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Conventional methods for obtaining pancreatic beta cells are based on simulating the embryonic development phase of endocrine cells via hierarchical differentiation of pluripotent stem cells (PSCs). Accordingly, we attempted to modify the protocols for obtaining insulin-secreting cells (ISCs) by sequential differentiation of a human embryonic stem cell (hESC), using the HS181 cell line. Furthermore, we hypothesize that actual pancreatic endocrine cells may arise from trans-differentiation of mature ductal cells after the embryonic developmental stage and throughout the rest of life. According to the hypothesis, ductal cells are trans-differentiated into endocrine and exocrine cells, undergoing a partial epithelial to mesenchymal transition (EMT). To address this issue, we developed two new protocols based on hESC differentiation to obtain ductal cells and then induce EMT in cells to obtain hormone-secreting islet-like cells (HSCs). The ductal (pre-EMT exocrine) cells were then induced to undergo partial EMT by treating with Wnt3a and activin A, in hypoxia. The cell derived from the latter method significantly expressed the main endocrine cell-specific markers and also beta cells, in particular. These experiments not only support our hypothetical model but also offer a promising approach to develop new methods to compensate beta cell depletion in patients with type 1 diabetes mellitus (T1DM). Although this protocol of generating islet-like cells from ductal cells has a potential to treat T1DM, this strategy may be exploited to optimize the function of these cells in an animal model and future clinical applications.

Receptor for advanced glycation end-products (RAGE) is a multiligand binding and single-pass transmembrane protein taken in diverse chronic inflammatory conditions. RAGE behaves as a pattern recognition receptor, which binds and is engaged in the cellular response to a variety of damage-associated molecular pattern molecules, as well as HMGB1, S100 proteins, and AGEs (advanced glycation end-products). The RAGE activation turns out to a formation of numerous intracellular signaling mechanisms, resulting in the progression and prolongation of colorectal carcinoma (CRC). The RAGE expression correlates well with the survival of colon cancer cells. RAGE is involved in the tumorigenesis, which increases and develops well in the stressed tumor microenvironment. In this review, we summarized downstream signaling cascade activated by the multiligand activation of RAGE, as well as RAGE ligands and their sources, clinical studies, and tumor markers related to RAGE particularly in the inflammatory tumor microenvironment in CRC. Furthermore, the role of RAGE signaling pathway in CRC patients with diabetic mellitus is investigated. RAGE has been reported to drive assorted signaling pathways, including activator protein 1, nuclear factor-κB, signal transducer and activator of transcription 3, SMAD family member 4 (Smad4), mitogen-activated protein kinases, mammalian target of rapamycin, phosphoinositide 3-kinases, reticular activating system, Wnt/β-catenin pathway, and Glycogen synthase kinase 3β, and even microRNAs.

Colorectal cancer (CRC) is one of the most common types of cancer which affects the colon and the rectum. Approximately one third of annual CRC mortality occurs due to the late detection of this type of cancer. Therefore, there is an urgent need for more powerful diagnostic and prognostic tools for identification and treatment of colorectal tumorigenesis. Non-coding RNAs (ncRNAs) have been implicated in the pathology of CRC and also linked to metastasis, proliferation, differentiation, migration, angiogenesis and apoptosis in numerous cancers. Recently, attention has turned towards ncRNAs as specific targets for diagnosis, prognosis and treatment of various types of cancers, including CRC. In this review, we have tried to outline the roles of ncRNAs, and their involvement in signaling pathways responsible for the progression of CRC.

RNA-binding protein, musashi1 (MSI1), is a main protein in asymmetric cell division of the sensory organ precursor cells, whereas its expression is reported to be upregulated in cancers. This protein is a critical element in proliferation of stem and cancer stem cells, which acts through Wnt and Notch signaling pathways. Moreover, MSI1 modulates malignancy and chemoresistance of lung cancer cells via activating the Akt signaling. Due to the main role of MSI1 in metastasis and cancer development, MSI1 would be an appropriate candidate for cancer therapy. Downregulation of MSI1 inhibits proliferation of cancer stem cells and reduces the growth of solid tumors in several cancers. On the other hand, MSI1 expression is regulated by microRNAs in such a way that several different tumor suppressor miRNAs negatively regulate oncogenic MSI1 and inhibit migration and tumor metastasis. The aim of this review is summarizing the role of MSI1 in stem cell proliferation and cancer promotion.

Background and aims: The most important cause of fertilization failure after assisted-reproduction techniques are lack of releasing sperm-associated oocyte activating factors (SAOAFs) and sperm DNA damage. PLCζ is one of the sperm factors that plays important role in oocyte activation. The aim of this study was to compare expression of PLCζ, protamine deficiency (maturity marker of sperm chromatin) and sperm DNA damage between fertile men and oligoasthenoteratozoospermic subfertile men. Methods: In this case-control study, semen samples were collected from 10 subfertile oligoasthenoteratozoospermic and 10 fertile men. Sperm parameters (concentration, motility, morphology), expression of PLCζ, DNA damage, and protamine deficiency were assessed by World Health Organization protocol, flow cytometery, TUNEL and chromomycin A3 staining, respectively. Results: Percentage of PLCζ- positive sperm, DNA integrity and protamine content significantly was lower in oligoasthenoteratozoospermic men compared to fertile men. In addition, significant correlations were observed between percentage of PLCζ- positive sperm with sperm parameters and percentage of protamine deficiency. A significant correlation was also observed between percentage of DNA fragmentation and protamine deficiency. Conclusion: In these infertile men, sperm chromatin integrity and percentage of PLCζ-positive sperm decreased extremely. This could be one cause of fertilization failure in these individuals. Therefore, the artificial oocyte activation is recommended for these type of infertile men.

Background and aims: The research reports indicate the effect of exercise as an important factor in the prevention of breast cancer. In the process of cancer development, the cytokines are playing the vital role as regulatory proteins. The aim of this study was to investigate the effect of a one-term moderate intense endurance exercise before tumor formation on serum concentration content of a cytokine, Interleukin-1 (IL-1) as well as life span in the breast cancer balb/c mouse model. Methods: This research was an experimental project. 17 female BALB/c mice were randomly distributed into two groups of experimental Group (exercise+tumor) and control Group (rest+tumor). Prior to the tumor formation, the EG mice performed a daily session of 35-40 minutes endurance exercise protocol for 12 consecutive weeks and 5 days a week. The exercise intensity began with 15.4 meter per minutes in the first week and then it was gradually raised up to 23.8 meters per minutes during the last week. In order to induce cancer, one day after the end of the exercise protocol, all animals in both groups received subcutaneous injections of T410 cell line. After the formation of cancerous tumor, the ELISA method applied to measure the serum content of IL-1 and the volum of tumor was measured by caliper. Results: Comparing the 2 groups, a significant difference (P=0.001) between the average serum IL-1 content was observed and also the volume of tumor in mice suffered from cancer in experimental group was significant (P=0.003). Conclusion: Moderate intense endurance exercise can play a preventive role in breast cancer causing reduction of the cancerous inflammation in the patients suffering from breast cancer.

Background and aims: Apelin is a protein which is expressed in myocardia cardiac tissue of mammals, as well as coronary artery, aorta and saphenous vein. Apelin has various cardiovascular functions and causes hypertension reduction. The aim of this study was to investigate the effect of endurance training on the cardiac apelin gene expression in left ventricular in Wistar male rats. Methods: In the current experimental study, 12 male Wistar rats were divided into control group and training groups. Training group was received exercise on a motor-driven treadmill at 28 m/min (0% grade) for 60 min/day, 5 days/week for 8 weeks. 24 hours after the last exercise session, the rats were anesthetized. After cutting chest, a part of heart tissue was separated. The RNA from each sample was converted into cDNA. Calculated relative expression of the interested gene was obtained. By dividing threshold of the gene to the cycle threshold of the house keeping gene, relative expression of the gene was calculated by 2-∆∆Ct equation. All data was analyzed statistically by independent t-test. Results: Data showed significant increase in the expression of Apelin in exercised group compared to control group after 8-week training (P

Objective(s): Phospholipase C ζ (PLCζ) is considered as a nominee for sperm associated oocyte activating factors and is located back-to-back with CAPZA3, an actin-capping protein controlling actin polymerization during spermiogenesis. They contain a common bidirectional promoter. The objective of this study was to identify individuals with parallel low expression of PLCζ and CAPZA3 mRNA, in hope of detecting genetic defects in this bidirectional promoter. Materials and Methods: Semen samples were collected from 24 fertile and 59 infertile individuals with total failed, low and high fertilization rate post intra-cytoplasmic sperm injection (ICSI), as well as globozoospermic individuals. Expression of PLCζ and CAPZA3 were assessed by Real time PCR. In addition, PLCζ was assessed by Western blot. Results: Significant correlations between PLCζ with CAPZA3 and also between these two genes with fertilization were observed. Individuals with low fertilization presented significantly lower expression of these two genes. Low expression of PLCζ was also verified by Western analysis. Sequence analysis of bidirectional promoter of these two genes in an individual with parallel low expression of both PLCζ and CAPZA3, revealed a mutation within the CAPZA3 predicted promoter, known as human regulatory factor X4 which is a testis-specific dimeric DNA-binding protein. In the opposite stand, in the same location, the mutation appears to be outside but in the vicinity of PLCζ, in a binding region predicate by Genomatix. Conclusion: Parallel assessment of CAPZA3 with PLCζ at mRNA level in individuals with inability to induce oocyte activation may help researcher to identify genetic defects associated with failed fertilization.

Background: Granulocyte colony-stimulating factor (G-CSF) is a cytokine which have many functions such as stimulating of hematopoiesis, proliferation and differentiation of granulocyte progenitor cells and production of bone marrow neutrophilic granulocyte colonies. Nowadays, the usage of human recombinant G-CSF (rh G-CSF) is for curing of chemotherapy- radiotherapy-induced neutropenia, and also in patients with bone marrow transplantation. The aim of this study was to produce a soluble type of G-CSF in E.coli and purification in new way. In this study, we achieved active G-CSF using the intein fusion system. Therefore, two copies of optimized Intein - GCSF genewas cloned into the pET22b vector to generate pET22b-G-CSF-intein2 (C-terminal fusion vector) and pET22b intein1-G-CSF (N-terminal fusion vector). The hG-CSF sequence with two different intein sequence from pTWIN1 vector are synthesized and then inserted into a pET22 expression vector under the control of T7 promoter and cloned in E. coli strain BL21 (DE3).The fused proteins formed misfolded proteins as inclusion bodies (IBs). The IBs were solved with urea solution and afterward proteins were saved for size-exclusion chromatography. Then , CBD-intein–GCSF was loaded onto chitin beads column equilibrated with 10mM Tris buffer, 500mM NaCl, pH 8.5, and was eluted from the column by incubation at 25°C under pH 6.5 for 16h based on intein C-terminal self-cleavage. SDS-Page, western blot, dot blot, size exclusion chromatography and in vivo assay of protein demonstrated that the bioactivity of recombinant GCSF was compared with standard GCSF. Results: After culturing and induction of recombinant E. coli with IPTG, we obtained a good expression of the hG-CSF, as determined by SDS-PAGE and confirmed by dot blotting and western blotting. Approximately 50% of the expressed G-CSF was soluble after the IPTG induction and temperature reduction. Up to 1.2 mg of G-CSF was achieved from a 1 L culture, and nearly75% of the GCSF-Intein2 was cleared by pH shift. Intein1-GCSF was not cleaved due to the inhibition of cleavage by the N-terminal amino acid of GCSF. The biological activity assay, in vivo, showed a higher biological effect than standard reference hG-CSF. Conclusion: The immunological and biological analyses showed that this protocol can be useful to develop bioproducts. In conclusion, the combination of different methods presented here permitted an simple and cost-effective protocol for rhG-CSF production.

Objective: The aim of this study was to produce a stable CHO cell line expressing tenecteplase.Materials and Methods: In the first step, the tenecteplase coding sequence was clonedin a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then,using lipofection, the CHO cells were co-transfected with constructed recombinant plasmidencoding tenecteplase and attB recognition sites and the integrase coding sequencecontaining pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistancegene (neo), stable cells were then selected using G418 as an antibiotic. Stabletransformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionalityof tenecteplase was evaluated on the cell culture media.Results: our results indicated that tenecteplase coding sequence was inserted into theCHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessmentindicated the presence of our functional tenecteplase in the cell culture medium.Conclusion: Considering the data obtained from this study, φC31 integrase can be usedfor the production of a stable cell line and it be used to introduce ectopic genes into mammaliancells.

Objective: Amino acid alignment analysis of deduced amino acid residues revealed a tripeptide(SKI) at the carboxy terminus of peroxisomal protein cDNA. In order to see the importanceof the above sorting signal, we have performed a site-directed mutagenesis to deleteSKI tripeptide and its transfection into CHO-K1 and P19 cells.Materials and Methods: In order to create the appropriate site-directed mutant, PCR wasdone with specific primers. Amplified PeP cDNAs either containing SKI or deleted ones wereconstructed downstream of EGFP cDNA under regulation of the cytomegalovirus (CMV)promoter in a pEGFP-C1 vector. Transfection of P19 and CHO cells was done with lipofectamine2000.Results: Gradient PCR showed that the best annealing temperature was 71.6 ºC. Transfectionof plasmids containing chimera of EGFP-PEP cDNAs into the CHO-K1 and P19 cellsshowed several punctuate structures, presumably peroxisomes, while SKI deletion showeda cytosolic mislocalization of the EGFP pattern.Conclusion: Taken together, these data strongly suggest that SKI, which is located at theC- terminus of the protein, is required for sorting this protein.

Background Single nucleotide polymorphism (SNPs) are considered as one of the underlying causes of male infertility. Proper sperm chromatin packaging which involves replacement of histones with protamines has profound effect on male fertility. Over 20 SNPs have been reported for the protamine 1 and 2. Materials and methods The aim of this study was to evaluate the frequency of two previously reported SNPs using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) approach in 35, 96 and 177 normal, oligozoospermic and azoospermic individuals. These SNPs are: 1. A base pair substitution (G) at position 197 instead of T in protamine type 1 Open reading frame (ORF) including untranslated region, which causes an Arg residue change to Ser residue in a highly conserved region. 2. cytidine nucleotide change to thymidine in position of 248 of protamine type 2 ORF which caused a nonsense point mutation. Results The two mentioned SNPs were not present in the studied population, thus concluding that these SNPs can not serves as molecular markers for male infertility diagnosis. Conclusion The results of our study reveal that in a selected Iranian population, the SNP G197T and C248T are completely absent and are not associated with male infertility and therefore these SNPs may not represent a molecular marker for genetic diagnosis of male infertility.

Background Globozoospermia is a rare syndrome with an incidence of less than 0.1% among infertile men. Researchers have recently identified a large deletion, about 200 kbp, encompassing the whole length of DPY19L2 or mutations in SPATA16 and PICK1 genes associated with globozoospermia. The aim of this study was to analyze the DPY19L2 gene deletion using polymerase chain reaction technique for the exons 1, 48, 11 and 22 as well as break point (BP) “a” in globozoospermic men. Materials and Methods In this experimental study, genome samples were collected from 27 men with globozoospermia (cases) and 36 fertile individuals (controls), and genomic analysis was carried out on each sample. Results Deletion of DPY19L2 gene accounted for 74% of individuals with globozoospermia. DPY19L2 gene deletion was considered as the molecular pathogenic factor for the onset of globozoospermia in infertile men. By quantitative real-time polymerase chain reaction (qPCR), we genotyped DPY19L2 deletion and identified carriers within the population. Conclusion This technique may be considered as a method for family counseling and has the potential to be used as a pre-implantation genetic diagnosis, especially in ethnic community with high rate of consanguineous marriages.

Background: P19 cells are mouse embryonic carcinoma cells which contain pluripotent ability, like stem cells, to differentiate into different cell lines. There are several properties for this cell line that make it a valuable cell model for study of developmental stages. Methods: At the first step, PTS2-EGFP coding sequence which was cloned in pUcD2.hygro vector was used for transfection in to P19 cells. As the plasmid contained hygromycin (Hygror) resistance gene, stable cells were selected using hygromycin as an antibiotic. Stable transformed cells were characterized by RT-PCR and immunostaining analyses. Findings: RT-PCR results indicated EGFP was expressed in these cells. Moreover immunocytochemical analysis of the cells confirmed the preserved pluripotency states of transfected cells. Conclusion: As P19 Cells are able to differentiate into several cell lines, using this stable cell line we will able to chase the molecular kinetics of peroxisomes. Key words: Lipofection, P19, Plasmid, RT-PCR, peroxisome.

Objective: Reporter gene transfer to mammalian cells receives a great deal of attention due to its importance for molecular biology, embryology and developmental biology studies. Among DNA transfer technologies to eukaryotic cells, lipofection is known as the most widely used because of its easy handling procedure, low cell mortality and the natural pathway it undertakes.Materials and Methods: In this study we have examined the transfectability of two cell types: CHO and Vero cells via Lipofection in four different treatments, with combination of exposure duration, 3 and 6 hrs, and different plasmid DNA concentration, 0.5 and 1μgs. A fusion protein expression vector, pUcD2. PTS2-EGFP was used to direct the EGFP protein to peroxisomes after expression of related cDNA. An SPSS analysis was preformed after counting the positive cells.Results: optimum gene expression was found when using 1 μg DNA treated for three hrs for CHO cells, and 1 μg DNA treated for six hrs for Vero cells.Conclusion: The result suggests that CHO lipofection efficiency is significantly increased by both the DNA concentration and exposure time increment; however, an increase in exposure time has less significant effect on low DNA concentration conditions. The same results have been observed for Vero cells. Optimum expression was obtained with highest DNA concentration.

Background In vitro simulation of developmental processes is an invaluable tool to shed light on the intrinsic mechanism of developmental biosystems such as central nervous system in mammals. Chick somites have been used to simulate the neural differentiation of human neural progenitor cells. In the present study, we aimed to indicate whether somites have the ability to express required neural differentiation factors at mRNA level. Methods Chick embryos were isolated from the yolk surface of the fertilized eggs and somites were subsequently isolated from embryos under a dissecting microscope. Total RNA of the somites was extracted and RT-PCR carried out with specific primers of cerberus, chordin, FGF8, follistatin and noggin. Results Data showed that five aforementioned factors were co-expressed after 7 days in vitro by somites. Conclusion We concluded that neural induction property of somites appeared by production of required neural differentiation factors including cerberus, chordin, FGF8, follistatin and noggin.

Objective Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the PPAR nuclear receptor superfamily. Although PPARγ acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPARγ in order to address its role in cardiac cell differentiation of mouse embryonic stem cells (mESCs). Materials and Methods In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction (qPCR) was carried out to estimate level of gene expression. Furthermore, the requirement of PPARγ in cardiac differentiation of mESCs, during cardiac progenitor cells (CPCs) formation, was examined by applying the respective agonist and antagonist. Results The obtained data revealed an elevation in the expression level of PPARγ during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPARγ inactivation via treatment with GW9662 (GW) reduced expression of CPC and cardiac markers. Conclusion We conclude that PPARγ modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis.