Association of Foxp3 rs3761548 polymorphism with cytokines concentration in gastric adenocarcinoma patients.

T regulatory cells (Tregs) and related-cytokines are effectively engaged in the process of tumor immune escape and functionally inhibit immune response against the tumor. This study aimed to investigate the association of Foxp3 gene single nucleotide polymorphism (SNP) (rs3761548) with serum IL-35, IL-10, and TGF-ß levels in gastric adenocarcinoma (GA) patients. The blood samples were obtained from 150 GA patients and 166 control subjects. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was done to genotyping of Foxp3 gene polymorphism (rs3761548). The serum cytokines levels were measured using the ELISA method. According to genotyping, the AA, and AC genotypes and A allele demonstrated significantly greater risk of GA. Considering the Lauren classification, our results revealed a greater risk of GA progression in patients with AC + AA genotype compared to CC genotype. Moreover, significantly increased levels of IL-10, IL-35, and TGF-ß were observed in GA patients compared to controls and also in diffuse-type compared to the intestinal type of GA patients. The IL-35, IL-10 concentrations in GA patients displayed significant differences between the participants with CC, AC and AA genotypes. Further analysis indicated the prognostic role of serum IL-35, IL-10, and TGF-ß levels in GA patients. Our results confirmed that the Foxp3 polymorphism (rs3761548) could influence the predisposition to GA and the serum IL-10, IL-35, and TGF-ß levels. Thus, this polymorphism might be involved in the GA progression through influencing Tregs function and the secretion of immunomodulatory cytokines.

Mutation analysis and clinical characterization of Iranian patients with mucopolysaccharidosis type I.

BACKGROUND: Mucopolysaccharidosis type I (MPSI) is a rare autosomal recessive disorder caused by a deficiency of α-l-iduronidase (IDUA) encoded by the IDUA gene. We examined the mutation spectrum of the IDUA gene to explain the clinical, biochemical, and molecular features in 21 Iranian patients with MPSI. METHODS: Sanger sequencing was used to measure the IDUA gene sequence in the coding region and exon-intron boundaries. We recorded the clinical findings of studied patients at the first diagnosis of disease and then during the treatment and follow-up. RESULTS: Five different missense disease-causing mutations were determined in our patient groups, indicating 90.48% of detection rate. The most widespread mutation was the p.Y109H, occurring in 15.625% of all alleles, which was reported for the first time in our study. Other frequent mutations were as follows: p.Ser157Pro (12.5%), p.Gly84Arg (12.5%), p.Asp257His (9.375%), and p.Asp301Glu (9.375%). Three ones of them were new missense mutations: p.Ser157Pro, p.Asp257His, and p.Asp301Glu. DISCUSSION: The results of this study explain the different spectrum of IDUA gene mutations in our patients with MPSI. We introduced here 32 different variants including four new variants: p.Y109H (15.625%), p.S157P (12.5%), p.D257H (9.375%), and p.D301E (9.375%). In this series, there was no relationship between the happening of clinical features and genotype variations and biochemical findings.

L-carnitine effectively improves the metabolism and quality of platelet concentrates during storage.

Human platelets undergo structural and biochemical alternations during storage which are collectively called platelet storage lesion (PSL). PSL is characterized as metabolic and functionally changes. It causes decrease in platelet recovery and survival. Here, we evaluated the effect of L-carnitine (LC) on the metabolism, function, and mitochondrial metabolic activity of platelet during storage. Platelet-rich plasma was used to prepare platelet concentrate (PC) in Iranian Blood Transfusion Organization. For this purpose, ten PC bags from healthy donors were stored at 22 °C with gentle agitation in the presence or absence of LC. The effects of LC (15 mM) on the platelet quality were assessed by analyzing the levels of glucose, lactate, ATP, and lactate dehydrogenase (LDH) activity. Platelet aggregations induced by arachidonate and ristocetin were analyzed by aggregometer. Platelet mitochondrial melablolic activity was measured by tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay; platelet count and mean platelet volume were also determined by a hematology analyzer during 5 days of PC storage. The results indicated that LC could significantly decrease lactate concentration and glucose consumption accompanied with the increased oxygen consumption in stored PC. LDH activity also less significantly increased in LC-treated PC on days 2 and 5 of storage. Platelet aggregation in response to the ristocetin and arachidonate was significantly higher in LC-treated PC than that in untreated PC on day 5 of storage. Finally, platelet mitochondrial metabolic activity less significantly decreased in LC-treated PC compared to the control group on days 2 and 5 of storage. It seems that LC would be a good additive to reduce PSL and improve the platelet metabolism and quality of the stored PC for platelet transfusion therapy.

Determination of Biological Variance and Validation of a Fluorometric Assay for Measurement of α-l-Iduronidase Activity in Dried Blood Spots Samples: The First Experience in Iran.

Methods for assaying lysosomal diseases in dried blood samples are very useful today due to its several advantages related to the stability of samples, its transportation, handled and analysis, and its potential use for newborn screening compared to traditional methods in leucocytes samples. For this reason, it is important to validate these assays before being used in routine laboratory. Because of different in biological markers based on ethnicity, we aimed this study to validation a DBS-based fluorometric assay for measurement of α-l-Iduronidase activity for diagnosis of MPS I patients in Iran. DBS samples were collected from 15 MPS I patients and 60 healthy age matched subjects. Diagnostic value, biological variance and α-l-Iduronidase activity were determined. DBS α-l-Iduronidase activity was significantly higher in male subjects than in female group. Using a cut-off level of 1.08 µmol/spot 20 h, sensitivity and specificity were 100 and 98 %. The linearity of test was proved and we showed that within-run and between run precision were 5.6 and 14.66 %. Measurement of α-l-Iduronidase activity in DBS samples is an accurate test for diagnosis of MPS I and because of its rapid shipping and simplicity to keeping, DBS-based enzyme activity could be considered as a useful diagnostic tool in this disease.

Generation of high-yield insulin producing cells from human bone marrow mesenchymal stem cells.

Allogenic islet transplantation is a most efficient approach for treatment of diabetes mellitus. However, the scarcity of islets and long term need for an immunosuppressant limits its application. Recently, cell replacement therapies that generate of unlimited sources of ß cells have been developed to overcome these limitations. In this study we have described a stage specific differentiation protocol for the generation of insulin producing islet-like clusters from human bone marrow mesenchymal stem cells (hBM-MSCs). This specific stepwise protocol induced differentiation of hMSCs into definitive endoderm, pancreatic endoderm and pancreatic endocrine cells that expressed of sox17, foxa2, pdx1, ngn3, nkx2.2, insulin, glucagon, somatostatin, pancreatic polypeptide, and glut2 transcripts respectively. In addition, immunocytochemical analysis confirmed protein expression of the above mentioned genes. Western blot analysis discriminated insulin from proinsulin in the final differentiated cells. In derived insulin producing cells (IPCs), secreted insulin and C-peptide was in a glucose dependent manner. We have developed a protocol that generates effective high-yield human IPCs from hBM-MSCs in vitro. These finding suggest that functional IPCs generated by this procedure can be used as a cell-based approach for insulin dependent diabetes mellitus.

Tandem Mass Analysis of Amino Acids and Acylcarnitine Profiles in Neonates with Congenital Hypothyroidism.

BACKGROUND: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder. This study aimed to investigate whether disturbances in amino acid metabolism and fatty acid oxidation existed in neonates with CH compared to healthy neonates. METHODS: In this case-control study, we evaluated the metabolomics of neonates with newly diagnosed CH and healthy neonates. Forty-three metabolites, including 13 amino acids and 30 acylcarnitines, were investigated. RESULTS: Two hundred neonates with CH and 209 healthy children were enrolled. The mean age of males and females was 4.8 ± 2.4 and 5.52 ± 3.2 days in the case group and 5.1 ± 2.6 and 4.7 ± 3.6 days in the control group, respectively. Of the metabolites, 34 were significantly different between the two groups. Five amino acids and four acylcarnitines did not differ significantly between groups. CONCLUSION: These findings pave the way for a better understanding of the relationship between alterations and the clinical manifestation of CH, which has the potential for identifying novel therapeutics.

Investigation of Zinc Supplement Impact on the Serum Biochemical Parameters in Pulmonary Tuberculosis: A Double Blinded Placebo Control Trial.

BACKGROUND: Zinc (Zn) is nutritionally essential trace element, and thus deficiency may severely affect human health. The results of cross-sectional studies indicate that micronutrient deficiencies are common in patients with tuberculosis. Our goal is to investigate whether Zn supplementation can increase the effects of anti-TB treatment or not. METHODS: Patients with newly diagnosed tuberculosis were divided in to 2 groups. One group (n= 37) received capsule contains 50 mg of elemental zinc (as zinc sulfate) for 6 months every other day (micronutrient group) and Group II (n= 37) received placebo. Both groups received the same anti-tuberculosis treatment recommended by the WHO. Clinical examination, BMI, chest X-ray, direct sputum examination, assessment of serum zinc levels (by atomic absorption spectrophotometry), and biochemical markers serum concentration (by using an RA1000 AutoAnalyzer) were carried out before and after 2- and 6-months anti-tuberculosis treatment. RESULTS: Plasma zinc concentrations in the micronutrient group was higher than placebo group After treatment. In the placebo group increasing in SGOT and SGPT concentrations were significantly higher than micronutrient group after 2 months of treatment (p< 0.05). The significant changes (p< 0.05) were observed on the serum levels of total protein, albumin. Alkaline phosphatase (ALP) levels, serum creatinine, uric acid and urea in groups were not significantly different. CONCLUSION: Zinc supplementation results in earlier sputum smear conversion in the micronutrient group during the first 6 weeks. Increased body weight and serum zinc and serum albumin and decrease in total protein was observed in the micronutrient group.

Downregulation of fatty acid oxidation by involvement of HIF-1α and PPARγ in human gastric adenocarcinoma and related clinical significance.

Lipid metabolism rewiring in gastric adenocarcinoma (GA) pathogenesis is still not clearly elucidated. This study aimed to describe the role of lipid catabolism in GA patient outcomes and possible therapeutic targets by analyzing the effect of hypoxia-inducible factor-1α (HIF-1α) on fatty acid oxidation (FAO). AGS cell line was cultured in normoxic and hypoxic conditions, and FAO-related genes were analyzed by real-time-PCR and Western-blot. The study group comprised 108 newly diagnosed GA patients and 152 control cases. Serum concentrations of medium and long-chain acyl-CoA dehydrogenases (MCAD and LCAD) proteins were measured using ELISA, and local expression of HIF-1α, carnitine palmitoyl transferase 1 (CPT1A) and peroxisome proliferator-activated receptor γ (PPARγ) was evaluated by immunohistochemistry. In addition, gene expression of PPARγ, CPT1A, LCAD, and MCAD was assessed by real-time-PCR. In vitro findings indicate HIF-1α upregulation and FAO-related genes and proteins reduction in the hypoxic culture of AGS cells. GA patients had significantly lower circulating levels of LCAD compared to controls. Higher protein expression of HIF-1α and downregulated CPT1A and PPARγ were observed in GA tissues versus controls. Gene expression of CPT1A, PPARγ, LCAD, and MCAD were repressed in GA tissues compared to controls. Moreover, reduced expression of CPT1A, PPARγ, and MCAD were correlated with HIF-1α upregulation in GA. Poor patient outcome was associated with lower PPARγ and LCAD expression in GA. HIF-1α upregulation in human GA patients and AGS cells was paralleled by downregulation of lipid catabolism genes potentially via reduced PPARγ-mediated FAO. This metabolic adaptation to hypoxic condition may play a role in GA pathogenesis and might have clinical and therapeutic value in GA patients.

Palmitate and inflammatory state additively induce the expression of PTP1B in muscle cells.

The factors responsible for up-regulation of PTP1B, a negative regulator of insulin signaling, in insulin resistance state are not well understood. We performed a series of experiments in C2C12 muscle cells to determine the role of palmitate and an inflammatory state in regulation of PTP1B. Palmitate (0.75mM) induced PTP1B mRNA and protein level only at 16h. The combination of palmitate and macrophages, accompanied by a great increase of TNF-alpha and IL-6 in the culture media, additively caused a higher level of PTP1B protein levels in the muscle. Higher concentrations of palmitate reduced insulin stimulated glucose uptake in myotubes. A specific inhibitor of PTP1B partly increased insulin stimulated glucose uptake in palmitate treated cells. In conclusion, our results showing the additive influence of palmitate and the inflammatory state in the expression of PTP1B imply the involvement of these factors in the overexpression of PTP1B in insulin resistance state. We further provided the evidence suggesting the mediatory role for PTP1B in palmitate induced insulin resistance in myotubes.

SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation.

PURPOSE: Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. MATERIAL AND METHODS: Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. RESULTS: Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 +/- 0.08. CONCLUSION: These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone.

Downregulation of IGF-IR expression by RNAi inhibits proliferation and enhances chemosensitization of human colon cancer cells.

PURPOSE: Colon cancer is the second leading cause of cancer death worldwide. Elevated expression of insulin-like growth factor-I receptor (IGF-IR) is a frequent genetic abnormality seen in this malignancy. For a better understanding of its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against IGF-IR in our study. The aim of this study was to examine the anti-proliferation and chemosensitization effects elicited by a decrease in the transcription and protein levels of IGF-IR by RNAi in SW480 colon cancer cells. METHODS: A plasmid-based polymerase III promoter system was used to deliver and express short interfering RNA (siRNA) targeting IGF-IR to reduce its expression in SW480 cells. Western blot analysis was used to measure the protein level of IGF-IR. We assessed the effects of IGF-IR silencing on cancer cell growth by a cell growth curve. The effect of the 5-fluorouracil (5-FU)-induced cell death by knockdown of IGF-IR was also investigated by methyl thiazolyl tetrazolium assay. RESULTS: Transfection of siRNA targeting IGF-IR was shown to reduce IGF-IR messenger RNA levels by 95%. Western blotting detected a similar inhibition of IGF-IR protein levels in those cells. The cells transfected with PKD-short hairpin RNA-IGF-IR-V2 significantly decreased cell growth and rendered cells more sensitive to chemotherapy. The highest proliferation inhibitory and chemosensitization ratios were 53 +/- 2% and 1.78, respectively. CONCLUSION: This study indicates that downregulation of IGF-IR results in significant inhibition of tumor growth in vitro. It also provides a promising strategy to chemotherapy efficacy in human tumors and forming a basis for future in vivo trials.

Antibody detection against HPV16 E7 & GP96 fragments as biomarkers in cervical cancer patients.

BACKGROUND & OBJECTIVE: Cervical cancer is the second most frequent cancer among females worldwide, especially human papilloma viruses (HPV) types 16 and 18. In viral systems the identification of serological markers would facilitate the diagnosis of HPV infections and virus-related disease. The aim of the present investigation was to determine and search for serologic markers in cervical cancer patients associated with HPV. METHODS: A total of 58 Iranian women with invasive cervical carcinoma including adenocarcinoma and squamous cell carcinoma (SCC) were included. Serum antibody response to HPV infections in patients was detected by Western blot and ELISA techniques based on recombinant HPV16E7 and the N-terminal and C-terminal fragments of gp96 (NT-gp96 and CT-gp96) proteins. These recombinant proteins were expressed in Escherichia coli as a His-tag protein and purified using affinity chromatography. RESULTS: The ELISA results indicated that patients with high antibody response to HPV16E7 had significant seroreactivity to CT-gp96 fragment. In Western blot analysis, a strong association between anti-E7, anti-NT-gp96 and anti-CT-gp96 reactivity and cervical cancer was obtained using purified recombinant proteins. In adenocarcinoma cases, no significant difference was observed in seroreactivities between normal and patients. INTERPRETATION & CONCLUSION: The evaluation of cervical cancer patients' seroreactivities against three recombinant proteins (rE7, rNT-gp96 and rCT-gp96) showed significantly higher levels of these markers in SCC only, but not in adenocarcinoma and control groups. Also, the usage of both techniques (ELISA and Western blotting) can provide more reliable tools for diagnosis of cervical cancer.

L-lysine protects C2C12 myotubes and 3T3-L1 adipocytes against high glucose damages and stresses.

Hyperglycemia is a hallmark of diabetes, which is associated with protein glycation and misfolding, impaired cell metabolism and altered signaling pathways result in endoplasmic reticulum stress (ERS). We previously showed that L-lysine (Lys) inhibits the nonenzymatic glycation of proteins, and protects diabetic rats and type 2 diabetic patients against diabetic complications. Here, we studied some molecular aspects of the Lys protective role in high glucose (HG)-induced toxicity in C2C12 myotubes and 3T3-L1 adipocytes. C2C12 and 3T3-L1 cell lines were differentiated into myotubes and adipocytes, respectively. Then, they were incubated with normal or high glucose (HG) concentrations in the absence/presence of Lys (1 mM). To investigate the role of HG and/or Lys on cell apoptosis, oxidative status, unfolded protein response (UPR) and autophagy, we used the MTT assay and flow cytometry, spectrophotometry and fluorometry, RT-PCR and Western blotting, respectively. In both cell lines, HG significantly reduced cell viability and induced apoptosis, accompanying with the significant increase in reactive oxygen species (ROS) and nitric oxide (NO). Furthermore, the spliced form of X-box binding protein 1 (XBP1), at both mRNA and protein levels, the phosphorylated eukaryotic translation initiation factor 2α (p-eIf2α), and the Light chain 3 (LC3)II/LC3I ratio was also significantly increased. Lys alone had no significant effects on most of these parameters; but, treatment with HG plus Lys returned them all to, or close to, the normal values. The results indicated the protective role of Lys against glucotoxicity induced by HG in C2C12 myotubes and 3T3-L1 adipocytes.

Clinical importance of FASN in relation to HIF-1α and SREBP-1c in gastric adenocarcinoma.

AIMS: Identifying alterations in lipid metabolism along gastric adenocarcinoma (GA) tumorigenesis pathways could lead to a new approach for potential diagnosis, efficient prediction and promising therapeutic strategies. This study aimed to identify the possible effect of HIF-1α on FASN and SREBP-1c regulation in GA. MAIN METHODS: AGS cell line was cultured in normoxic and hypoxic conditions, and HIF-1α, FASN and SREBP-1c gene expression were analyzed by qRT-PCR and Western blot. Serum HIF-1α, FASN and insulin concentration were measured in 112 GA patients and 156 control cases by ELISA, and immunohistochemical method was employed to analyze SREBP-1c expression. Tissue mRNA expression of SREBP-1c, FASN and HIF-1α were determined by qRT-PCR. KEY FINDINGS: In vitro findings indicate upregulation of HIF-1α, FASN and SREBP-1c gene and protein expression in the hypoxic culture of AGS cells. High circulating levels of HIF-1α and FASN were significantly observed in GA patients compared to the controls. HIF-1α, SREBP-1c and FASN gene expression were higher in GA vs. controls. In addition, SREBP-1c protein level was enhanced in GA tissues compared to controls. Furthermore, elevated serum levels of HIF-1α and FASN and expression of HIF-1α, SREBP-1c and FASN genes were associated with unfavorable clinicopathological features such as diffuse type tumor and poor survival. SIGNIFICANCE: The results by correlating increased levels of FASN to those of HIF-1α and SREBP-1c are consistent with a possible up-regulation of FASN upon induction of HIF-1α through SREBP-1c.

HSP27 expression in the human peripheral blood mononuclear cells as an early prognostic biomarker in coronary artery disease patients.

BACKGROUND: Coronary artery disease (CAD), is one of the leading causes of death globally. CAD risk factors, such as smoking, dyslipidemia, and obesity, are mainly associated with increased oxidative stress. Heat Shock Protein-27 (HSP27) has a protective role in conditions of oxidative stress. The aim of the current study was to investigate the relationship between HSP27 mRNA copy numbers in the peripheral blood mononuclear cell (PBMCs) and the degree of CAD progression. METHODS: A total of 103 subjects aged 49-71 years were recruited; Patients with CAD were categorized into two groups: patients having <50% stenosis (Angio-) and ≥50% stenosis (Angio+). The mRNA copy numbers of HSP-27 in PBMCs, anthropometric-parameters, fasting blood glucose (FBG), and the fasted serum lipid profile were evaluated. RESULTS: Angio+ patients had a significantly higher level of TC and LDL-C values compared with Angio- patients and the control group (p < 0.05). The HSP27 expression in PBMCs was significantly increased in Angio+ and Angio- subjects, compared to the control group. Moreover, there was a significant association between the FBG, TC, LDL-C and TG among the groups (p < 0.05). CONCLUSION: It was shown that the increased expression of HSP27 in PBMCs of CAD patients is significantly correlated with CAD severity in Angio+ subjects, which can be used as an early prognostic biomarker, indicating the degree of overall oxidative stress in patients. In order to verify this statement, it is suggested to measure Pro-oxidant- Antioxidant Balance (PAB) test by the same design in subsequent studies.

Palmitate-induced impairment of autophagy turnover leads to increased apoptosis and inflammation in peripheral blood mononuclear cells.

Previous works have linked high concentrations of palmitate to cellular toxicity by autophagy modulation. However, the ways in which palmitate regulates inflammation and apoptosis in peripheral blood mononuclear cells (PBMCs), has not been well characterized. In the present study, we therefore aimed to investigate the role autophagy in inflammatory responses and apoptotic death of PBMCs treated with palmitate. 0.5mM palmitate increased the level of LC3-II at 6h, peaked at 12h and then decreased at 24h. The protein level of p62 was significantly increased at 6h and 12h, suggesting an impairment of autophagic flux in palmitate-treated PBMCs. Inhibiting autophagy with chloroquine (CQ) and 3-Methyladenine (3-MA) significantly augmented palmitate-induced PBMCs apoptotic death as demonstrated by increased cleaved PARP level and increased the percentage of apoptotic (YO-PRO-1 positive and PI negative) cells. Furthermore, CQ pretreatment exacerbated palmitate-induced TNF-α and IL-6 mRNA expression in PBMCs. Moreover, induction of autophagy by pretreatment of PBMCs with rapamycin resulted in a distinct increase of palmitate-induced apoptosis. The induction of autophagy also led to a further increase in palmitate-induced expression of TNF-α and IL-6. These results indicate that the excess palmitate could impair autophagy, hence contributing to palmitate-induced-inflammation and apoptosis in PBMCs. Therefore, dysregulated autophagy in PBMCs may provide a novel mechanism that connects diet-induced obesity to low grade inflammation in patients with type 2 diabetes.

1484insG polymorphism of the PTPN1 gene is associated with insulin resistance in an Iranian population.

BACKGROUND: Protein tyrosine phosphatase 1B (PTP1B), encoded by the PTPN1 gene, efficiently dephosphorylates the insulin receptor, and attenuates insulin signaling. Recently, a 1484insG variant of the PTPN1 gene has been associated with an increased risk of metabolic syndrome in an Italian population that has not been confirmed in the subsequent studies. The purpose of this study was to investigate the association of 1484insG polymorphism of the PTPN1 with obesity, insulin resistance, type 2 diabetes and other cardiovascular-related traits in an Iranian population. METHODS: The genotypes of 1484insG variant were determined by PCR-RFLP method in 696 unrelated subjects including 412 subjects with normal glucose tolerance and normal fasting glucose and 284 type 2 diabetic patients. RESULTS: The allelic frequency of 1484insG polymorphism among type 2 diabetic patients and non-diabetic subjects was 4.9 and 4.1%, respectively (p = 0.475). In type 2 diabetic patients, among quantitative traits, no significant difference in anthropometric and biochemical parameters was seen between the wild-type and heterozygous 1484insG genotypes in male and female groups and in non-diabetic subjects, male carriers of 1484insG allele had significantly higher fasting insulin (p = 0.003), cholesterol (p = 0.012), LDL-C (p = 0.037), apo B (p = 0.015), and HOMA-IR (p = 0.011) levels compared to the individuals carrying the wild-type genotype. Among non-diabetic female individuals, only body mass index was significantly higher in 1484insG subjects compared to the wild-type individuals (p = 0.007). CONCLUSIONS: Our results from a sample of Iranian type 2 diabetes cases and controls provide evidence that the 1484insG genotype of the PTPN1 gene may be associated with insulin resistance and other cardiovascular risk factors in non-diabetic male subjects.

Identification of mutations in the rpoB encoding the RNA polymerase beta subunit in rifampicine-resistant Mycobacterium tuberculosis strains from Iran.

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Iran. 91 sputum were collected from suspected tuberculosis patients, 34 Rif-r isolates (87%) were identified as M. tuberculosis. Polymerase chain reaction (PCR) amplification and DNA sequencing methods were performed. 411 bp fragments of rpoB gene were sequenced and mutations in 81 bp regions were analyzed. 60 mutations and 13 micro deletions were identified in 29 RIF-r MBT (85%). Among 60 mutations, 6 silent and 54 missense were identified. Missense mutations produced 23 types of amino acid substitutions. In 5 RIF-r MBT isolates (15%) no mutations were found in the core region of the rpoB gene. All silent mutations were localized in codon 507. Most frequent mutations detected from Iranian strains were in codons 523 and 526. Five alleles in codon 526 and 3 alleles in triplets in each codons 507, 508, 513 were found. 6 (19%) strains harboured single mutations 6 (18%) placed in codons 526, 510 while the rest of isolates 23 (69%) had multiple mutations: Double 11 (34%), triple 7 (22%), and quartile mutations 1 (3%) and 4 (12%) of strains harboured 5 mutations respectively.

katG mutations in isoniazid-resistant strains of Mycobacterium tuberculosis isolates from Belarusian patients.

The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Mutations in different codons causing resistance to isoniazide in the gene of catalase peroxides (katG) in Belarusian strains was determined. 42 strains of Rif-r and Inhr(MDR) were isolated in different regions of Belarus. Culture susceptibility testing of all 42 strains revealed resistance to streptomycin (90%), 16 strains (43%) were resistant to etambutol. DNA Extraction, Standard PCR identification and katG gene amplification were performed. The most affected codons of katG gene were 315(95%), 316(16.2%), and 309(14.5%). Four types of mutations were identified in codon 315: AGC-->ACC (n=36)32.4%, AGC-->AGG (n=1) 0.9%, AGC-->AAC (n=2) 1.8%, AGC-->GGC (n=1) 0.9%. One type of mutation was found in codon 316: GGC-->AGC (n=18)16.2%, four types of mutations were detected in codon 309: GGT-->GGT (n=7)6.3%, GGT-->GCT (n=4)3.6%, GGT-->GTC (n=3)2.7%, GGT-->GGG (n=1)0.9%. Mutations in codon 309 make up 34%, in codon 316 (37%) and other types of mutations 29% of all detected mutations. In 2 isolated strains mutation were identified in codons 463, 35 and, in codons 454, 357 respectively and 2 isolates, there were not found any mutations. Concluding, all INH-r MBT had resistance-associated nucleotide changes mostly in codons 315.

Britannin induces apoptosis through AKT-FOXO1 pathway in human pancreatic cancer cells.

Induction of apoptosis in cancerous cells is considered as a promising treatment option for cancer therapy. The present study was designed to evaluate the anticancer properties of Britannin and its possible mechanisms of action in human pancreatic cancer cells. Apoptosis induction by Britannin was confirmed by annexin V-FITC/PI staining, Hoechst 33258 staining and caspase-3 activity assay in both AsPC-1 and Panc-1 cells. Additionally, by using western blot and Real-time PCR, we observed that Britannin induced apoptosis by decreasing the expression of BCL-2 and increasing the expression of BAX. Moreover, Britannin increased reactive oxygen species (ROS) generation in different intracellular sites of pancreatic cancer cells. Using western blot analysis, we observed that Britannin decreased the phosphorylated AKT and induced the nuclear accumulation of FOXO1 and also up regulation of FOXO-responsive target BIM in both pancreatic cancer cell lines. Taken together, we found that Britannin is able to induce mitochondrial apoptotic pathway through ROS production and modulation of the AKT-FOXO1 signaling axis in AsPC-1 and Panc-1 human pancreatic cancer cells. Our results can help to illuminate the molecular mechanisms underlying Britannin-induced cell death in pancreatic cancer cell lines and may potentially serve as an anticancer agent for the treatment of pancreatic cancer.