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.

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.

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.

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.

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.

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.

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.

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.

Chitotriosidase Activity and Gene Polymorphism in Iranian Patients with Gaucher Disease and Sibling Carriers.

OBJECTIVE: Chitotriosidase (CT) activity is a useful biomarker for diagnosis and monitoring of Gaucher disease (GD). Its application is limited by some variants in the CT gene. Two main polymorphisms are 24 bp duplication and G102S led to reduce CT activity. The aim of this study was to determine these variants influencing on plasma CT activity. MATERIALS & METHODS: Blood samples were collected from 33 patients with GD, 15 sibling carriers and 105 healthy individuals serving as controls. CT activity was measured using 4-methylumbelliferyl-ß-D-N,N',N″triacetylchitotrioside substrate in plasma samples. The CT genotypes of 24 bp duplication and G102S variants were determined using PCR and PCR-RFLP. RESULTS: Untreated GD patients had a significantly higher CT activity compared to treated patients (P = 0.021). In addition, chitotriosidase activity in carriers was higher rather than controls. Allele frequencies of 24 bp duplication in GD patients, sibling carriers and controls were 0.21, 0.266 and 0.29 and for G102S were 0.318, 0.366 and 0.219, respectively. Different G102S genotypes had not significant effect on CT activity. Chitotriosidase activity has a positive correlation with age in normal group, carriers, and negative correlation with hemoglobin in GD patients. Using cut-off level of 80.75 nmol/ml/h, sensitivity and specificity of CT activity were 93.9% and 100%, respectively. CONCLUSION: Chitotriosidase activity is a suitable biomarker for diagnosis and monitoring of GD. Determination of 24 bp duplication is helpful for more accurate monitoring the GD patient's therapy. However, it seems that, specifying of the G102S polymorphism is not required for Iranian GD patients.

Preparation of monoclonal antibodies against mannosylated lipoarabinomannan (ManLAM), a surface antigen of BCG vaccine produced in Iran.

BACKGROUND: Bacille Calmette-Guerin (BCG) vaccine is the only vaccine that is used against Mycobacterium tuberculosis, but its efficacy is limited in mycobacterium-endemic regions. One of the major antigens present on the cell envelope of the vaccine that suppresses the immune system is mannosylated lipoarabinomannan (ManLAM). MATERIALS AND METHODS: In this study, we immunized 4-week-old mice with sonicated BCG vaccine injected intraperitoneally two times at an interval of 2 weeks and with ManLAM antigen injected intravenously and then extracted the spleen cells of the immunized mice. They were fused with SP2/0 myeloma cells. RESULTS: Five cell line clones producing antibody against ManLAM antigens were prepared and each clone was tested for immunoreactivity against sonicated BCG and purified ManLAM by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The clones designated H13F33E11 and H23D91G4 reacted strongly with ManLAM. Immunoblotting using monoclonal antibodies (MAbs) H13F33E11 and H23D91G4 showed that these MAbs bind to ManLAM with a molecular weight of 35 kDa. CONCLUSIONS: In this study, we produced a monoclonal antibody of immunoglobulin G3 (IgG3) subclass. This MAb can be used for purification of ManLAM in culture media and detection of the antigen in patient's urine and for increasing the efficacy of BCG vaccine.

Allelic Frequency of a 24-bp Duplication in Exon 10 of the CHIT1 Gene in the General Iranian Population.

BACKGROUND: The human chitinase chitotriosidase enzyme, which is encoded by the CHIT1 gene, is produced by macrophages, and may be important in immune responses to chitin-containing organisms, such as fungi. Plasma chitotriosidase activity is used to diagnose and monitor some forms of lysosomal storage disorders, such as Gaucher's and Niemann-Pick disease. However, homozygous duplication of a 24-bp region in exon 10 of the CHIT1 gene eliminates enzyme activity and may complicate disease monitoring. The high prevalence of this mutation highlights the need to determine its frequency in different populations and screen patients for this mutation to verify whether chitotriosidase activity is a reliable marker of lysosomal storage disease. This study investigated the allele frequency of the 24-bp duplication in the general Iranian population. METHODS: To identify the 24-bp duplication in exon 10 of the CHIT1 gene (H allele), genotyping of DNA extracted from peripheral blood leukocytes of 577 healthy Iranians was performed using polymerase chain reaction (PCR) amplification and high resolution melting (HRM) PCR techniques. RESULTS: In this study, heterozygous and homozygous duplications were detected in 183 (31.7%) and 35 (6.1%) subjects, respectively. In addition, the allelic frequency was 21.9% (95% confidence interval). CONCLUSION: Our study indicates that genotype analysis by HRM-PCR is a fast, reliable, and highly accurate screening approach for identifying the 24-bp duplication in CHIT1 exon 10. Due to the wide range of duplication frequencies among different ethnic groups, new biomarkers are necessary for assessing genetic characteristics of lysosomal storage disorders in different populations.

Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1.

OBJECTIVE: Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce ß-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. MATERIALS AND METHODS: We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. RESULTS: After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. CONCLUSION: MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation.

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.

Synthesis of a stabilized 177Lu-siRNA complex and evaluation of its stability and RNAi activity.

PURPOSE: Serum and intracellular instability limits the therapeutic applications of short interfering RNA (siRNA) as a radiopharmaceutical. Chemical modifications like phosphorothioate (PS) substitution and 2'-O-methoxy (2'-O-Me) modifications could eliminate such limitations. In this study, the effects of PS and 2'-O-Me modifications at the backbone of siRNA on serum stability and RNA interference activity were investigated. MATERIALS AND METHODS: Fully PS and 2'-O-Me-modified type 1 insulin-like growth factor receptor (IGF-1R) siRNA was radiolabeled with lutetium-177 ((177)Lu) through p-SCN-Bn-DTPA as a chelator. After purification with Vivaspin and PD-10 columns, the radiolabs were examined for stability in serum by instant thin-layer chromatography and polyacrylamide gel electrophoresis. The level of IGF-1R in response to the modified and labeled IGF-1R siRNA was examined using RT-PCR and ELISA assay in colon cancer cells. The effects of such siRNA on the prevention of proliferation of colon cancer cells and its apoptosis were investigated using MTT assay and Annexin-V/propidium iodide double staining, respectively. Cellular accumulation quantities of the labeled and modified IGF-1R siRNA were determined using a γ-counter by taking advantage of (177)Lu as a γ-emitter. RESULTS: Both the modified (177)Lu-siRNA complex and the modified nonlabeled siRNA showed significant stability in serum. The levels of IGF-1R mRNA and protein significantly decreased with both the labeled and nonlabeled IGF-1R siRNAs, but no such reduction in IGF-1R was observed with luciferase siRNA (P<0.01). Proliferation decreased significantly and apoptosis increased in the cells treated with modified (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or labeled luciferase siRNA (P<0.001). CONCLUSION: Uniform chemically modified siRNAs can form stable complexes with Lu that pronounce its cytotoxic effect through apoptosis in colon cancer cells.

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.

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.

Validation of Urinary Glycosaminoglycans in Iranian patients with Mucopolysaccharidase type I: The effect of urine sedimentation characteristics.

OBJECTIVE: The first line-screening test for mucopolysaccharidosis is based on measurement of urinary glycosaminoglycans. The most reliable test for measurement of urine glycosaminoglycans is the 1,9-dimethyleneblue colorimetric assay. Biological markers are affected by ethnical factors, for this reason, the World Health Organization recommends that the diagnostic test characteristics should be used to determine results for different populations. This study determines the diagnostic value of 1,9-dimethyleneblue tests for diagnosis of mucopolysaccharidosis type I patients in Iran. MATERIALS & METHODS: In addition to routine urine analysis, the qualitative and quantitative measurements of urine glucosaminoglycans were performed with the Berry spot test and 1,9-dimethyleneblue assay. Diagnostic values of the tests were determined using the ROC curve. RESULTS: Urine total glycosaminoglycans were significantly higher in male subjects than in female subjects. Glycosaminoglycan concentration was markedly decreased in specimens with elevated white blood cell and epithelial cells count. Using a cut-off level of 10.37 mg/g creatinine, sensitivity, and specificity were 100% and 97.22%, respectively, for a 1,9-dimethyleneblue colorimetric assay. CONCLUSION: Urine glycosaminoglycans concentration significantly differs in our studied population. In addition to determine diagnostic validity of the 1,9-dimethyleneblue test, our results demonstrate the usefulness of measuring glycosaminoglycans for early screening of mucopolysaccharidosis type I Iran.

Demonstration of dose dependent cytotoxic activity in SW480 colon cancer cells by ¹77Lu-labeled siRNA targeting IGF-1R.

PURPOSE: The radiolabeling of targeting biomolecules with gamma emitter radionuclides for tracing and beta emitters for therapy involves the conjugation of such biomolecules to the chelating agents to form complexes with the radionuclide of interest. In this study, radioconjugate of IGF-1R siRNA with lutetium-177 ((177)Lu) was produced, and the anti-proliferation and apoptosis effects elicited by this (177)Lu-siRNA complex in the SW480 colon cancer cells were evaluated. METHODS: IGF-1R and Luciferase siRNAs were conjugated with p-SCN-Bn-DTPA, and then radiolabeled with (177)Lu. The effects of labeled and non-labeled IGF-1R siRNAs on IGF-1R expression were assessed with RT-PCR analysis and ELISA assay. IGF-1R siRNAs induced cell death and apoptosis were investigated using MTT assay and Annexin-V/propidium iodide (PI) double staining, respectively. RESULTS: Combined purification using Vivaspin and PD-10 columns resulted in a radiochemical purity of 97.32% ± 1.97%. Knockdown effect of the labeled IGF-1R siRNA was not significantly different from the non-labeled duplex of the same sequence (P<0.05), but it was significant compared to the Luciferase siRNAs (P<0.001). Proliferation decreased significantly, but apoptosis increased in the cells treated with the (177)Lu-IGF-1R siRNA in comparison with either (177)Lu or IGF-1R siRNA (P<0.001). CONCLUSION: Radioconjugate of IGF-1R siRNA, p-SCN-Bn-DTPA and (177)Lu was successfully produced and characterized as radiopharmaceutical. The present study demonstrates the involvement of (177)Lu-labeled IGF-1R siRNA in the inhibition of cell growth and induction of apoptosis in colon cancer cells.