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This study aimed to develop a local 3 D-printed bioactive graft using poly-caprolacton (PCL) as a drug carrier and 3-O-acetyl-β-boswellic acid (β-ABA) as an anticancer compound. β-ABA-loaded 3 D-printed scaffold was fabricated and physically characterized. The results indicated more desirable mechanical and physical properties of the β-ABA-loaded PCL mat in comparison with the PCL scaffold. Following sustained release of β-ABA, the β-ABA-loaded PCL scaffold revealed selective cytotoxic activity against melanoma cells, while the PCL + ABA with the bolus delivery of β-ABA was toxic against fibroblast cells. Followed by the induction of apoptosis in melanoma cells at the gene level, the result of the western blot showed that the β-ABA-loaded scaffold significantly up-regulated P53 and down-regulated BCL2, with an increment in the ratio of Bax/BCL2. The selective anti-cancer properties of β-ABA-loaded 3 D printed scaffold against melanoma cells indicated that this scaffold could be potentially used as a bioactive graft to improve the melanoma treatment.

Sepsis is a life-threatening disorder that leads to the induction of inflammatory responses and organ failure. Phage therapy is a new approach to controlling infections resistant to common treatments, including sepsis. Several studies have shown the effect of lytic bacteriophages on infection control by reducing the bacterial load. The present study deals with lysogenic bacteriophage M13 on the inflammatory responses caused by cecal ligation and puncture (CLP) induced sepsis in a mouse model.Bacteriophage M13 harvested from ER2738, titrated, and confirmed by TEM analysis. In-vitro toxicity and immunomodulatory effect of bacteriophage M13 were assessed on splenocytes by measurement of cell viability and the production level of cytokines, nitric oxide (NO), and reactive oxygen species (ROS). For in-vivo experiments, eight weeks old male C57BL/6 mice were randomly divided into three groups: CLP + NS (treated with normal saline), CLP + M13 (treated with an intraperitoneal injection of 109 PFU/ml of bacteriophage M13), and sham+ NS (induced surgery but without ligation and puncture, treated with NS). The mice were sacrificed at different time points post-surgery (6,24,48, and 72, n = 10 for each time point of each group). The kidney, liver, and lungs were harvested for histopathological analysis, and blood was obtained for cytokine and liver enzyme assay. The spleen was used to assess the bacterial load using colony forming unit (CFU) assay. The rectal temperature and survival were evaluated during the study.According to the in-vitro results, 109 PFU/ml of bacteriophage M13 was not toxic and did not affect the level of cytokine, NO, and ROS production by splenocytes, but it reduced the inflammatory response of splenocytes in responses to LPS. In-vivo studies indicated that the amount of pro-inflammatory cytokines, liver enzymes, bacterial load, and organ failure were decreased in the CLP + M13 group compared to CLP + NS, whereas the survival rate was increased.These experiments demonstrated that bacteriophage M13 could lessen the consequences related to sepsis in CLP mice and can be considered a therapeutic approach in sepsis.

Widespread investigation has revealed the promising ability of suicidal genes in the treatment of glioma tumors; nevertheless, promoting their effects relies on the ability to apply suitable vehicles and techniques. In this study, the safety and feasibility of using bone marrow-derived mesenchymal stem cells (MSCs) in combination with prodrug for treatment of patients with primary and secondary glioblastoma multiform (GBM) was assessed. Five GBM patients were recruited. Following gross total resection of the tumor and adjuvant radiotherapy and chemotherapy, intracerebral injection of autologous MSCs transduced with lentivirus containing herpes simplex virus thymidine kinase (HSV-TK) was performed followed by intravenous administration of ganciclovir for 2 weeks. The treatment was well tolerated by all patients. Mild-to-moderate fever, headache, and cerebrospinal fluid leukocytosis were evident in three, two, and one patient, respectively. The progression-free survival (PFS) and overall survival (OS) of patients were 95.79 ± 51.40 and 128.85 ± 48.81 weeks, respectively. The 1-year PFS and OS were 60% and 100%, respectively, among our patients, and two patients had more than 3 years of OS and more than 2 years of PFS. It seems that intracerebral administration of bone marrow MSC containing the HSV-TK gene in combination with intravenous ganciclovir would be safe and feasible in the treatment of patients with GBM.

Among medicinal plants, Acridocarpus orientalis (AO) possesses a remarkable anti-cancer potential, possibly because of its anti-oxidant property. In this study, the leaf and stem extracts from AO were assessed to find the bioactive compound with selective anti-cancer properties. The MTT viability and live and dead assays revealed that around 80% and 98% of 4T1 cells survival were declined after 48 h incubation with leaf and stem extracts, respectively. The leaf extract increased stem cell proliferation by 20% whereas the stem extract inhibited around 22% of stem cells proliferation after 48 h treatment. The live and dead assay of MSCs confirmed that 40% of the MSCs died when treated with AO stem extract. On the other hand, there were no dead cells after two days of treatment with the leaf extract. Followed by the induction of cell cycle arrest in G0/G1-phase, the real-time PCR demonstrated apoptosis properties in 4T1 cells through overexpression of Bax and down-regulation of BCL2 genes. Interestingly, within the pure compounds isolated from AO leaf extract, Morin was responsible for the inhibition of 4T1 cells proliferation as well as MSCs expansion, predicting to play an essential role in the treatment of cancer. The promising in vitro anti-cancer and stem cell-inductive properties of morin isolated from AO extract may provide a great potential to produce selective herbal derived drugs.

Glioblastoma is the most lethal malignancy of the brain and is resistant to conventional cancer treatments. Gene-therapy approaches like using tumor suppressor miRNAs are promising in the treatment of glioblastoma. They control the expression of oncogenes and influence tumor features and behaviors. Therefore, in the present study, it was predicted that miR-142 regulates oncogenic epidermal growth factor receptor (

Glioblastoma is the most common malignant primary brain tumor among adults and one of the most lethal cancers. It is characterized by the deregulation of signaling pathways involving proliferation, growth, survival, and other factors. MicroRNAs (miRNAs) play a role in the regulation of genes by affecting the 3′ untranslated region (UTR) of mRNA and affect many cell functions. The present study showed that miR-129 decreased the expression of retinoblastoma and p53 signaling pathways’ genes, including CDK4, CDK6, and MDM2. The real-time PCR data indicated that expression of CDK4 in U251 and U87 cell lines declined by 69.8% and 47% (p

This study was designed to evaluate the effect of different concentrations of conjugated equine estrogens (CEE) on the ovarian epithelium of female CD1 mice.Twenty-four female mice at 7 months with irregular estrus cycles were randomly divided into four groups of 6 mice each. Group one was considered as a control group and received a daily dose of 0.5ml of propylene glycol, for three weeks, while those in the treatment groups received a daily dose of 14μg/kg, 28μg/kg and 56μg/kg conjugated equine estrogens, respectively.The results from this study showed a strong correlation between elevated concentrations of CEE and histological changes in ovarian surface epithelium (OSE). They also showed that administration of high-dose estrogen created the conditions for excessive proliferation of OSE which may progress into the development of cysts in the ovaries.This study concluded that high concentrations of CEE may increase the chances of developing epithelial ovarian cancer.

Background and purpose: The formation of postoperative intra-abdominal adhesion band formation may lead to severe complications. This study aimed to evaluate the preventive effect of local administration of frankincense n-hexane extract (FHE) on the formation of postsurgical adhesion bands. Materials and methods FHE was extracted from the resin of a Boswellia sacra tree and its components were identified by gas chromatography-mass spectrometry (GC-MS). In an animal model, the expression levels of TNF-α and TGF-β1 cytokines after application of FHE were assessed to check the inflammatory and fibrotic cues, respectively. Results Following FHE compound analysis, in vivo experiments demonstrated that intraoperative local administration of FHE resulted in the prevention of adhesion band formation. The adhesion grades in the FHE-treated group were significantly lower than those in the negative control (NC) and the positive control (Interceed). The infiltration of inflammatory cells observed by histopathology revealed a significant anti-inflammatory potential of FHE. Furthermore, the gene expression results proved that significant suppression of TNF-α and TGF-β1 was responsible for its antiadhesion properties. Conclusions The study reported the potential of FHE as an ointment for the prevention of adhesion bands.

The aim of this study was to evaluate a bioactive multilayer wound dressing, based on chitosan and alginate. To enhance healing potential, Dracaena Cinnabari and Aloe Vera were loaded as separate layers into the scaffold. The bare and bioactive multilayered scaffolds were fabricated by an iterative layering freeze-drying technique. Following of topographical, chemical, and physical assessment, the performance of the scaffolds was evaluated in vitro and in vivo. The results revealed adequate attachment, and proliferation of human foreskin fibroblasts, indicating excellent biocompatibility of the bioactive scaffold. In vivo, the performance of the multi-layered scaffold loaded with the bioactive materials was comparable with Comfeel plus®. The wounds treated with the bioactive scaffold exhibited superior hypergranulation, fibroblast maturation, epithelization, and collagen deposition, with minimal inflammation, and crust formation. It is concluded that the synergism of extracellular matrix-mimicking multi-layered scaffolding with Aloe Vera and Dracaena Cinnabari could be considered as a supportive wound dressing.

In situ prolonged delivery of drugs at the site of tumor can be satisfactorily accelerated patient recovery. We compared the effect of temozolomide while incorporated by polycaprolactone nanofibers on the apoptotic behavior of U87 glioma cells. After biocompatibility evaluation of nanofibers by scanning electron microscope and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide analysis, the apoptosis of U87 cells was evaluated using p53, Bcl2 and Bax genes expression. It was found that nanofiber-temozolomide group showed a greater ability to induce apoptosis as well as have a significantly diminished initial burst release of drug compared with other groups and have promising potential in treating cancer.

Glioma is an aggressive and lethal type of brain tumor that originates from glial cells. Glioblastoma cells confer considerable resistance to induction of apoptosis, which may be due to overexpression of anti-apoptotic proteins, or the reduction of the level of some pro-apoptotic proteins. MicroRNAs (miRNAs) can affect the cell biology pathways, including replication, autophagy, necrosis, and apoptosis by regulating gene expression. In this study, using bioinformatics methods, we selected the anti-apoptotic genes, BCL2L1 and MCL1, and microRNA that targeted them (miR-342). In the next step, the Lentiviral particles that contain miR-342 (LV-miR-342) were synthesized in HEK293T cell lines. Glioblastoma cell lines, U251 and U87, were transduced with LV-miR-342. The gene expression and apoptosis induction were then assayed by real-time PCR and flow cytometry respectively. The present study showed that increasing the expression of miR-342 reduced the expression of the anti-apoptotic genes, BCL2L1 and MCL1. The results of luciferase assay reports confirmed that miR-342 targeted BCL2L1 and MCL1. In addition, flow cytometry analysis indicated that miR-342 overexpression induced apoptosis in glioblastoma cells. As well as, Western blotting results confirmed a decrease in BCL2L1 protein following overexpression of miR-342 in glioblastoma cells. These findings may provide a novel therapeutic target for the treatment of glioblastoma.

Glioblastoma is a lethal and incurable cancer. Tumor suppressor miRNAs are promising gene therapy tools for cancer treatment. In silico, we predicted miR-424 as a tumor suppressor. It had several target genes from the epidermal growth factor receptor (ERBB) signaling pathway that are overactive in most glioblastoma cases. We overexpressed miR-424 by lentiviral transduction of U-251 and U-87 glioblastoma cells confirmed with fluorescent microscopy and real-time quantitative PCR (qRT-PCR). Then the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay and scratch wound migration assay were performed to investigate the miR-424 tumor suppressor effect in glioblastoma. miR-424's effect on glioblastoma apoptosis and cell-cycle arrest was verified using Annexin V- phosphatidylethanolamine (PE) and 7-minoactinomycin D (7-AAD) apoptosis assay and cell-cycle assay. miR-424 predicted target genes mRNA and protein level were measured after miR-424 overexpression in comparison to the control group by qRT-PCR and western blotting, respectively. We confirmed miR-424 direct target genes by dual-luciferase reporter assay. miR-424 overexpression significantly suppressed cell proliferation and migration rate in glioblastoma cells based on the MTT and scratch assays. Flow cytometry results confirmed that miR-424 promotes apoptosis and cell-cycle arrest in glioblastoma cells. Predicted target genes of miR-424 from the ERBB pathway were downregulated by miR-424 overexpression. qRT-PCR and western blotting showed that KRAS, RAF1, MAP2K1, EGFR, PDGFRA, AKT1, and mTOR mRNA expression levels and KRAS, RAF1, MAP2K1, EGFR, and AKT1 protein level, respectively, had significantly decreased as a result of miR-424 overexpression in comparison to the control group. Dual-luciferase reporter assay confirmed that miR-424 directly targets RAF1 and AKT1 oncogenes. Overall, miR-424 acts as tumor suppressor miRNA in glioblastoma cells.

Background The development of anti-cancer drugs with the ability to inhibit brain metastasis through the blood-brain barrier (BBB) is substantially limited due to the lack of reliable in vitro models. Main methods In this study, the Geltrex™-based Transwell® and microfluidic BBB models were applied to screen the effect of β-boswellic acid (β-BA) on the metastasis of MDA-MB-231 cells through the BBB in static and dynamic conditions, respectively. Major results The toxicity assay revealed that β-BA deteriorates MDA-MB-231 cells, while β-BA had no detectable toxic effects on human umbilical vein endothelial cells (HUVECs) and astrocytes. Trans-endothelial electrical resistance evaluation showed sustainable barrier integrity upon treatment with β-BA. Vimentin expression in HUVECs, evaluated using western blot, confirmed superior barrier integrity in the presence of β-BA. The obtained results were confirmed using an invasion study with a cell tracker and a scanning electron microscope. β-BA significantly inhibited metastasis by 85%, while cisplatin (Cis), a positive control, inhibited cancer cell migration by 12% under static conditions. Upon applying a dynamic BBB model, it was revealed that β-BA-mediated metastasis inhibition was significantly higher than that mediated by Cis. Conclusions and implications In summary, the current study proved the anti-metastatic potential of β-BA in both static and dynamic BBB models. This article is protected by copyright. All rights reserved.

In this study, prevention of the adhesion bands and inflammatory features has been investigated using poly (lactic-co-glycolic acid)-ibuprofen (PLGA-IB) nanofibrous meshes in a mice model. To find the optimized membrane for prevention of postoperative adhesion bands, we have compared PLGA-IB group with PLGA, IB, and control groups in a mice adhesion model. Two scoring adhesion systems were used to represent the outcome. According to the results obtained in this study, the PLGA-IB nanofiber membrane showed a greater reduction in adhesion band than other groups. In conclusion, among FDA-approved polymers and drugs, PLGA-IB meshes could be applicable as a potential candidate for prevention of postoperative abdominal inflammation and adhesion bands formation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:990-997, 2016.

Recent advances in small RNA research have implicated microRNAs (miRNAs) as important regulators of proliferation and development. The miR-371-373 cluster is prominently expressed in human embryonic stem cells (ESCs) and rapidly decreases after cell differentiation. MiR-371-373 cluster was investigated as one of the key factors of stem cell maintenance and pluripotency in unrestricted somatic stem cells (USSCs) using a lentivirus system. Gene expression showed a dual effect on proliferation, which revealed a transient cell cycle progression and consequent repression in pluripotency factors and cell cycle genes. Cell proliferation analysis with CFU, MTT, and DNA content assays further confirmed the dual effect of cluster after prolonged exposure. Analyzing the course of action, it seems that miR-371-373 cluster acts as an onco/tumor suppressor-miR. MiR371-373 cluster acts by modulating the function of these factors and limiting the excessive cell cycle propagation upon oncogenic stimuli to protect cells from replicative stress, but also activate CDK inhibitors and transcriptional repressors of the retinoblastoma family to cause cell cycle arrest. In contrast to the previous studies, we believe that miR-371-373 cluster functions as a self-renewal miRNA to induce and maintain the pluripotent state but also to potentially inhibit dysregulated proliferation through cell cycle arrest. It seems that miR-371-373 cluster presents with a dual effect in this cellular context which may possess different actions in various cells. This not only expands the basic knowledge of the cluster but may offer a great chance for therapeutic interventions.

To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH) activity in sheep oocytes with cytoplasmic lipid content, maturational competence, developmental competence to the blastocyst stage, and gene expression of certain molecular markers., Purpose To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH)activity in sheep oocytes with cytoplasmic lipid content, maturational competence,developmental competence to the blastocyst stage, and gene expression of certainmolecular markers.Methods Before brilliant cresyl blue (BCB) staining test, oocytes were classified ashigh, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after thetest as having low or high G6PDH-activity (BCB+ and BCB-, respectively). Aftermaturation in vitro, a group of oocytes were subjected to IVF followed by in vitroembryo culture and another group was used for evaluation of expression of candidategenes.Results The cleavage and blastosyst rates were lowest (P < 0.05) in LCLC group,intermediate (P < 0.05) in MCLC group and highest (P < 0.05) in HCLC group. More(P < 0.05) oocytes in HCLC group were BCB+, and higher (P < 0.05) maturation,cleavage, and blastocyst rates were seen for BCB+ oocytes than the BCB- oocytes.Our gene expression data indicated that mRNA transcript abundance of ITGB2, pZP3,BMP15, and GDF9 genes were similar between BCB oocytes groups. However, theexpression of ATP1A1 was higher (P < 0.05) for BCB+ oocytes compared to BCBoocytes.In addition, BAX transcript abundance was similar (P > 0.05) among BCB+,BCB-, and control groups, before and after maturation in vitro.Conclusion Activity of G6PDH in sheep oocytes is highly associated with lipid content,and compared with the morphological parameters, might be a more precise andobjective predictor for subsequent developmental competence in vitro.

We have investigated the combination effects of bioceramics and poly(lactide-co-glycolide) (PLGA) on bone reconstruction in calvarial critical size defects using a rat model. Willemite (Zn2SiO4) ceramics were prepared and coated on the surface of electrospun fabricated scaffolds. After scaffolds and nanoparticles characterization, osteoconductivity of the construct was analyzed using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. Eight weeks after implantation, no sign of inflammation was observed at the site of the osseous defect. The results showed that the ceramics supported bone regeneration and highest bone reconstruction were observed in willemite-coated PLGA. This suggests that electrospun PLGA nanofibers coated with BG are potential candidate implants for bone tissue engineering applications.

Many studies have reported that miR-302-367 cluster acts in different ways in various cell types. For instance, this cluster is shown to have a potential role in stemness regulation in embryonic stem cells (ESCs). On the other hand, this cluster inhibits the tumorigenicity of human pluripotent stem cells by coordinated suppression of CDK2 and CDK4/6 cell cycle pathways. Indeed, this cluster has a significant posttranscriptional impact on cell cycle progression. Previous reports have shown the participation of miR-302-367 cluster in cell cycle regulation of hESCs, MCF7, HepG2, and Teta-2 embryonal teratocarcinoma cells, but its effect on unrestricted somatic stem cells (USSCs) as a new source of human somatic stem cells from the umbilical cord blood remains to be elucidated. Therefore, in this study, we aimed to investigate the effect of miR-302-367 cluster on cell proliferation by MTT assay, cell cycle analysis, and colony formation assay. In addition, the expression of candidate cell cycle regulatory performance and tumor suppressor genes was determined. In this study, for the first time, we found that miR-302-367 cluster not only did not reprogram human USSCs into a pluripotent ESC-like state, but also inhibited the proliferation of human USSCs. Moreover, analyzing the cell cycle curve revealed a significant apoptotic phase upon viral introduction of miR-302-367. Our gene expression study revealed the overexpression of candidate genes after transduction of USSCs with miR-302-367 cluster. In conclusion, the controversial role of miR-302-367 in different cell types may provide better understanding for its role in stemness level and its antitumorigenicity potential in different contexts.

Keratin-based biomaterials can be considered as the beneficial platform for designing suitable wound dressers. One of the most common natural materials that are composed of keratin and lipid is snakes shed skin. In this new study, shed skin of two different Omani snakes; Bitis arietans snake (Puff adder, “P”) and Telescopus dhara snake (Arabian Cat snake, “C”); were examined as wound dresser. The presence of lipid and keratin were assessed using Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR). 3- Layered filamentous morphology of the P shed skin and the 3-layered compact structure of C shed skin were analyzed by Scanning Electron Microscopy (SEM). X-Ray powder Diffraction (XRD) analysis proved two main peaks corresponding to the α-helix and β-sheet of the protein. Crystallinity index (CI) of P and C shed skins were obtained from XRD peaks 42.85 and 28.57%. The in vivo and histopathological results indicated that skin reconstruction was effectively improved under P shed skin treatment as compared with negative and positive control (PC) and C groups. Superior histopathological scores were the beneficial properties of P group over the positive control. The synergistic effect of natural extracellular matrix-mimicking structure and keratin beneficial proteins for wound healing could develop a natural substrate for wound healing in the clinical setting.

Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.

Expression of miR-122 is highly specific to hepatocytes of the liver. This miRNA is involved in lipid hemostasis of the tissue; however, there is no comprehensive understanding of its function in lipid hemostasis.Since hepatocytes are responsible for part of Triacylglycerol (TAG) synthesis in the body, we hypothesized that miR-122, as the most abundant miRNA in the tissue, might regulate TAG metabolism by targeting key enzymes that are involved in its production pathway. A systematic computational analysis of putative targets of miR-122 identified CTDNEP1 and LPIN1 genes in the TAG pathway. We used dual-luciferase reporter assay, quantitative RT-PCR as well as western blot to confirm the repressive effect of miR-122 on CTDNEP1 and LPIN1 in TAG pathway.Real time PCR on liver needle biopsies with hepatosteatosis showed that miR-122 is up-regulated in hepatosteatosis. Surprisingly, the protein and RNA level of identified targets of miR-122 are also up-regulated in clinical samples, probably as a disproportionate feedback response to the high level of miR-122.Our findings suggest that up-regulation of miR-122 can trigger the compensatory response of LPIN1 and CTDNEP1 in hepatosteatosis.

We have investigated the combination effects of bioceramics and poly(lactide-co-glycolide) (PLGA) on bone reconstruction in calvarial critical size defects using a rat model. Willemite (Zn2SiO4) ceramics were prepared and coated on the surface of electrospun fabricated scaffolds. After scaffolds and nanoparticles characterization, osteoconductivity of the construct was analyzed using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. Eight weeks after implantation, no sign of inflammation was observed at the site of the osseous defect. The results showed that the ceramics supported bone regeneration and highest bone reconstruction were observed in willemite-coated PLGA. This suggests that electrospun PLGA nanofibers coated with BG are potential candidate implants for bone tissue engineering applications.

To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts., Purpose To associate the glucose-6-phosphate dehydrogenase (G6PDH) activity inimmature goat oocyte with intracellular glutathione (GSH) content, developmentalcompetence after parthenogenetic activation and relative abundance of Bax and Bcl-2genes transcripts using .Methods Goat oocytes were exposed to brilliant cresyl blue (BCB) staining andcategorized into BCB+ (blue cytoplasm) and BCB− (colorless cytoplasm) groups. Agroup of oocytes were not exposed to BCB stain and was considered as a control.After maturation in vitro, a group of oocytes were used for determination of nuclearstatus and intracellular GSH content and another group were subjected toparthenogenetic activation followed by in vitro embryo culture.Results We found that BCB+ oocytes yielded a significantly higher maturation rate thanthe BCB− and control oocytes. Moreover, BCB+ oocytes showed increased inintracellular level of GSH than BCB− and control oocytes. Furthermore, BCB+ oocytesproduced more blastocysts than BCB− and control oocytes. The expression of antiapoptoticgene Bcl-2 and pro-apoptotic gene Bax in mature oocyte, their surroundingcumulus cells and blastocyst was affected by BCB staining test.Conclusions These results strongly suggest that BCB+ oocytes have a higher meioticand cytoplasmic maturity (as determined by intracellular GSH content), and that BCBstaining can be used to select developmentally competent oocytes for in vitro embryoproduction.

The self-renewal and differentiation status of a stem cell is very important in the applications concerning regenerative medicine. Proliferation capacity, differentiation potentials and epigenetic properties of stem cells differ between sources. Studies have shown the high potentials of stem cells in iPS reprogramming. To examine this; we have compared the stem-ness and differential potential of four adult stem cells from common sources. We show a correlation between pluripotency and differentiation status of each stem cell with available data on the reprogramming efficiency. Four human adult stem cells including, adipose tissue-mesenchymal stem cells (AT-MSC), bone marrow mesenchymal stem cells (BM-MSCs), nasal septum derived multipotent progenitors (NSP) and umbilical cord blood stem cells (USSCs) were isolated and characterized. The self- renewal and differentiation potentials of each stem cell were assessed. Stem-ness transcription factors and the propagation potentials of all cells were analyzed. Furthermore the differentiation potentials were evaluated using treatment with induction factors and specific MicroRNA profile. Real-time PCR results showed that our stem cells express innate differentiation factors, miR145 and Let7g, which regulate the stem-ness and also the reprogramming potentials of each stem cell. To complete our view, we compared the propagation and differentiation potentials by correlating the stem-ness gene expression with differentiation MicroRNAs, also the direct effect of these factors on reprogramming. Our results suggest that the potentials of adipose tissue stem cells for GMP (Good Manufacturing Practice) compliant starting material are adequate for clinical applications. Our results indicate a low risk potential for AT-MSCs as starting material for iPS production. Although let7g and mir145 are well known for their differentiation promoting effects, but function more of a fine tuning system between self-renewal and differentiation status.

The miR-17-92 cluster is composed of seven miRNAs (microRNAs; miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1). Previous studies have indicated that this cluster is involved in cell proliferation and their overexpression has been seen in several types of cancer. We have assessed the overexpression effects of miR-17-92 on the expression of several genes associated with cell-cycle regulation. The human miR-17-92 gene was cloned into a transposone-based vector, piggyBac and transfected into HEK-293T [HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40)] cell line. Gene expression analysis indicated that up-regulation of this cluster causes significant changes in the expression of several cell-cycle related genes, including CDK2 (cyclin-dependent kinase 2), cyclin-D2, c-Myc and CREB (cAMP-response-element-binding protein). Other methods of transcripts assessment confirmed miR-17-92 overexpression enhances cell proliferation.