Anacardic acid reduces acetylation of H4K12 in mouse Oocytes during vitrification

Objective: Over the last years, vitrification has been widely used for oocyte cryopreservation, in animals and humans; however, it frequently causes minor and major epigenetic modifications. The effect of oocyte vitrification on levels of acetylation of histone H4 at lysine 12 [AcH4K12], and histone acetyltransferase [Hat] expression, was previously assessed; however, little is known about the inhibition of Hat expression during oocyte vitrification. This study evaluated the effect of anacardic acid [AA] as a Hat inhibitor on vitrified mouse oocytes

Materials and Methods: In this experimental study, 248 mouse oocytes at metaphase II [MII] stage were divided in three experimental groups namely, fresh control oocytes [which were not affected by vitrification], frozen/thawed oocytes [vitrified] and frozen/thawed oocytes pre-treated with AA [treatment]. Out of 248 oocytes, 173 oocytes were selected and from them, 84 oocytes were vitrified without AA [vitrified group] and 89 oocytes were pretreated with AA, and then vitrified [treatment group]. Fresh MII mouse oocytes were used as control group. Hat expression and AcH4K12 levels were assessed by using real-time quantitative polymerase chain reaction [PCR] and immunofluoresce staining, respectively. In addition, survival rate was determined in vitrified and treatment oocytes

Results: Hat expression and AcH4K12 modification significantly increased [4.17 +/- 1.27 [P.0.001] and 97.57 +/- 6.30 [P<0.001], respectively] in oocytes that were vitrified, compared to the fresh oocytes. After treatment with AA, the Hat mRNA expression and subsequently H4K12 acetylation levels were significantly reduced [0.12 +/- 0.03 [P.0.001] and 89.79 +/- 3.20 [P.0.05], respectively] in comparison to the vitrified group. However, the survival rate was not significantly different between the vitrified [90.47%] and treatment [91.01%] groups [P>0.05]

Conclusion: The present study suggests that AA reduces vitrification risks caused by epigenetic modifications, but does not affect the quality of vitrification. In fact, AA as a Hat inhibitor was effective in reducing the acetylation levels of H4K12

Melatonin modifies histone acetylation during in vitro maturation of mouse oocytes

Objective: We evaluated the effect of melatonin, as a potent antioxidant agent, on glutathione [GSH] and reactive oxygen species [ROS] levels, as well as histone H3 lysine 9 [H3K9], and H4 lysine 12 [H4K12] acetylation when added to oocytes culture medium

Materials and Methods: In this experimental study, two in vitro and in vivo groups were used. In the in vitro group, cumulus oocyte complexes [COCs] from the ovaries of B6D2F1 mice were cultured in maturation medium containing two doses of melatonin [10-9 and 10-6 M] and without melatonin [control group treated with dimethyl sulfoxide [DMSO]] for 22-24 hour. The cumulus expansion and nuclear status were monitored by an inverted microscope. Next, COCs were isolated from the oviducts of superovulated mice and studied as the in vivo group. In in vitro and in vivo matured oocytes, GSH and ROS levels were assessed by monochlorobimane [MCB] and 2-7-dichlorodihydrofluorescein diacetate [H2DCFDA] staining, respectively. Changes in histone acetylation were examined by immunofluorescent staining with specific antibodies against acetylated H3K9 and H4K12

Results: The H4K12 acetylation and ROS levels were significantly higher in the oocytes matured in the in vitro group compared to the in vivo group [P<0.05]. Furthermore, glutathione levels in the in vitro group were considerably lower than that of the in vivo group [P<0.05]. Melatonin at the concentration of 10-6 M had the most substantial effect on nuclear maturation and histone acetylation as well as glutathione and ROS levels in the in vitro group [P<0.05]

Conclusion: Exogenous melatonin improves the competence of mouse oocytes during in vitro maturation [IVM]

Granulocyte-colony stimulating factor may improve pregnancy outcome in patients with history of unexplained recurrent implantation failure: an RCT

Background: Family of colony-stimulating factors [CSF] have an essential role on early cross talk between embryo and uterine endometrium

Objective: The aim of this study was to evaluate the effects of the single dose of Granulocyte-CSF [G-CSF] injection on clinical outcome of assisted reproductive technology cycle in patients with repeated implantation failures

Materials and Methods: This randomized control trial study was performed on 52 infertile women who referred to the clinic with the history of more than three previous In vitro fertilization/Intracytoplasmic sperm injection-embryo transfer failures. All patients were stimulated with standard long protocol. All embryos were transferred on day five in blastocyst stage in both groups. The treated group received 300 Mug [0.5 ml] recombinant human G-CSF subcutaneously which was injected 30 min before blastocyst embryo transfer

Results: There was not statistically significant differences in abortion rate in G-CSF and control group [p=0.09]. G-CSF treated group showed higher clinical pregnancy rate in comparison with control group [56.2% vs. 40.0%] but it was not statistically significant [p=0.09]. Although live birth rate in G-CSF group was higher than control group [53.1% vs. 35.0%] but there wasn't statistically significant difference in the overall live birth rate between the two groups [p=0.10]. G-CSF group had a twin pregnancies while in control group there was no twin pregnancy

Conclusion: Our result demonstrates the possibility that pregnancy outcome is better in women with repeated unexplained In vitro fertilization failure who are treated with G-CSF

Adipose derived stem cells exert immunomodulatory effects on natural killer cells in breast cancer

Objective: Adipose derived stem cells [ASCs], as one of the important stromal cells in the tumor microenvironment, are determined with immunomodulatory effects. The principle aim of this study was to evaluate the immunosuppressive effects of ASCs on natural killer [NK] cells

Materials and Methods: In this experimental study, we assessed the expressions of indolamine 2, 3-dioxygenase [IDO1], IDO2 and human leukocyte antigen-G5 [HLA-G5] in ASCs isolated from breast cancer patients with different stages as well as normal individuals, using quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR]. Immunomodulatory effects of ASCs on the expression of CD16, CD56, CD69, NKG2D, NKp30, NKG2A and NKp44 was also assessed in peripheral blood lymphocytes [PBLs] by flow-cytometry

Results: Our result showed that IDO1, IDO2 and HLA-G5 had higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals [P>0.05]. mRNA expression of these molecules were higher in ASCs isolated from breast cancer patients with stage III tumors than those with stage II. The indirect culture of ASCs isolated from breast cancer patients and normal individuals with activated PBLs significantly reduced NKG2D+ and CD69+ NK cells [P<0.05]

Conclusion: Results of the present study suggest more evidences for the immunosuppression of ASCs on NK cells, providing conditions in favor of tumor immune evasion

Multiple sclerosis gene therapy using recombinant viral vectors: overexpression of IL-4, IL-10 and leukemia inhibitory factor in wharton's jelly stem cells in the EAE mice model

Objective: Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 [IL-4], IL-10 and leukemia inhibitory factor [LIF] in Wharton's jelly stem cells [WJSCs] in the experimental autoimmune encephalomyelitis [EAE] mice model

Materials and Methods: In this experimental study, a DNA construction containing IL- 4, IL-10 and LIF was assembled to make a polycistronic vector [as the transfer vector]. Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 [HEK-293T] cells with helper plasmids which produced recombinant lentiviral viruses [rLV]. WJSCs were transduced with rLV to make recombinant WJSC [rWJSC]. In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction [qPCR], enzyme-linked immunosorbent assay [ELISA] and western blot [WB] analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2×10[5] rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions

Results: Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with >90% efficiency. Recombinant viruses were produced and results of titration showed 2-3×10[7] infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group

Conclusion: These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis [MS]. In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy

Comparison of the expression of hepatic genes by human Wharton's jelly mesenchymal stem cells cultured in 2D and 3D collagen culture systems

Background: Human Wharton's jelly mesenchymal stem cells [HWJMSCs] express liver-specific markers such as albumin, alpha-fetoprotein, cytokeratin-19, cytokeratin-18, and glucose-6-phosphatase. Therefore, they can be considered as a good source for cell replacement therapy for liver diseases. This study aimed to evaluate the effects of various culture systems on the hepatocyte-specific gene expression pattern of naive HWJMSCs

Methods: HWJMSCs were characterized as MSCs by detecting the surface CD markers and capability to differentiate toward osteoblast and adipocyte. HWJMSCs were cultured in 2D collagen films and 3D collagen scaffolds for 21 days and were compared to control cultures. Real time RT-PCR was used to evaluate the expression of liver-specific genes

Results: The HWJMSCs which were grown on non-coated culture plates expressed cytokeratin-18 and -19, alpha-fetoprotein, albumin, glucose-6-phosphatase, and claudin. The expression of the hepatic nuclear factor 4 [HNF4] was very low. The cells showed a significant increase in caludin expression when they cultured in 3D collagen scaffolds compared to the conventional monolayer culture and 2D collagen scaffold

Conclusion: Various culture systems did not influence on hepatocyte specific marker expression by HWJMSCs, except for claudin. The expression of claudin showed that 3D collagen scaffold provided the extracellular matrix for induction of the cells to interconnect with each other

DNA methylation and histone acetylation patterns in cultured bovine adipose tissue-derived stem cells [BADSCs]

Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer [SCNT]. We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells [BADSCs] would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases [DNMT1, DNMT3a, DNMT3b] and histone deacetyltransferses [HDAC1, HDAC2, HDAC3] in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression [OCT4] and acetylation of H3K9 [H3K9ac] in BADSCs cultures and different passages in vitro. In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction [q-PCR], and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 [P3], 5 [P5] and 7 [P7]. The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages

relationship between intestinal parasites and some immune-mediated intestinal conditions

Over the last decades, the incidence of infestation by minor parasites has decreased in developed countries. Infectious agents can also suppress autoimmune and allergic disorders. Some investigations show that various protozoa and helminthes are connected with the main immune-mediated intestinal conditions including celiac disease [CD], inflammatory bowel diseases [IBD] and irritable bowel syndrome [IBS]. Celiac disease is a digestive and autoimmune disorder that can damage the small intestine and characterized by a multitude gastrointestinal [GI] and extra GI symptoms. IBD [including ulcerative colitis and Crohn's disease] is a group of inflammatory conditions of the small intestine and colon. The etiology of IBD is unknown, but it may be related to instability in the intestinal microflora that leading to an immoderate inflammatory response to commensal microbiota. Irritable bowel syndrome [IBS] is a common, long-term condition of the digestive system. Bloating, diarrhoea and/or constipation are nonspecific symptoms of IBS. Various studies have shown that some intestinal parasites can effect on immune system of infected hosts and in some cases, they are able to modify and change the host's immune responses, particularly in autoimmune disorders like celiac disease and IBD. The main objective of this review is to investigate the relationship between intestinal parasites and different inflammatory bowel disorders

effect of melatonin on maturation, glutathione level and expression of HMGB1 gene in brilliant cresyl blue [BCB] stained immature oocyte

Nutrients and antioxidants in the medium of immature oocyte have a profound effect on maturation, fertilization and development of resulting embryos. In this study the effects of melatonin as an antioxidant agent on maturation, glutathione level and expression of high mobility group box-1 [HMGB1] gene were evaluated in immature oocytes of mice stained with brilliant cresyl blue [BCB]. In this experimental study, immature oocytes were harvested from ovaries of Naval Medical Research Institute [NMRI] mice. Oocytes were stained with 26 Micro M BCB for 90 minutes and transferred to in vitro maturation medium containing varying doses of melatonin [10[-12], 10[-9], 10[-6], 10[-3] M] and without melatonin, for 22-24 hours. Maturation was monitored using an inverted microscope. Glutathione was assessed by monochlorobimane [MCB] staining and HMGB1 expression in mature oocyte was analyzed using real-time polymerase chain reaction [PCR]. Melatonin in the concentration of 10[-6] M had the most effect on maturation and HMGB1 expression of BCB+ oocytes [p<0.05]. Meanwhile melatonin had no effects on glutathione levels. Additionally in immature BCB- oocytes, compared to the control group, melatonin did not affect cytoplasm maturation [p>0.05]. In vitro treatment with melatonin increases the maturation and HMGB1 expression in BCB+ immature oocytes and has no significant effect on glutathione levels

Histone modification of embryonic stem cells produced by somatic cell nuclear transfer and fertilized blastocysts

Nuclear transfer-embryonic stem cells [NT-ESCs] are genetically identical to the donor's cells; provide a renewable source of tissue for replacement, and therefore, decrease the risk of immune rejection. Trichostatin A [TSA] as a histone deacetylase inhibitor [HDACi] plays an important role in the reorganization of the genome and epigenetic changes. In this study, we examined whether TSA treatment after somatic cell nuclear transfer [SCNT] can improve the developmental rate of embryos and establishment rate of NT-ESCs line, as well as whether TSA treatment can improve histone modification in NT-ESCs lines. In this experimental study, mature oocytes were recovered from BDF1 [C57BL/6xDBA/2] mice and enucleated by micromanipulator. Cumulus cells were injected into enucleated oocytes as donor. Reconstructed embryos were activated in the presence or absence of TSA and cultured for 5 days. Blastocysts were transferred on inactive mouse embryonic fibroblasts [MEF], so ESCs lines were established. ESCs markers were evaluated by reverse transcription-polymerase chain reaction [RT-PCR]. Histone modifications were analyzed by enzyme linked immunosorbent assay [ELISA]. Result of this study showed that TSA treatment after SCNT can improve developmental rate of embryos [21.12 +/- 3.56 vs.8.08 +/- 7.92], as well as establishment rate of NT-ESCs line [25 vs.12.5]. We established 6 NT-ESCs in two experimental groups, and three embryonic stem cells [ESCs] lines as control group. TSA treatment has no effect in H3K4 acetylation and H3K9 tri-methylation in ESCs. TSA plays a key role in the developmental rate of embryos, establishment rate of ESC lines after SCNT, and regulation of histone modification in NT-ESCs, in a manner similar to that of ESCs established from normal blastocysts

Transcript analysis of heat shock protein 72 in vitrified 2-cell mouse embryos and subsequent in vitro development

The aim of the study was to compare the effects of two different concentrations of cryoprotectants by cryotopvitrification on survival, developmental capacity and Heat shock protein 72 [Hsp72] expression of two-cell mouse embryos. In this experimental study, transcript analysis of Hsp72 gene was performed on non-vitrified and vitrified 2-cell mouse embryos via a nested quantitative polymerase chain reaction [nqPCR] subsequent to normalization with Hprt1 as the reference gene. The different cryoprotectant combinations were 15% [vit[1]:7.5% of each ethylene glycol [EG] and dimethyl sulfoxide [DMSO], 30% [vit[2]:15% EG + 15% DMSO] and control group with no cryoprotectants. Vitrified and fresh 2-cell embryos were cultured to obtain cleavage and blastocyst formation rates. The results were analyzed via one-way analysis of variance and the mean values were compared with least significant difference [LSD] [p< 0.05]. The relative expression of Hsp72 in vit2 [30% v/v] was significantly higher than vit[1] [15% v/v]. Survival rates were the same for both vitrification treatments and significantly lower than the control group. Cleavage and blastocyst rates in vit[1] were significantly higher than vit[2] while those in two vitrified groups were significantly lower than the control group. Our developmental data demonstrated that vit[1] treatment [7.5% EG and 7.5% DMSO] was more efficient than vit[2] [15% EG and 15% DMSO] in mouse embryos. The cryotopvitrification with two concentrations of cryoprotectants caused the relative changes of Hsp72 transcript level, but the stability of the gene in vit[1] was significantly higher than vit[2] and closer to the fresh 2-cell embryos

Effect of follicular fluid and platelet-activating factor on lactate dehydrogenase C expression in human asthenozoospermic samples

Background: Application of follicular fluid [FF] and plateletactivating factor [PAF] in artificial insemination improves sperm motility. Lactate dehydrogenase C [LDH-C] is a key enzyme for sperm motility. In this study, the effects of FF and PAF on the sperm motility index and LDH-C expression were investigated. Moreover, LDH-C expression was compared between asthenozoospermic and normozoospermic samples

Methods: The expression of LDH-C was examined by quantitative real-time polymerase chain reaction [q-RT PCR] and western blotting after it was treated with optimized concentrations of FF and PAF in twenty asthenozoospermic samples. Also, LDH-C expression was evaluated in five normozoospermic samples

Results: Samples with 75% FF and 100 nM of PAF had an increase in their percentages of progressive and slowly motile sperms and a decrease in their percentages of non-progressive and non-motile sperms. Moreover, LDH-C mRNA transcripts were not changed following PAF and FF treatment, and LDH-C protein was detected in highly progressive motile specimens treated with FF in the asthenozoospermic samples. Furthermore, LDH-C expression was more detectable in the normal sperms

Conclusion: Our results indicated that PAF had more beneficial effects than FF on sperm motility in the asthenozoospermic samples [P=0.0001], although the LDH-C expressions of the sperms were not changed significantly in both groups. We found no association between LDH-C expression and sperm motility after FF and PAF actions. This finding, however, requires further investigation. The fact that LDH-C protein was detected in the normozoospermic, but not asthenozoospermic, samples could be cited as a reason for the infertility in these patients

IL-23/IL-27 ratio in peripheral blood of patients with breast cancer

Background: Interleukin [IL]-23 and IL-27 are two IL-12-related cytokines which their function may dramatically influence the inflammatory response to tumor development. IL-12 and IL-27 seem to have antagonistic roles with IL-23 in tumor site. In this study, IL-23 and IL-27 mRNA expressions were analyzed in peripheral blood of patients with breast cancer and healthy volunteers using quantitative real-time PCR

Methods: Peripheral blood samples were collected from 50 women with breast cancer and 50 healthy ones. The total RNA was extracted from peripheral blood after lysis with ammonium chloride and TRizol reagent and the cDNA was synthesized. The expression of IL-23 and IL-27 gene transcripts was determined with real-time polymerase chain reaction [qRT-PCR] using Syber Green PCR Master Mix

Results: It is found that IL-23 and IL-27 transcripts had significantly higher expression in peripheral blood of patients compared with the healthy controls. The ratio of IL-23 transcript expression to IL-27 was 3.4 fold lower in the studied patients compared with the normal individuals

Conclusion: It is concluded that the over expression of IL-23 and IL-27 gene transcript in peripheral blood of breast cancer patients may be an immune response against tumor development and the inflammatory response plays a critical role in tumor development via up regulating the corresponding cytokines. However, the IL-23/IL-27 ratio may play an important role in cytokine-based immunotherapy against cancer. Further research should be carried out to assess these cytokines in a larger sample size

Echinococcus granulosus genotypes in Iran

Hydatidosis, caused by Echinococcus granulosus is one of the most important zoonotic diseases, throughout most parts of the world. Hydatidosis is endemic in Iran and responsible for approximately 1% of admission to surgical wards. There are extensive genetic variations within E. granulosus and 10 different genotypes [G1-G10] within this parasite have been reported. Identification of strains is important for improvement of control and prevention of the disease. No new review article presented the situation of Echinococcus granulosus genotypes in Iran in the recent years; therefore in this paper we reviewed the different studies regarding Echinococcus granulosus genotypes in Iran.

Molecular identification of giardia lamblia; is there any correlation between diarrhea and genotyping in Iranian population?

The aim of this study is to investigate the molecular identification of Giardia lamblia in patients with diarrhea. Giardiasis caused by Giardia lamblia is a common intestinal disease. Although this parasitic infection found in mammals including human, pets and livestock, but few species within the genus Giardia can infects humans. G. lamblia have seven complex genotypes termed [A-H]. Genotype A and B the main causes of human infections. Sixty seven microscopically positive G. Lamblia samples were collected from clinical laboratories in Isfahan province between June 2013 and February 2014. Extraction of genomic DNA was performed for 65 concentrated cysts and 2 cultured trophozoites. Partial sequences of tpi including 148-bp and 81-bp were amplified for detection the genotypes A and B using RFLP- PCR protocol respectively. PCR results showed that out of 67 patients with giardiasis infection, genotype A [148 bp] was detected in 40 isolates [59.70%] compared to genotype B [81 bp] isolated was detected in 25 isolates [37.31%]. Also two isolates [2.98%] had mix infection infected with genotype A and B. By comparing the frequency of genotype A [81.8%] and genotype B [13.6%], we found that genotype A is six times higher prevalence than genotype B in patients with diarrhea. We suggest that using sensitive techniques and larger sample for detection of G. lamblia genotypes and their subtypes would be necessary for investigation the immune system respond and correlation with diarrhea in the future studies in Iran

New single nucleotide variation in the promotor region of androgen receptor [AR] gene in hypospadic patients

Hypospadias is one of the most common congenital abnormalities in the male which is characterized by altered development of urethra, foreskin and ventral surface of the penis. Androgen receptor gene plays a critical role in the development of the male genital system by mediating the androgens effects. In present study, we looked for new variations in androgen receptor promotor and screened its exon 1 for five single nucleotide polymorphisms [SNP] in healthy and hypospadias Iranian men. In our study, at first DNA was extracted from patients [n=100] and controls [n=100] blood samples. Desired fragments of promoter and exon 1 were amplified using polymerase chain reaction. The promoter region was sequenced for the new variation and exone 1 screened for five SNPs [rs139767835, rs78686797, rs62636528, rs62636529, rs145326748] using restriction fragment length polymorphism technique. The results showed a new single nucleotide variation [CT] at -480 of two patients' promoter region [2%]. None of the mentioned SNPs were detected in patients and controls groups [0%].This finding indicates that new single nucleotide polymorphism in androgen receptor promoter may have role in etiology of hypospadias and development of this anomaly

Lentiviral mediating genetic engineered mesenchymal stem cells for releasing IL-27 as a gene therapy approach for autoimmune diseases

Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 [Th17] and interleukin [IL]-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells [MSCs] that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation and autoimmune diseases. In this experimental study, we isolated adipose-derived MSCs [AD-MSCs] from lipoaspirate and subsequently characterized them by differentiation. Two subunits of IL-27 [p28 and EBI3] were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 [Epstein-Barr virus induced gene 3] were determined by real time polymerase chain reaction [PCR]. MSCs were transduced by a pCDH-CMV-p28-IRESEBI3- EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression. Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28 and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27. The results showed transduction of functional IL-27 to AD-MSCs by means of a lentiviral vector. The lentiviral vector did not impact MSC characteristics

[Comparison of intracytoplasmic sperm injection outcome of oligoasthenoteratozoospermic and azoospermic men]

Background: With introduction of intracytoplasmic sperm injection with testicular sperm extraction or precutaneouse epididymal sperm aspiration, effective treatment was provided for azoospermic men. The aim of present study was to compare clinical outcome following intracytoplasmic sperm injection using extracted testicular/epididymal sperm or ejaculated severe oligoasthenoteratozoospermic sperm

Methods: After retrospective evaluation of more than four hundred medical records of patients undergoing intracytoplasmic sperm injection Mehr medical institute [between 2011-2012], 45 cycles with severe eligoasthenoteratozoospermia and 34 cycles with azoospermia were included. Patients were treated with gonadotropin releasing hormone agonist. The clinical characteristics and intracytoplasmic sperm injection outcome such as the rate of fertilization, implantation and clinical pregnancy were compared between the two groups. Results were presented as mean +/- standard deviation and number [percent]

Differences between variables were analyzed using student's t test and the chi-square test was used to examine differences between categorical variables. P value less than 0.05 were considered as statistically significant

Results: Mean of female age [29+/-4.9 vs. 30.2+/-5.8], body mass index [26.9+/-5.3 vs. 26.9+/-3.8], estradiol level on human chorionic gonadotropin administration day [1375.6+/-843.9 vs. 1181.8+/-673.1], total number of retrieved oocytes [9.7+/-5.3 vs. 9.2+/-5.9] and metaphase II oocytes [7.7+/-5.1 vs. 7.5+/-5.4] were similar between the two groups. Of 436 and 313 retrieved oocytes, respectively 232 and 163 oocytes were fertilized in oligoasthenoteratozoospermic and azoospermic groups [53.2% vs. 52.1%, P=0.214]. There were not statistical differences between groups in number of transferred top quality embryos [1.5+/-1.2 vs. 1+/-1.2, P=0.09], implantation rate [22.7% vs. 16.9%, P=0.238] and clinical pregnancy rate [21 [47.7%] vs. 11 [35.4%], P=0.199]

Conclusion: Intracytoplasmic sperm injection with precutaneouse epididymal sperm aspiration and testicular sperm extraction are effective methods to treat azoospermic men and its clinical outcome were comparable to ejaculated sever oligoasthenoteratozoospermic cycles. It can be concluded that the influence of sperm quality and origin on intracytoplasmic sperm injection outcome are the same

Genetic Diversity of Echinococcus granulosus in Center of Iran

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.

Non-viral transfection methods optimized for gene delivery to a lung cancer cell line

Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. In the current study, calcium phosphate [CaP], DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein [GFP]. Transgene expression was detected by fluorescent microscopy and flow cytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 [SDF-1] gene and measured its expression by real-time PCR. Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% [CaP], 8.2% [DEAE-dextran], 4.9% [superfect], 34.1% [electroporation], and 40.1% [lipofection]. Lipofection had the highest intense SDF-1 expression of the analyzed methods. This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods