Cell Journal (Yakhteh)
Year:2018 | Volume:19 | Issue:4|Papers Count:19

Objective: Reactive oxygen species (ROS) is an apoptosis inducer in pancreatic β-cells that stimulates p53/p65 mediated microRNA (miR) -145 expression.Punica granatum L. (pomegranate) is an antioxidant fruit that attenuates ROS generation. This study examines the effects of pomegranate fruit aqueous extract (PGE) on the levels of ROS, p53, p65, miR-145, and its target insulin receptor substrate 1 (irs-1) mRNA in Alloxan-diabetic male Wistar rats.Materials and Methods: In this experimental study, diabetic rats received different doses of PGE. The effects of the PGE polyphenols were examined through a long-term PGE treatment period model, followed by an evaluation of the plasma and tissue contents of free fatty acids (FFAs), triglycerides (TG), and glycogen compared with diabetic controls (DC) and normal controls (NC). We used real-time polymerase chain reaction (PCR) to investigate the modulation of p53, p65, miR-145, and irs-1 expression levels.Results: There was a noticeable reduction in fasting blood glucose (FBG) and ROS generation compared to DC.We observed marked decreases inp53, p65, miR-145 expression levels followed by an elevated level of irs-1, which contributed to improvement in insulin sensitivity.Conclusion: PGE administration downregulated miR-145 levels in Alloxan-diabetic Wistar rats by suppression of ROS-mediatedp53 and p65 overexpression.

Objective: We examined the protective effects of ethanolic extract of Lippia citriodora (L. citriodora) on rats subjected to chronic constriction injury (CCI) of sciatic nerve and possible mechanisms of actions.Materials and Methods: In this experimental study, the extract was administered 50, 100 and 200 mg/kg, Intraperitoneally (I.P) from the surgery time for 14 consecutive days. The changes in the spinal cord levels of apoptotic factors, microglia and astroglia markers during the time course of study were assessed by western blotting on days 3, 7 and 14 post-CCI.Results: CCI rats developed neuropathy evident from a marked mechanical allodynia, cold allodynia and thermal hyperalgesia on days 3, 5, 7, 10 and 14 post-CCI. A significant increase in the levels of Iba (a marker of microglia activation) and Bax (a proapoptotic factor) was observed three days after nerve injury. The levels of Iba remained high on day 7. In contrast, there was no difference in glial fibrillary acidic protein (GFAP) contents between sham and CCI animals. Treatment with the extract significantly attenuated behavioral changes associated with neuropathy. Bax/Bcl-2 and Iba1 were decreased in CCI animals treated with the extract.Conclusion: The results support the evidence that microglial activation and apoptosis are correlated with pain behaviors. It is suggested that anti-allodynic and anti-hyperalgesic effects, elicited byL. citriodora, might have some degrees of association with the inhibition of microglia activation and apoptotic pathways.

Objective: Cerebrospinal fluid (CSF) plays an important role in cortical development during the fetal stages. Embryonic CSF (E-CSF) consists of numerous neurotrophic and growth factors that regulate neurogenesis, differentiation, and proliferation. Mesenchymal stem cells (MSCs) are multi-potential stem cells that can differentiate into mesenchymal and non-mesenchymal cells, including neural cells. This study evaluates the prenatal and postnatal effects of CSF on proliferation and neural differentiation of bone marrow MSCs (BM-MSCs) at gestational ages E19, E20, and the first day after birth (P1).Materials and Methods: In this experimental study, we confirmed the mesenchymal nature of BM-MSCs according to their adherence properties and surface markers (CD44, CD73 and CD45). The multi-potential characteristics of BMMSCs were verified by assessments of the osteogenic and adipogenic potentials of these cells. Under appropriatein vitroconditions, the BM-MSCs cultures were incubated with and without additional pre- and postnatal CSF. The MTT assay was used to quantify cellular proliferation and viability. Immunocytochemistry was used to study the expression of MAP-2 and β-III tubulin in the BM-MSCs. We used ImageJ software to measure the length of the neurites in the cultured cells.Results: BM-MSCs differentiated into neuronal cell types when exposed to basic fibroblast growth factor (b-FGF).Viability and proliferation of the BM-MSCs conditioned with E19, E20, and P1 CSF increased compared to the control group. We observed significantly elevated neural differentiation of the BM-MSCS cultured in the CSF-supplemented medium from E19 compared to cultures conditioned with E20 and P1 CSF group.Conclusion: The results have confirmed that E19, E20, and P1 CSF could induce proliferation and differentiation of BM-MSCs though they are age dependent factors. The presented data support a significant, conductive role of CSF components in neuronal survival, proliferation, and differentiation.

Objective: Motor neuron differentiation from human embryonic stem cells (hESCs) is a goal of regenerative medicine to provide cell therapy as treatments for diseases that damage motor neurons. Most protocols lack adequate efficiency in generating functional motor neurons. However, small molecules present a new approach to overcome this challenge.The aim of this research is to replace morphogen factors with a cocktail of efficient, affordable small molecules for effective, low cost motor neuron differentiation.Materials and Methods: In this experimental study, hESCs were differentiated into motor neuron by the application of a small molecule cocktail that consisted of dorsomorphin, A8301, and XAV939. During the differentiation protocol, we selected five stages and assessed expressions of neural markers by real-time polymerase chain reaction (PCR), immunofluorescence staining, and flow cytometry. Motor neuron ion currents were determined by whole cell patch clamp recording.Results: Immunofluorescence staining and flow cytometry analysis of hESC-derived neural ectoderm (NE) indicated that they were positive for NESTIN (92.68%), PAX6 (64.40%), and SOX1 (82.11%) in a chemically defined adherent culture. The replated (hESC) -derived NE differentiated cells were positive for TUJ1, MAP2, HB9 and ISL1. We evaluated the gene expression levels with real-time reverse transcriptase-PCR at different stages of the differentiation protocol.Voltage gated channel currents of differentiated cells were examined by the whole-cell patch clamp technique. The hESC-derived motor neurons showed voltage gated delay rectifier K+, Na+and Ca2+inward currents.Conclusion: Our results indicated that hESC-derived neurons expressed the specific motor neuron markers specially HB9 and ISL1 but voltage clamp recording showed small ionic currents therefore it seems that voltage gated channel population were inadequate for firing action potentials.

Objective: Cell proliferation is known to be controlled by many networks of regulatory proteins. These multiple and complicated mechanisms of control are still being investigated. The aim of the present study is to determine the different properties of adult rat brain thermostable protein complex (TPC) which affect cell proliferation.Materials and Methods: This experimental study used brain, kidney and liver tissue from adult (150-170 g) and adolescent (7, 10, 21, 28 days) white rats, adult pigeons and mice. Brain TPC was isolated by alcohol extraction, and primary antibodies Ki67 and GAD65/67 were used for immunohistochemistry, evaluation of transcriptional activity of the tissues and determination of the mitotic index.Results: The results show that brain TPC from rats reversibly decreases cell proliferation by inhibiting transcription.The evidence suggests that TPC is not species-specific, but expresses tissue specificity with regards to terminally differentiated cells. Rat brain TPC inhibits mitotic activity of the progenitor cells in the dentate gyrus of adolescent rats, and corresponding with this decrease in the mitotic index the number of Ki67 positive cells increases. Simultaneously, the number of GAD65/67-positive cells in the hippocampus decreases by approximately threefold.Conclusion: These results indicate that rat brain TPC causes the reversible suppression of cell proliferation through the inhibition of transcription. Inhibitory effects of rat brain TPC leads to an increase the number of cells in the cell cycle, in tissues of adolescent rats.

Objective: Recent studies have reported dysregulated expression of matrix metalloproteinases (MMPs), especially MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, -2(TIMP-1, TIMP-2), and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in activated macrophages of patients with inflammatory diseases. Therefore, MMP-2, MMP-9, and their regulators may represent a new target for treatment of inflammatory diseases. Probiotics, which are comprised of lactic acid bacteria, have the potential to modulate inflammatory responses. In this experimental study, we investigated the anti-inflammatory effects of cell-free supernatants (CFS) fromLactobacillus acidophilus (L.acidophilus)and L. rhamnosus GG (LGG) in phorbol myristate acetate (PMA) -differentiated THP-1 cells.Materials and Methods: In this experimental study, PMA-differentiated THP-1 cells were treated with CFS from L.acidophilus, LGG and uninoculated bacterial growth media (as a control). The expression of MMP-2, MMP-9, TIMP-1, andTIMP-2 mRNAs were determined using real-time quantitative reverse transcription polymerase chain reaction (RTPCR).The levels of cellular surface expression of CD147 were assessed by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography.Results: Our results showed that CFS from both L. acidophilus and LGG significantly inhibited the gene expression of MMP-9(P=0.0011 and P=0.0005, respectively), increased the expression of TIMP-1 (P<0.0001), decreased the cell surface expression of CD147 (P=0.0307 and P=0.0054, respectively), and inhibited the gelatinolytic activity of MMP-9 (P=0.0003 and P<0.0001, respectively) in PMA-differentiated THP-1 cells. Although, MMP-2 expression and activity and TIMP-2 expression remained unchanged.Conclusion: Our results indicate that CFS from L. acidophilus and LGG possess anti-inflammatory properties and can modulate the inflammatory response.

Objective: Aggregation of the TAU proteins in the form of neurofibrillary tangles (NFTs) in the brain is a common risk factor in tauopathies including Alzheimer’s disease (AD). Several strategies have been implemented to target NFTs, among which chaperones, which facilitate the proper folding of proteins, appear to hold great promise in effectively inhibiting TAU polymerization. The aim of this study was to analyze the impact of the chaperone Artemin on TAU aggregationin vitro.Materials and Methods: In this experimental study, recombinant TAU- or Artemin proteins were expressed in E.coli bacteria, and purified using ion-exchange and affinity chromatography. Sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) was used to run the extracted proteins and check their purity. Heparin was used as an aggregation inducer. The interaction kinetics of TAU aggregation and disassembly was performed using thioflavin T (ThT) fluorescence analysis and circular dichroism (CD) spectroscopy.Results: Ion-exchange and affinity chromatography yielded highly pure TAU and Artemin proteins for subsequent analyses. In addition, we found that heparin efficiently induced TAU fibrillization 48 hours post-incubation, as evidenced by ThT assay. Importantly, Artemin was observed to effectively block the aggregation of both physiologic- and supraphysiologic TAU concentrations in a dose-dependent manner, as judged by ThT and CD spectroscopy analyses.Conclusion: Our collective results show, for the first time, that the chaperone Artemin could significantly inhibit aggregation of the TAU proteins in a dose-dependent manner, and support Artemin as a potential potent blocker of TAU aggregation in people with AD.

Therapeutic angiogenesis is employed to induce vascular network formation and improve functional recovery in ischemia. The aim of this study is to find an appropriate method to recover local ischemic conditions.Materials and Methods: In this experimental survey, 20 male Wistar rats weighing approximately 200-250 g were randomly divided into four experimental groups respectively: ischemia group in which the femoral artery was transected; phosphate buffer solution group (PBS) in which the femoral artery transected location was immersed with PBS; chitosan (CHIT) group in which the transected location was immersed in a 50 mL CHIT solution; and mast cell transplanted group in which the transected location was immersed with a mixture of 50 mL CHIT and 50 mL PBS that contained 1×106 mast cells.Results: On day 14 after surgery, mean numbers of blood vessels of different sizes in the CHIT/mast cell group significantly increased compared to the other experimental groups (P<0.05).Conclusion: Our data suggest that mast cell reconstitution could offer a new approach for therapeutic angiogenesis in cases of peripheral arterial diseases.

Objective: Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates.Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristicsin vitro.Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages.Materials and Methods: In this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase (ALP) activity, calcium content, and osteogenic gene expression were evaluated in both BMMSCs and BlCs cultures at days 7, 14, and 21.Results: qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs (P<0.05). Alizarin red staining (ARS), calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points.Conclusion: Characteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages.

Objective: Implantation failure is an obstacle in assisted reproduction techniques (ART). Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor (HB-EGF) expression in murine endometrium.Materials and Methods: In this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in GTM medium with or without 10-9 M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal (IP) injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P<0.05 was considered statistically significant.Results: Melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein (P<0.001) in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels (P<0.001). Compared with the control group (2.6 ± 0.5), the average number of implantation sites in the melatonin group (4.6 ± 0.5, P<0.05) and calcitonin group (7 ± 1, P<0.001) significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group (8.6 ± 0.5, P<0.001). Calcitonin significantly enhanced calcitonin receptor mRNA (P<0.001) and protein (P<0.05) in the uteri of naturally pregnant and pseudo-pregnant mice.Conclusion: Melatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudopregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice.

Objective: Vitrification is increasingly used in assisted reproductive technology (ART) laboratories worldwide. In this study the effect of vitrification on the expression and modifications of H3 histones ofIgf2 and Oct4 was investigated in blastocysts cultured from vitrified and non-vitrified two-cell embryos.Materials and Methods: In this experimental study, two-cell embryos were cultured in KSOM medium to reach the blastocyst stage. Expression ofIgf2 and Oct4 and modifications of H3 histones in regulatory regions of both genes were compared within vivo blastocysts, which comprise the control group. To gene expression evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the ChIP assay method were carried out to assess expression and histone modifications of the two genes.Results: The expression level of Igf2 was significantly higher in both experimental groups than the control group. In the regulatory region ofIgf2, H3K9 methylation decreased whereas H3K9 acetylation increased in the experimental group compared with the control group. In contrast, the expression level ofOct4 was significantly lower in experimental groups. TheOct4 gene promoter showed a significant increase in H3K9 methylation and decrease in H3K9 acetylation (P<0.05).Conclusion: According to our results, both vitrification and cultivation conditions may lead to changes in expression level and modification of histones inIgf2 and Oct4. However, these effects were the same in vitrified and non-vitrified groups. Indeed, the embryo is most affected by culture environment andin vitro culture. Therefore, vitrification may be used as a low-risk technique for embryo cryopreservation in ART.

Objective: Re-vitrification of embryos immediately after thawing or after a culture period could be used to preserve the extra embryos after embryo transfer. This study aims to clarify the effect of re-vitrification onBax and Bcl-2 gene expressions of compact and early blastocyst stage embryos.Materials and Methods: This experimental study was performed on mouse embryos. We collected 8-cell stage embryos (n=400) from female mature mice, 60-62 hoursafter injection of human chorionic gonadotropin (hCG). The embryos were divided into 5 groups: fresh (n=80), vitrified at the 8-cell stage (n=80), vitrified at the blastocyst stage (n=80), vitrified at the 8-cell stage, and re-vitrified at the compact (n=80) and early blastocyst stages (n=80). Embryos were vitrified by cryolock. We analyzed the developmental rates of the vitrified-warmed embryos with the chi-square test. Quantitative polymerase chain reaction (qPCR) data were analyzed with SPSS version 16 using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P<0.05 were considered statistically significant.Results: The expanded blastocyst formation rate showed a significant difference in re-vitrified embryos compared with fresh embryos (P<0.05). However, this result was similar between the two re-vitrified groups. Our data showed a significant difference in expression of theBax and Bcl-2 genes between re-vitrified and fresh embryos (P<0.05).Expressions of theBax and Bcl-2 genes showed no significant difference between the two re-vitrified groups.Conclusion: Based on our study, re-vitrification could affect developmental rate and expressions of the Bax and Bcl2 genes.

Objective: Ovarian reserve is defined as the capacity of the ovary to provide fertile oocytes. Diminished ovarian reserve (DOR) is a disorder in which ovaries are prone to go through early menopause. Where this loss of function occurs before the age of 40, it results in the premature ovarian failure (POF) disease. Throughout folliculogenesis, the follicle-stimulating hormone receptor (FSHR) starts a signaling cascade in the granulosa cells where its inactivation leads to the arrest of follicle maturation and therefore adversely affects ovarian reserve. The aim of this study was to investigate the association of genetic variation (polymorphisms and inactivating mutations) ofFSHR with POF and DOR.Materials and Methods: This case-control study comprised 84 POF, 52 DOR and 80 fertile Iranian women. To determine the presence of the 566C>T mutation and the -29G>A polymorphism inFSHR, PCR-RFLP method was used. SSCP-sequencing was used to identify any allelic variants in exon 10. The expression of humanFSHR at the transcript level was also compared between DOR and fertile controls by real time-polymerase chain reaction (PCR).Results: The 566C>T polymorphism was normal in all the cases. All genotypes of -29G>A and 919G>A (exon 10) polymorphisms were observed. Statistically significant differences were seen in the genotypic distribution of both polymorphisms when comparing the control group with the DOR patient group. A decrease was observed inFSHR expression of DOR patients compared with the control group but was not significant.Conclusion: We conclude that the -29G>A and 919G>A polymorphisms in FSHR may be associated with DOR. Although these polymorphisms had significant differences at the genic level, no significant variation was found at the transcript level

Objective: Polycystic ovary syndrome (PCOS), an ovarian-pituitary axis androgen disorder, is a common endocrine disease in women. Obesity-induced androgenesis and imbalance of adipokine secretion may lead to some metabolic features of PCOS. The beneficial effects of polyphenolic compounds such as quercetin have been reported, however, the underlying molecular mechanism is not entirely understood. In the present study, we investigated the effect of quercetin supplementation on the expression of adiponectin receptors at the transcript level in peripheral blood mononuclear cells (PBMC) samples of PCOS patients.Materials and Methods: In this randomized clinical trial, 84 PCOS subjects were randomly assigned to two groups; the treatment group received 1 g quercetin (two 500 mg capsules) daily for 12 weeks and the control group received placebo.To examine the effect of quercetin supplementation on PCOS patients in addition to biochemical and anthropometric assessments, the expression ofADIPOR1 and ADIPOR2 at the transcript level and AMPK level were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and ELISA assays respectively.Results: Oral quercetin supplementation significantly increased ADIPOR1 and ADIPOR2 transcript expression by 1.32- and 1.46-fold respecetively (P<0.01). In addition, quercetin supplementation enhanced AMPK level by 12.3% compared with the control group (P<0.05).Conclusion: Oral quercetin supplementation improves the metabolic features of PCOS patients by upregulating the expression of adiponectin receptors and AMPK (Registration Number: IRCT2013112515536N1).

Objective: low intensity ultrasound (continues and pulsed) is a form of energy. Spermatogonial stem cells (SSCs) are at the base of male fertility. This study investigated the effects of low intensity ultrasound stimulation (LIUS) and low intensity pulsed ultrasound stimulation (LIUPS) on the expression of germ cell-specific and pluripotency genes in SSCs in vitro.Materials and Methods: In this experimental study, isolated SSCs from neonatal male mice were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). In addition, to confirm identification of SSCs, PLZF protein was detected positively in SSCs derived colonies. SSCs were stimulated by LIUS and LIUPS for 5 days, followed by assessment of expression ofintegrin-α6 (Itga6) and β1 (Itgβ1), as two germ cell-specific genes, and Oct-4, as a pluripotency gene, on day 21st by quantitive reverse transcriptase-polymerase chain reaction (qRT-PCR). To investigate the proliferation rate and colonization of SSCs in different groups, counting whole number of the cells and colonies as well as analysis of the respective diameters were performed on days 7th, 14th and 21st. Data was analyzed by ANOVA test.Results: LIUS and LIUPS treatment of mouse SSCs increased expression of Itga6 and Itgβ1 genes in the experimental groups, compared to the control group (P<0.05), whereas there was no significant difference between the groups, regarding the expression ofOct-4 gene. These treatments maintained survival rate, while they increased proliferation rate and colonization of SSCs during the first week of culture. However, within the second week, proliferation rate and colonization were decreased in the experimental groups.Conclusion: These results suggested that LIUS and LIUPS treatment had good effect on SSCs proliferation and colonization, based on the gene-specific marker expression during 21 days culturein vitro.

Objective: The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions ofKlf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells.Materials and Methods: In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown ofKlf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice.Results: Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown ofKlf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells withoutKlf4 and c-Myc expression failed to form teratomas.Conclusion: The pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms ofKlf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.

Objective: This study aimed to identify several potentially key genes associated with the pathogenesis of Takayasu’s arteritis (TA). This identification may lead to a deeper mechanistic understanding of TA etiology and pave the way for potential therapeutic approaches.Materials and Methods: In this experimental study, the microarray dataset GSE33910, which includes expression data for peripheral blood mononuclear cell (PBMC) samples isolated from TA patients and normal volunteers, was downloaded from the publicly accessible Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified in PBMCs of TA patients compared with normal controls. Gene ontology (GO) enrichment analysis of DEGs and analysis of protein-protein interaction (PPI) network were carried out. Several hub proteins were extracted from the PPI network based on node degrees and random walk algorithm. Additionally, transcription factors (TFs) were predicted and the corresponding regulatory network was constructed.Results: A total of 932 DEGs (372 up- and 560 down-regulated genes) were identified in PBMCs from TA patients.Interestingly, up-regulated and down-regulated genes were involved in different GO terms and pathways. A PPI network of proteins encoded by DEGs was constructed and RHOA, FOS, EGR1, and GNB1 were considered to be hub proteins with both higher random walk score and node degree. A total of 13 TFs were predicted to be differentially expressed. A total of 49 DEGs had been reported to be associated with TA in the Comparative Toxicogenomics Database (CTD). The only TA marker gene in the CTD database wasNOS2, confirmed by three studies. However, NOS2 was not significantly altered in the analyzed microarray dataset. Nevertheless, NOS3 was a significantly down-regulated gene and was involved in the platelet activation pathway.Conclusion: RHOA, FOS, and EGR1 are potential candidate genes for the diagnosis and therapy of TA.

Objective: Umbilical cord blood is used for transplantation purposes in regenerative medicine of hematological disorders. MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as cancer development and incidence. There is a new relation betweenp53 (tumor suppressor gene) andmiR-145 (suppressor of cell growth) upregulation. In this study, we have assessed how adipose-derived stem cells (ADSCs) affect the expansion of hematopoietic stem cells (HSCs), as well asmiR-145 and p53 expressions.Materials and Methods: In this experimental study, we cultured passage-3 isolated human ADSCs as a feeder layer. Flow cytometry analysis confirmed the presence of ADSC surface markers CD73, CD90, CD105.Ex vivo cultures of cordblood CD34+cells were cultured under the following 4 culture conditions for 7 days: i. Medium only supplemented with cytokines, ii. Culture on an ADSCs feeder layer, iii. Indirect culture on an ADSCs feeder layer (Thin CertTM plate with a 0.4 mm pore size), and iv. Control group analyzed immediately after extraction. Real-time polymerase chain reaction (PCR) was used to determine the expressions of thep53 and miR-145 genes. Flow cytometry analysis of cells stained by annexin V and propidium iodide (PI) was performed to detect the rate of apoptosis in the expanded cells.Results: ADSCs tested positive for mesenchymal stem cell (MSC) markers CD105, CD90, and CD73, and negative for HSC markers CD34 and CD45. Our data demonstrated the differentiation potential of ASCs to osteoblasts by alizarin red and alkaline phosphatase staining. MTT assay results showed a higher proliferation rate of CD34+cells directly cultured on the ADSCs feeder layer group compared to the other groups. Direct contact between HSCs and the feeder layer was prevented by a microporous membrane p53 expression increased in the HSCs group with indirect contact of the feeder layer compared to direct contact of the feeder layer.p53 significantly downregulated in HSCs cultured on ADSCs, whereas miR-145 significantly upregulated in HSCs cultured on ADSCs.

Identification of molecular markers which can predict the outcome of sperm retrieval non-invasively in patients with non-obstructive azoospermia (NOA) are valuable in clinical andrology. Jumonji domain-containing 1a (JMJD1A) is a significant epigenetic regulator during spermatogenesis, which plays an important role in the differentiation of post-meiotic germ cells into mature spermatozoa. We therefore aimed to examine the potential association between JMJD1Aexpression and the outcome of sperm retrieval in patients with NOA. Testicular biopsy specimens from 50 NOA patients with either successful sperm retrieval (sperm+, n=22) or failed sperm retrieval (sperm-, n=28) were collected and then examined forJMJD1A expression by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, conventional clinical parameters including luteinizing hormone, follicle-stimulating hormone, testosterone, age, and testicular volume were compared between the two NOA groups. The expression ofJMJD1A in the sperm+group was significantly higher than in the sperm- group (P<0.001), however, no significant difference was observed between the two groups in clinical parameters. The receiver operating characteristic (ROC) curve ofJMJD1A expression in predicting the sperm retrieval outcome showed a sensitivity of 90.91% and a specificity of 89.29% with significant discriminatory ability between the sperm+and sperm- groups [area under the ROC curve (AUC)=0.91]. This study demonstrates a significant association between the expression ofJMJD1A and the success of sperm recovery in patients with NOA, and thus suggests thatJMJD1A expression quantification in testicular biopsies may be a valuable biomarker along with conventional parameters in predicting the presence of spermatozoa.