Synergistic effects of mesenchymal stem cell-derived extracellular vesicles and dexamethasone on macrophage polarization under inflammatory conditions.

The undesirable inflammation and the excessive M1 macrophage activity may lead to inflammatory diseases. Corticosteroids and stem cell therapy are used in clinical practice to promote anti-inflammatory responses. However, this protocol has limitations and is associated with numerous side effects. In this study, the synergistic anti-inflammatory effects of dexamethasone (Dex) and mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) were evaluated to enhance the polarization of M1 inflammatory macrophages into the anti-inflammatory (M2) phenotype. Hence, we designed different combinations of Dex and EVs using three methods, including EVs isolated from Dex-preconditioned MSCs (Pre-Dex-EVs), EVs loaded with Dex (L-Dex-EVs), and EVs and Dex co-administration (Dex + EVs). All designed EVs had a significant effect on reducing the expression of M1-related genes (iNOS, Stat1, and IRF5), cytokines (IL6 and TNF-a), and CD markers (CD86) in lipopolysaccharide-stimulated macrophages. On the other hand, these combinations promoted the expression of alternative-activated M2-related genes (Arg-1, Stat6, and IRF4), cytokine (IL10), and CD markers (CD206).The combination of Dex and MSC-EVs enhances the effectiveness of both and synergistically promotes the conversion of inflammatory macrophages into an anti-inflammatory state.

Avastin-Loaded 3D-Printed Alginate Scaffold as an Effective Antiadhesive Barrier to Prevent Postsurgical Adhesion Bands Formation.

Postoperative adhesion can cause complications, such as pain and organ blockage, in the abdominal regions. To address this issue, surgical techniques and antiadhesive treatments are applied. Given the significant role of vascularization in adhesion band formation, Avastin (Ava) that targets vascular endothelial growth factor (VEGF) can be applied to prevent peritoneal adhesion bands. Moreover, Alginate (Alg), a natural polysaccharide, is a promising physical barrier to prevent adhesion bands. Incorporating Ava into Alg hydrogel in a form of 3D-printed scaffold (Alg/Ava) has potential to suppress inflammation and angiogenesis, leading to reduce peritoneal adhesion bands. Following physical, morphological, and biocompatibility evaluations, the efficacy of Alg and Ava alone and their combination in Alg/Ava on the formation of postsurgical adhesions is evaluated. Upon confirming physical stability and sustained release of Ava, the Alg/Ava scaffold effectively diminishes both the extent and strength of adhesion bands. Histopathological examination shows that the reduction in fibrosis and inflammation is responsible for preventing adhesion bands by the Alg/Ava scaffold. Additionally, the cytokine assessment reveals that this is due to the inhibition in the secretion of VEGF and Interleukin 6 suppressing vascularization and inflammatory pathways. This study suggests that a 3D-printed Alg/Ava scaffold has great potential to prevent the postsurgical adhesion bands.

Lanthanide complexes facilitate wound healing by promoting fibroblast viability, migration and M2 macrophage polarization.

A tridentate ligand LH3 ((2-hydroxy-3-methoxybenzylidene)-2-(hydroxyimino)propanehydrazide) comprising o-vanillin, hydrazone and oxime donor groups has been employed to prepare a series of tetranuclear Ln(III) complexes. The reaction of ligand LH3 with Ln(NO3)3 [Ln = Sm, Eu, Gd, Tb, Dy, Ho, Er] in MeOH yielded Ln4(LH)6(MeOH)2 (Ln = Sm(1), Eu(2), Gd(3), Tb(4), Ho (6) and Er (7))] whereas the corresponding reaction with Dy(NO3)3 afforded Dy4(LH)4(LH2)2(OH)2 (5). All complexes were characterized by various analytical techniques including single crystal X-ray diffraction, IR spectroscopy, UV-Vis spectroscopy, and elemental analysis. To investigate the potential of these lanthanide complexes for wound healing applications, their effects on fibroblast viability, migration, and M2 macrophage polarization were evaluated. The cytotoxicity assessment revealed that complexes 2(Eu), 4(Tb), 5(Dy), and 7(Er) significantly enhanced fibroblast viability compared to the negative control (NC). In vitro wound healing assay demonstrated that complexes 2(Eu) and 7(Eu) substantially promoted fibroblast migration compared to the NC. Moreover, complex 2(Eu) exhibited significant anti-inflammatory effects by reducing the phagocytic ability of lipopolysaccharide (LPS)-stimulated macrophage cells and attenuating nitric oxide (NO) production. In conclusion, among the series of complexes tested, complex 2(Eu) displayed the most potent anti-inflammatory effect on macrophage cells, while simultaneously promoting fibroblast viability and migration. This unique combination of properties renders complex 2 (Eu) highly promising for wound healing applications.

BACTERIOPHAGE M13 MODULATES THE SEPSIS-RELATED INFLAMMATORY RESPONSES AND ORGAN DAMAGE IN A CLP MODEL.

ABSTRACT: Background: 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. Methods Bacteriophage M13 harvested from ER2738, titrated, and confirmed by transmission electron microscopy 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, and reactive oxygen species. For in vivo experiments, 8-weeks-old male C57BL/6 mice were randomly divided into the following three groups: CLP + NS (treated with normal saline), CLP + M13 (treated with an intraperitoneal injection of 10 9 PFU/mL of bacteriophage M13), and sham + NS (induced surgery but without ligation and puncture, treated with NS). The mice were killed at different time points after 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 assay. The rectal temperature and survival were evaluated during the study. Results According to the in vitro results, 10 9 PFU/mL of bacteriophage M13 was not toxic and did not affect the level of cytokine, nitric oxide, and reactive oxygen species production by splenocytes, but it reduced the inflammatory response of splenocytes in responses to LPS. In vivo studies indicated that the amount of proinflammatory cytokines, liver enzymes, bacterial load, and organ failure were decreased in the CLP + M13 group compared with CLP + NS, whereas the survival rate was increased. Conclusions 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.

Selective anti-cancer activity against melanoma cells using 3-O-acetyl-ß-boswellic acid-loaded 3D-Printed scaffold.

This study aimed to develop a local 3 D-printed bioactive graft using poly-caprolacton (PCL) as a drug carrier and 3-O-acetyl-ß-boswellic acid (ß-ABA) as an anticancer compound. ß-ABA-loaded 3 D-printed scaffold was fabricated and physically characterized. The results indicated more desirable mechanical and physical properties of the ß-ABA-loaded PCL mat in comparison with the PCL scaffold. Following sustained release of ß-ABA, the ß-ABA-loaded PCL scaffold revealed selective cytotoxic activity against melanoma cells, while the PCL + ABA with the bolus delivery of ß-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 ß-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 ß-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.

Comparative study of the cytotoxicity, apoptotic, and epigenetic effects of Boswellic acid derivatives on breast cancer.

This study aimed to compare the effect of Boswellic acid derivatives on the viability, apoptosis, and epigenomic profiling of breast cancer. According to the viability assays, 3-O-acetyl-11-keto-ß-Boswellic acid (AKBA) showed more toxicity against MDA-MB-231 cells when compared with the 3-O-acetyl-ß-Boswellic acid (ABA). In contrast, ABA revealed less toxicity against MCF-10A. Cell cycle and apoptosis assays determined the maximum apoptotic effect of AKBA on MCF-7, and MDA-MB-231 cells. Interestingly, ß-Boswellic acid (BA) and ABA did not promote the apoptosis in MCF-10A cells. Transwell migration assay indicated the greatest normalized inhibition (around 160%) in the migration of MDA-MB-231 cells induced by AKBA. The expression of P53, BAX, and BCL2 genes in cancerous cell lines has affirmed that both AKBA and ABA could induce the maximal apoptosis. Western-blot investigation demonstrated that the maximum over-expression of P53 protein (1.96 times) was caused by AKBA in MDA-MB-231 cells, followed by ABA in MCF-7 cells. The BCL2 protein expression was in agreement with the previously reported results. The global DNA methylation in both cancerous cells was reduced by ABA. These results suggest that ABA represented more epigenetic modulatory effect while AKBA shows more cytotoxic and apoptotic effect against breast cancer cell lines.

Microscopic Evaluation of Ovarian Surface Epithelium Following Treatment with Conjugated Estrogens in a Mouse Model.

BACKGROUND: This study was designed to evaluate the effect of different concentrations of conjugated equine estrogens (CEE) on the ovarian epithelium of female CD1 mice. METHODS: 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. RESULTS: 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. CONCLUSION: This study concluded that high concentrations of CEE may increase the chances of developing epithelial ovarian cancer.

An engineered microfluidic blood-brain barrier model to evaluate the anti-metastatic activity of ß-boswellic acid.

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 ß-boswellic acid (ß-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 ß-BA deteriorates MDA-MB-231 cells, while ß-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 ß-BA. Vimentin expression in HUVECs, evaluated using western blot, confirmed superior barrier integrity in the presence of ß-BA. The obtained results were confirmed using an invasion study with a cell tracker and a scanning electron microscope. ß-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 ß-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 ß-BA in both static and dynamic BBB models.

Intracerebral Administration of Autologous Mesenchymal Stem Cells as HSV-TK Gene Vehicle for Treatment of Glioblastoma Multiform: Safety and Feasibility Assessment.

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.

The miR-142 Suppresses U-87 Glioblastoma Cell Growth by Targeting EGFR Oncogenic Signaling Pathway.

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 (EGFR) signaling pathway via TargetScan and miRWalk online tools. Its differential expression level was reduced in glioblastoma according to the previous microarray results, and its predicted target genes were upregulated, as shown by the Expression Atlas. The miR-142 was overexpressed in U-87 glioblastoma cells via lentiviral transduction, and the way it influences proliferation and migration of cells was investigated through MTT assay and wound healing assay. Apoptosis rate was also measured via the Annexin V assay, and cell-cycle analysis was done. Then, real-time polymerase chain reaction (real-time PCR) and western blotting were performed to assess fold changes in mRNA and protein levels of the miR-142 predicted targets. Direct target genes of miR-142 were confirmed through a dual-luciferase reporter assay. The miR-142 significantly suppressed cell proliferation and migration and induced apoptosis and cell-cycle arrest in U-87 glioblastoma cells. This was accompanied by a decrease in expression of SHC adaptor protein 4 (SHC4), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), v-akt murine thymoma viral oncogene homolog 1 (AKT1), Kirsten rat sarcoma viral oncogene homolog (KRAS), and mitogen-activated protein kinase 8 (MAPK8) oncogenes at mRNA and protein levels in glioblastoma cells. Also, AKT1 was demonstrated as a direct target of miR-142. Overall, miR-424 acts as tumor suppressor miRNA in glioblastoma cells.

Secondary metabolites from acridocarpus orientalis inhibits 4T1 cells and promotes mesenchymal stem cells (MSCs) proliferation.

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.

MicroRNA-129 Inhibits Glioma Cell Growth by Targeting CDK4, CDK6, and MDM2.

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 < 0.05), respectively, and expression of CDK6 and MDM2 in U251 cells decreased by 55.3% (p < 0.0001) and 34.7% (p < 0.05), respectively. Luciferase assays confirmed that overexpression of miR-129 decreased the expression of the CDK4 gene by 58.9% (p < 0.01), CDK6 by 35.7% (p < 0.0001), and MDM2 by 49% (p < 0.001). Moreover, cell cycle assays showed a decrease of the G2-phase population to 10% and pre-G2 arrest in U87 cells (p < 0.05). Additionally, wound healing assays indicated that miR-129 overexpression inhibits cell growth of glioblastoma cells. These findings introduced novel targets for miR-129 in glioblastoma cells.

Inhibiting the expression of anti-apoptotic genes BCL2L1 and MCL1, and apoptosis induction in glioblastoma cells by microRNA-342.

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.

Two Triacylglycerol Pathway Genes, CTDNEP1 and LPIN1, are Down-Regulated by hsa-miR-122-5p in Hepatocytes.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: Our findings suggest that up-regulation of miR-122 can trigger the compensatory response of LPIN1 and CTDNEP1 in hepatosteatosis.

The role of miR-17-92 cluster in the expression of tumor suppressor genes in unrestricted somatic stem cells.

The miR-17-92 cluster consisted of seven miRNAs (mir-17-5p, -17-3p, -18a, -19a, -20a, -19b-1, and -92a-1). Previous studies have shown this cluster has been over-expressed in several cancers. The aim of this study was to evaluate the over-expression impacts of miR-17-92 on stem cells. In the current work, the effect of miR-17-92 cluster which was cloned in Lentiviral vector has been investigated on unrestricted somatic stem cells (USSCs). Tumor suppressor genes (p53, p15, RBL1, SMAD2, SMAD4, and MAPK-1) expression, especially p53, was considerably reduced. These data show the potential of miR-17-92 for oncogenesis regulation in stem cells. In conclusion, the role of miR-17-92 in USSCs may provide a better understanding of its function in tumorigenesis and for the possible use in cell therapy of the anti-mir-17-92 cluster.

MiR-371-373 cluster acts as a tumor-suppressor-miR and promotes cell cycle arrest in unrestricted somatic stem cells.

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.

Coating of electrospun poly(lactic-co-glycolic acid) nanofibers with willemite bioceramic: improvement of bone reconstruction in rat model.

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.

Mir-302 cluster exhibits tumor suppressor properties on human unrestricted somatic stem cells.

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.

Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte.

PURPOSE: 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. METHODS: Goat oocytes were exposed to brilliant cresyl blue (BCB) staining test and categorized into BCB(+) (blue-cytoplasm), and BCB(-) (colorless-cytoplasm) groups. A group of oocytes were not exposed to BCB test and was considered as a control group. After maturation in vitro, a group of oocytes were used for determination of nuclear status and intracellular GSH content while another group was subjected to parthenogenetic activation followed by in vitro embryo culture. RESULTS: We found that BCB(+) oocytes not only yielded higher rate of maturation, but also showed an increased level of intracellular GSH content than BCB(-) and control oocytes. Furthermore, BCB(+) oocytes produced more blastocysts than BCB(-) and control oocytes. Our data revealed that the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes were interacted with G6PDH-activity in mature oocyte, their surrounding cumulus cells, and blastocyst-stage embryos. CONCLUSIONS: The results of this study demonstrate that selection of goat oocytes based on G6PDH-activity through the BCB test improves their developmental competence, increases intracellular GSH content, and affects the expression of the apoptosis-related genes.