The Role of the PI3K/AKT/mTOR Signalling Pathway in Male Reproduction.

Male fertility is closely related to the normal function of the hypothalamicpituitary- testicular axis. The testis is an important male reproductive organ that secretes androgen and produces sperm through spermatogenesis. Spermatogenesis refers to the process by which spermatogonial stem cells (SSCs) produce highly differentiated spermatozoa and is divided into three stages: mitosis, meiosis and spermiogenesis. Spermatogenesis requires SSCs to strike a proper balance between self-renewal and differentiation and the commitment of spermatocytes to meiosis, which involves many molecules and signalling pathways. Abnormal gene expression or signal transduction in the hypothalamus and pituitary, but particularly in the testis, may lead to spermatogenic disorders and male infertility. The phosphoinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway is involved in many stages of male reproduction, including the regulation of the hypothalamus-pituitarygonad (HPG) axis during spermatogenesis, the proliferation and differentiation of spermatogonia and somatic cells, and the regulation of sperm autophagy and testicular endocrine function in the presence of environmental pollutants, particularly endocrinedisrupting chemicals (EDCs). In the PI3K/AKT/mTOR signalling pathway, mTOR is considered the central integrator of several signals, regulating metabolism, cell growth and proliferation. In particular, mTOR plays an important role in the maintenance and differentiation of SSCs, as well as in regulating the redox balance and metabolic activity of Sertoli cells, which play an important role in nutritional support during spermatogenesis.

Significant association between Let-7-KRAS rs712 G > T polymorphism and cancer risk in the Chinese population: a meta-analysis.

Association between let-7-KRAS rs712 polymorphism and cancer risk was inconsistent. We therefore conducted this meta-analysis to clarify the association between let-7-KRAS rs712 polymorphism and cancer risk with STATA 14.0 software. A systemic literature search in online databases (PubMed, Embase, CNKI and Wanfang database) was preformed to obtain relevant articles. A total of 13 case-control studies involving 3,453 patients and 4,470 controls were identified up to May 16, 2015. The pooled results indicated that significantly increased risk were observed in Chinese population in T vs. G (OR = 1.21, 95% CI = 1.03-1.42) and TT vs. GG + GT genetic models (OR = 1.69, 95% CI = 1.17-2.42). Sensitivity analysis was conducted and the result without heterogeneity showed significant associations in all five genetic models. Subgroup analyses of cancer type indicated a similar result in digestive cancer (for T vs. G: OR = 1.41, 95% CI = 1.26-1.57; GT vs. GG: OR = 1.24, 95% CI = 1.07-1.43; TT vs. GG: OR = 2.53, 95% CI = 1.86-3.44; GT + TT vs. GG: OR = 1.36, 95% CI = 1.19-1.56; TT vs. GG + GT: OR = 2.35, 95% CI = 1.73-3.19). In summary, these evidences demonstrate that let-7-KRAS rs712 G > T polymorphism might be associated with digestive system cancer risk in the Chinese population.

The effects of LSD1 inhibition on self-renewal and differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited self-renewal and can generate nearly all cells in the body. Changes induced by different LSD1 activities on the regulation of hiPSC self-renewal and differentiation and the mechanism underlying such changes were determined. We used two different LSD1 inhibitors (phenelzine sulfate and tranylcypromine) and RNAi technique to inhibit LSD1 activity, and we obtained hiPSCs showing 71.3%, 53.28%, and 31.33% of the LSD1 activity in normal hiPSCs. The cells still maintained satisfactory self-renewal capacity when LSD1 activity was at 71.3%. The growth rate of hiPSCs decreased and cells differentiated when LSD1 activity was at approximately 53.28%. The hiPSCs were mainly arrested in the G0/G1 phase and simultaneously differentiated into endodermal tissue when LSD1 activity was at 31.33%. Teratoma experiments showed that the downregulation of LSD1 resulted in low teratoma volume. When LSD1 activity was below 50%, pluripotency of hiPSCs was impaired, and the teratomas mainly comprised endodermal and mesodermal tissues. This phenomenon was achieved by regulating the critical balance between histone methylation and demethylation at regulatory regions of several key pluripotent and developmental genes.

Detection of circulating tumor cells in breast cancer with a refined immunomagnetic nanoparticle enriched assay and nested-RT-PCR.

Early detection of circulation tumor cells (CTCs) in breast cancer patients has great clinical relevance. Currently, immunomagnetic microparticles enriched assays require Fe3O4 inner cores, making it difficult to improve sensitivity. In this study, we prepared magnetic nanoparticles with carbon-coated pure iron (Fe@C) acted as the core, Conjugating with EpCAM monoclonal antibody for immunomagnetic nanoparticles(IMPs). IMPs were used in conjunction with immunocytochemistry (ICC) to develop a refined immunomagnetic nanoparticles enriched assay (IMPEA) for detection of circulating tumor cells (CTCs) in breast cancer patients. Compared with nested RT-PCR, this method achieved the same sensitivity, but with a significantly reduced false-positive rate. This method will help find hidden micrometastases, establish clinical stage, and guide individual treatment post-surgery, suggesting potentially significant value in the clinic. FROM THE CLINICAL EDITOR: This team of investigators prepared magnetic nanoparticles with carbon-coated pure iron as core and conjugated them with EpCAM monoclonal antibody to form immunomagnetic nanoparticles for circulating tumor cell (CTC) detection. Compared with nested RT-PCR, this method achieved the same sensitivity, but with a significantly reduced false-positive rate, paving the way to the development of a tool that enables enhanced detection of micrometastases and post-surgical treatment monitoring.

Protective effect of rat pancreatic progenitors cells expressing Pdx1 and nestin on islets survival and function in vitro and in vivo.

To maintain islets survival and function is critical in successful pancreatic transplantation. Pancreatic progenitors cells (PPCs) with lineage potentials, giving rise to exocrine, endocrine, and duct cells, reside in developing and adult pancreas. As tissue-specific stem cells, they can produce pancreatic tissue-specific matrix factors to promote islets survival and function. The aim of our research was to investigate the protective effect of rat pancreatic-duodenal homeobox 1 (Pdx1)(+)/nestin(+) PPCs on islets. In vitro, co-culturing islets with Pdx1(+)/nestin(+) PPCs prolonged the former survival from 7 to 14 days. Furthermore, with high glucose (300.8 mg/dl) stimuli, the yield of insulin in co-cultures was significantly higher than that in control group (single islets group). In vivo, co-transplanting islets and Pdx1(+)/nestin(+) PPCs for 3 days, the blood glucose of diabetic rat was significantly decreased to normal level and sustained for 2 weeks. Without Pdx1(+)/nestin(+) PPCs in islets transplantation, hyperglycemia was reversed at day 7 and recovered at day 15. Pathology analysis showed that islets had remnants in co-transplantation at day 21, as complete graft rejection in alone islets transplantation. Our study showed that Pdx1(+)/nestin(+) PPCs displayed the ability of preserving islets viability and function in vitro and prolonging their survival in vivo.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells.

Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic ß cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic ß cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.

Detection of micrometastases in peripheral blood of non-small cell lung cancer with a refined immunomagnetic nanoparticle enrichment assay.

Fe(3)O(4) particles are currently used as the core of immunomagnetic microspheres in the immunomagnetic enrichment assay of circulating tumor cells (CTCs). It is difficult to further improve the sensitivity of CTC detection or to improve tumor cell-type identification and characterization. In the present study, we prepared immunomagnetic nanoparticles with nanopure iron as the core, coated with anti-cytokeratin 7/8 (CK7/8) monoclonal antibody. These immunomagnetic nanoparticles (IMPs) were used in conjunction with immunocytochemistry (ICC) to establish a refined immunomagnetic nanoparticle enrichment assay for CTC detection in non-small cell lung cancer (NSCLC). The assay was compared with nested reverse transcription polymerase chain reaction (RT-PCR) to detect CK19 mRNA and lung specific X protein (LUNX) mRNA. Human lung adenocarcinoma cell line A549 was used for sensitivity and specificity evaluation. Peripheral blood samples were collected from each group for CTC detection. The average diameter of the immunomagnetic nanoparticles was 51 nm, and the amount of adsorbed antibodies was 111.2 µg/mg. We could detect down to one tumor cell in 5 × 10(7) peripheral blood mononuclear cells. The sensitivity was consistent with that of nested RT-PCR; however, the false positive rate was significantly reduced. The modified assay combined with ICC did not differ from nested RT-PCR in sensitivity, but it had significantly increased specificity. This approach could, therefore, contribute to identification of micrometastases, re-defining clinical staging, and guiding individual postoperative treatments. The technique shows considerable potential clinical value and further clinical trials are warranted.

[The effect of co-transplantation of bone marrow-derived mesenchymal stem cells and islet on maturation and function of bone marrow-derived dendritic cells in recipient mice].

AIM: To investigate the effect of transplantation of bone marrow-derived mesenchymal stem cells (MSCs), or co-transplantation of islet and MSCs, on maturation and function of bone marrow-derived dendritic cells (DCs) in recipient mice. METHODS: Bone marrow MSCs were isolated from BALB/c mice and co-cultured with bone marrow mononuclear cells (BMCs) from C57BL/6 mice at indicated ratios. The co-cultures were treated with recombinant mice granulocyte-macro-phage colony-stimulating factor (rmGM-CSF) and recombinant mice IL-4 (rmIL-4) for 7 days to induce the differentiation of DCs, with lipopolysaccharide (LPS) favoring the maturation of DCs. The differentiation markers and antigen uptake capability of DCs were analyzed by FCM. Production of Interleukin-12 in the supernatants of the DC cultures was quantified by ELISA. BALB/c-derived MSCs and islet were co-transplanted to the capsule of kidney in allogeneic C57BL/c mice. The recipient mice were assayed for their tissue morphology, blood glucose level, and the in vitro differentiation ability of their BMC into mature and functional DCs. RESULTS: The transplantation of MSCs prevented BMC from differentiating into mature DCs, as shown by down-regulated surface markers of DCs including CD11c, CD83, CD86 and I-Ab; (P<0.05), impaired antigen uptake and decreased IL-12 secretion (P<0.01). Co-transplantation of MSCs and islet inhibited immune rejection in the allogeneic recipient mice. CONCLUSION: Transplantation of MSCs inhibits maturation and function of monocyte-derived dendritic cells in the recipient mice, resulting in immune tolerance for the allogeneic islet.

[The effect of murine mesenchymal stem cells on the differentiation and function of allogenetic bone marrow-derived dendritic cells.].

AIM: To study the effect of murine mesenchymal stem cells (MSCs) on the differentiation, maturation and function of allogenetic bone marrow-derived dendritic cells (DCs) and to investigate the mechanism of MSCs displaying immunoregulatory activity. METHODS: BALB/c mice BMCs were isolated and cultured in vitro and then coclutured with C57BL/6 murine bone marrows cells (BMCs) at different ratios in vitro to induce DCs generation. The phenotype of cells was analyzed by flow cytometry. Endocytosis was measured as the cellular uptake of fluorescein isothiocyanate (FITC)-dextram amd was quantified by flow cytometry. The level of IL-12 secretion in the cell culture supernatants was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: Decreased expression of CD11c, CD14, CD83, CD86 and I-A(b);, down-regulated endocytosis capacity and interleukin-12 (IL-12) secretion of DCs were all observed when MSCs cocultured with BMCs at a higher ratio (1:10). CONCLUSION: Murine MSCs could supress the differentiation and maturation of DCs derived from allogeneic BMCs in vitro.