(2) and glutathione were measured. Supplementation of the culture media with BSO caused a dose-dependent decrease in MSC proliferation, whereas NAC or silymarin elevated the proliferation (p0.05). Treatment of MSC with NAC or silymarin caused a significant decrease in COX-2 levels. However, COX-2 levels in cells treated with high levels of NAC (1.0 mM) were significantly lower than those in MSCs treated with high levels of silymarin (100 μM). BSO (1.0 and 5.0 μM) caused a significant increase in COX-2 on days 2, 7 and 14. BSO caused a significant increase in iNOS, whereas NAC or silymarin decreased cellular iNOS. Overall result show that glutathione, iNOS and COX-2 in proliferating MSCs are affected by silymarin treatment. It appears that glutathione is the main target of silymarin, and in consequence iNOS and COX-2 are affected in response to silymarin treatment.

The effects of dietary essential oils prepared from caraway seeds on colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in rats has been studied. The number of aberrant crypt foci (ACF) and aberrant crypt (AC) induced by DMH were found to be significantly inhibited in colon of rats treated with essential oils in diet (0.01 and 0.1%). To find out the mechanism(s) by which the essential oils reduced colon premalignancies, plasma, liver, and colon tissues were collected and analyzed for parameters related to oxidative stress and xenobiotic metabolizing enzymes. Lack of influence of caraway extracts on hepatic lipid peroxidation products, superoxide dismutase (SOD), catalase (CAT) and ferric reducing ability of plasma (FRAP) may suggest that the oils do not interfere with these factors. However, it was clearly shown that DMH-related changes in hepatic and colonic cytochrome P4501A1 (CYP1A1) and glutathione S-transferae (GST) activities were recovered in liver but not in colon tissue in animals treated with caraway oil preparations. In conclusion, histopathological and biochemical data clearly showed that inhibition of colon premalignant lesions induced by DMH is mediated by interference of caraway oil components in the activities of the main hepatic xenobiotic metabolizing enzymes.

Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodies against it are widely employed for diagnostic purposes. The rarity of CA 125 antigenic domains and its highly glycosylated structure, however, is a problem that may prevent immunized mice from developing a diversified population of anti-CA 125 antibodies. In this study a prime-boost strategy, which potentially could augment the humoral immune responses against rare and poorly immunogenic determinants, was used for immunization of mice and monoclonal antibodies (mAbs) were produced by hybridoma technology. Reactivity of mAbs was then assessed by ELISA, western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3 cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the rest were IgM. Two-third of clones recognized cognate antigen in fixed and living cells and had strong immunoreactivity in IHC staining. In Western blotting, our antibodies recognized CA 125 as high molecular weight antigen mostly migrated in the 3% stacking gel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar migration pattern that reconfirmed their specificities. The mAbs produced in this study are invaluable tools in diagnosis and research fields for assessment of CA 125 expression in cancerous ovarian tissues.