Pathobiology Research
Year:2016 | Volume:19 | Issue:2|Papers Count:7

Currently, fertility and survival of future generations is emphasized in societies. Hence, male infertility is a major concern for community health worldwide. In Europe, 14% of young couples have infertility problems. Iranian couples have an infertility rate above World average, at approximately 20.2%. Hormonal factors, genetics, and psychological problems cause 40%-50% of infertility in men. Since there are increasing numbers of cancer patients in industrialized societies and anti-cancer treatments highly eliminate germ cells, hence, cancer treatments reduce male fertility. Recognition of primordial germ cells, to understand their migration process and understanding effective factors in their differentiation can open new avenues for primary studies that follow production of germ cells from other cell sources such as mesenchymal stem cells (MSCs). Therefore, finding a way to distinguish germ cells from MSCs, as well as their preservation and proliferation in a culture system can provide the base for spermatogenesis in an in vitro culture. Isolation and differentiation of germ cells from different cell sources such as umbilical cord using morphogens (bone morphogenetic protein and retinoic acid) is an efficient method for infertility research. We investigate some of the effective factors in differentiation of umbilical cord MSCs to germ cells.

Objective: Squamous cell carcinoma comprises approximately 94% of all oral cancers.One reason for this cancer is human papilloma virus (HPV) and its different genotypes.Determining the most common genotypes can assist with control and prevention of squamous cell carcinoma.Methods: We obtained 70 paraffin blocks from the Oncology Department at Imam Khomeini Hospital, Tehran, Iran. All samples included the histopathological report of dysplastic lesions. Samples were deparaffinated. Four primers were designed for PCR of samples. Positive samples were sequenced.Results: We have observed that 8 samples were HPV posetive, from which there were 3 HPV6 and 5 HPV16. HPV6 is one source for genital warts that can be spread by skin contact or oral-sexual behavior. There were 3 positive samples found in women, whereas the remainder were from men (2% more in men). People between the ages of 30 to 45 have increased sensitivity for HPV compared to the other group. People up to 60 years of age are also sensitive. All samples have been collected from different cities in Iran, however most of the positive samples were found in Tehran and Islam Shahr.Conclusion: These data confirmed that HPV infection with high risk types (6, 16) could be one of the risk factors for oral cancer which could be spread by genital warts.

Objective: Aging can affect adaption of the heart tissue’s apoptotic system to aerobic exercise and induction of doxorubicin. Therefore, this study aims to evaluate the effect of pretreatment of aerobic training on doxorubicin-induced left ventricular apoptosis gene expression in a model of aging rats.Methods: We randomly assigned 42 adult Wistar male rats to 6 groups (n=7 per group): control of young, control of aging, aging+saline, aging+doxorubicin, aging+aerobic exercise+saline, and aging+aerobic exercise+doxorubicin. Aging was induced by an intraperitoneal (i.p.) injection of D- galactose (100 mg/kg). The training protocol included treadmill running with a gradual increase from 25 min/day to 54 min/day at a velocity of 15 m/min to 20 m/min, 5 days/week for 6 weeks. During the two ultimate training weeks, the animals underwent a 15-day i.p. doxorubicin regimen that consisted of 1 mg/kg of doxorubicin per day. The rats were sacrificed 48 hours after the last training and injection session. A portion of the left ventricle was analyzed by real-time PCR for Bax and Bcl-2 gene expressions.Results: ANOVA indicated that doxorubicin injection induced a significant increase in expression of Bax and Bax/Bcl-2 ratio and an insignificant decrease in Bcl-2 gene expression. On the other hand, aerobic training before and during the induction of doxorubicin prevented an increase in the Bax/Bcl-2 ratio and the doxorubicin-induced decrease inBcl-2 gene expression.Conclusion: Due to the significant decrease in Bax/Bcl-2 ratio in the hearts of the trained rats that underwent doxorubicin treatment, we conclude that aerobic exercise training prior to and during treatment with doxorubicin as a non-pharmacological strategy, probably protects cardiomyocytes from doxorubicin-induced apoptosis.

Objective: Vaccination is the most effective tool to prevent influenza virus morbidity and mortality. Despite surface antigen mutability, the influenza virus inner proteins are remarkably conserved among various strains. The high similarity of NP protein sequences among various strains of influenza type A from one side, and the potency of IL-18 molecular adjuvant in shifting immune response toward Th1 from the other side, has persuaded us to evaluate the possibility to clone and express the influenza type A NP gene in a bicistronic vector that harbored the miceIL-18 gene. We sought to assess their immunogenic activities in a susceptible mouse model.Methods: Initially, we extracted genomic RNA from the influenza virus PR8. After cDNA synthesis, the target gene encoding NP was amplified by PCR after which the NP gene was cloned in the pJET1.2/blunt TA vector. The accuracy of cloned gene was confirmed by PCR, enzymatic digestion and sequencing. The pJET1.2-NP plasmid and pIRES-Igk/mIL18/Fc plasmids were simultaneously digested byBstXI/NotI enzymes. We inserted the digested NP fragment into the pIRES-Igk/mIL18/Fc plasmid using T4 ligase.Transformation into DH5α and colony selection was done. Gene cloning was confirmed by PCR, enzymatic double-digestion and sequencing. Eventually, by transfection of the constructed mIL-18-pIRES2-NP plasmid into BHK-21 and RAW264.7 cell lines, we assessed the expressions ofNP and IL-18 by indirect immunofluorescence and ELISA, respectively.Results: Electrophoresis of the PCR product, enzymatic digestion, and sequencing showed that the influenzaNP gene successfully cloned into pIRES-Igk/mIL18/Fc to generate the mIL-18-pIRES2-NP plasmid. Indirect immunofluorescence and ELISA indicated the successful expressions ofNP and mIL-18 from the produced plasmid in eukaryotic cell lines.Conclusion: The results of the present study confirm expressions of NP and IL-18 genes from mIL-18-pIRES2-NP. This is a proper candidate for influenza A gene vaccine in future investigations.

Objective: RNA interference (RNAi) is considered a potential approach to knock down target genes and their functional assessment. Lentiviral vectors serve as an efficient tool to transduce foreign genes in a wide variety of mammalian cells. Fibronectin type I domain-containing 5 (Fndc5) is a glycosylated membrane protein whose transcript levels increase during neural and cardiac differentiation of mouse embryonic stem cells (mESCs). Here, we report the efficacy of Fndc5 gene silencing in mESCs using lentiviral vectors that express shRNA.Methods: Two distinct shRNA sequences that targeted Fndc5 coding sequence (CDS) and one scramble shRNA sequence as a negative control were designed and commercially synthesized. Synthetic shRNA oligonucleotides were cloned into a lentiviral inducible vector after annealing downstream of the tetracycline inducible H1 promoter. The recombinant lentiviral vector was packaged in HEK293T cells, then mESCs were transduced by lentiviral particles. Expression of shRNA in transduced cell lines was induced by doxycycline treatment for 48 h.Results: Evaluation of transcript levels of Fndc5 by real-time PCR showed a significant decrease in transduced cells by a mixture of two shRNAs.Conclusion: Taken together, lentiviral-mediated RNAi that targeted the Fndc5 gene could be considered an efficient tool to silence gene expression in the transduced cell line to study the function of Fndc5.

Objective: Free-living amoebae, including Acanthamoeba constitute a large group of amoebae that live in fresh water, salty and bitter, moist soil, carious plants, and some on the stool. They are medically important agents. It is a medically important agent. The aim of the present study is to determine the effects of aqueous and alcoholic extracts of Artemisia annuaon trophozoites and cysts of Acanthamoeba in vitro.Methods: In this experimental research, after genotyping the clinical isolate, we prepared the aqueous and alcoholic extracts of Artemisia annua. Various concentrations (1.25, 2.5, 5, and 10 mg/ml) of the aqueous and alcoholic extracts of this plant as well as artemisinin were tested at three different times (24, 48 and 72 h) on trophozoites and cysts of Acanthamoebain vitro. We evaluated viability of the parasite by trypan blue, MTT, and flow cytometry analyses.Results: We observed the anti-acanthamoeba activity of different concentrations of the Artemisia extract. In the presence of 10 mg/ml alcoholic extract in medium culture after 72 h, we observed that 30.51% trophozoites and 91.40% cysts of Acanthamoeba were viable. However, in the presence of 10 mg/ml aqueous extract of Artemisia annua, only 58.25% trophozoites and 81.53% cysts were alive in the in medium culture after 72 h.Conclusion: Aqueous and alcoholic extracts of Artemisia annua had dose- and time dependent anti-acanthamoeba activity.

Objective: Myokines derived from skeletal muscle modulate metabolic, inflammatory, and other processes. Myonectin, a novel myokine, expression and circulating levels are tightly regulated by metabolic state and exercise. This study aims to investigate the effect of 4 weeks of endurance training on gene expression in myonectin muscle activity and plasma insulin resistance in adult male rats.Methods: We divided 16 Wistar rats (mean weight: 230 ± 15 g; age: 8 weeks) into two groups: (1) endurance training and (2) control. Animals in the exercise group received 4 weeks of endurance training (5 sessions per week) that included running on a treadmill for 45 minutes. The control group did not undertake any exercise. We measured soleus muscle homogenates and myonectin gene expression by real-time PCR. The enzyme linked immunosorbent assay (ELISA) method measured insulin resistance. The data were analyzed by the independent t-test. Differences were considered significant at P<0.05.Results: The endurance training groups had a significant increase in myonectin gene expression compared with the control group (P=0.048). Insulin resistance had no significant decrease comparison with the control group (P=0.500).Conclusion: The results of the present study suggest that endurance training increased the body’s metabolism through the secretion of myonectin.