Human UC-MSC-derived exosomes facilitate ovarian renovation in rats with chemotherapy-induced premature ovarian insufficiency.

Premature ovarian insufficiency (POI) induced by chemotherapy is an intractable disorder with a considerable incidence that commonly results in insufficient fertility and concomitant complications in female patients. Due to limitations in the current progress in POI diagnosis and treatment, there is an urgent need to develop novel remedies to improve ovarian function and protect fertility. The ameliorative effect of human umbilical cord mesenchymal stem cells (hUCMSCs) and exosomes derived from them in POI treatment could be a new hope for patients. Herein, we identified exosomes from hUCMSCs (hUCMSC-Exos). Then, systematic infusion of hUCMSC-Exos was accomplished via tail intravenous injection to investigate the feasibility of the treatment of rats with chemotherapy-induced POI by intraperitoneal injection of cyclophosphamide (CTX) and busulfan (BUS). Ovarian functions in the indicated group were evaluated, including oestrous cycle, serum sex hormone levels, follicle counts, ovarian pathological changes, proliferation and apoptosis of granulosa cells (GCs), and reproductive ability testing. Furthermore, the potential influence of hUCMSC-Exos on ovarian tissues was illuminated by conducting RNA-seq and multifaceted bioinformatics analyses. POI rats with hUCMSC-Exos transplantation exhibited a decrease in follicle-stimulating hormone (FSH) and apoptosis of GCs but an increase in oestradiol (E2), anti-Müllerian hormone (AMH), and the number of ovarian follicles and foetuses in the uterus. And the immunomodulation- and cellular vitality-associated gene sets in rats had also undergone moderate changes. Our data indicated the feasibility of hUCMSC-Exos in improving ovarian function and protecting fertility in chemotherapy-induced POI rats. HUCMSC-Exos can improve the local microenvironment of ovarian tissue in POI rats by participating in immune regulation, cellular viability, inflammation regulation, fibrosis and metabolism, and other related signal pathways.

Tet2-mediated DNA demethylation regulates the proliferation and apoptosis of human leukemia K562 cells.

TET2 is a member of the TET protein family which is responsible for active DNA demethylation through catalyzing the successive oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), and mutations of Tet2 frequently lead to hematological malignancies. However, the relationship between Tet2-mediated demethylation and hematological malignancies is unclear. The human leukemia K562 cell line is an immortalized leukemia line that serves as an in vitro model of erythroleukemia. In this study, we investigated the effect of Tet2-mediated demethylation on the apoptosis and proliferation of human leukemia K562 cells and found that knockdown of Tet2 promoted and inhibited K562 cell proliferation and apoptosis, respectively, while upregulation of TET2 enzymatic activity via alpha-ketoglutaric acid (α-KG) had the opposite effects. Therefore, the Tet2 gene acts as a potential target for the treatment of leukemia, and small molecules that target the Tet2 gene may be used to screen antitumor drugs for hematological malignancies.

APP mediates tau uptake and its overexpression leads to the exacerbated tau pathology.

Alzheimer's disease (AD), as the most common type of dementia, has two pathological hallmarks, extracellular senile plaques composed of ß-amyloid peptides and intracellular neurofibrillary tangles containing phosphorylated-tau protein. Amyloid precursor protein (APP) and tau each play central roles in AD, although how APP and tau interact and synergize in the disease process is largely unknown. Here, we showed that soluble tau interacts with the N-terminal of APP in vitro in cell-free and cell culture systems, which can be further confirmed in vivo in the brain of 3XTg-AD mouse. In addition, APP is involved in the cellular uptake of tau through endocytosis. APP knockdown or N-terminal APP-specific antagonist 6KApoEp can prevent tau uptake in vitro, resulting in an extracellular tau accumulation in cultured neuronal cells. Interestingly, in APP/PS1 transgenic mouse brain, the overexpression of APP exacerbated tau propagation. Moreover, in the human tau transgenic mouse brain, overexpression of APP promotes tau phosphorylation, which is significantly remediated by 6KapoEp. All these results demonstrate the important role of APP in the tauopathy of AD. Targeting the pathological interaction of N-terminal APP with tau may provide an important therapeutic strategy for AD.

Covalent Coupling-Stabilized Transition-Metal Sulfide/Carbon Nanotube Composites for Lithium/Sodium-Ion Batteries.

Transition-metal sulfides (TMSs) powered by conversion and/or alloying reactions are considered to be promising anode materials for advanced lithium-ion batteries (LIBs) and sodium-ion batteries (SIBs). However, the limited electronic conductivity and large volume expansion severely hinder their practical application. Herein, we report a covalent coupling strategy for TMS-based anode materials using amide linkages to bind TMSs and carbon nanotubes (CNTs). In the synthesis, the thiourea acts as not only the capping agent for morphology control but also the linking agent for the covalent coupling. As a proof of concept, the covalently coupled ZnS/CNT composite (CC-ZnS/CNT) has been prepared, with ZnS nanoparticles (∼10 nm) tightly anchored on CNT bundles. The compact ZnS-CNT heterojunctions are greatly beneficial to facilitating the electron/ion transfer and ensuring structural stability. Due to the strong coupling interaction between ZnS and CNTs, the composite presents prominent pseudocapacitive behavior and highly reversible electrochemical processes, thus leading to superior long-term stability and excellent rate capability, delivering reversible capacities of 333 mAh g-1 at 2 A g-1 over 4000 cycles for LIBs and 314 mAh g-1 at 5 A g-1 after 500 cycles for SIBs. Consequently, CC-ZnS/CNT exhibits great competence for applications in LIBs and SIBs, and the covalent coupling strategy is proposed as a promising approach for designing high-performance anode materials.

A Novel Apolipoprotein E Antagonist Functionally Blocks Apolipoprotein E Interaction With N-terminal Amyloid Precursor Protein, Reduces ß-Amyloid-Associated Pathology, and Improves Cognition.

BACKGROUND: The ɛ4 isoform of apolipoprotein E (apoE4) is a major genetic risk factor for the development of sporadic Alzheimer's disease (AD), and its modification has been an intense focus for treatment of AD during recent years. METHODS: We investigated the binding of apoE, a peptide corresponding to its low-density lipoprotein receptor binding domain (amino acids 133-152; ApoEp), and modified ApoEp to amyloid precursor protein (APP) and their effects on amyloid-ß (Aß) production in cultured cells. Having discovered a peptide (6KApoEp) that blocks the interaction of apoE with N-terminal APP, we investigated the effects of this peptide and ApoEp on AD-like pathology and behavioral impairment in 3XTg-AD and 5XFAD transgenic mice. RESULTS: ApoE and ApoEp, but not truncated apoE lacking the low-density lipoprotein receptor binding domain, physically interacted with N-terminal APP and thereby mediated Aß production. Interestingly, the addition of 6 lysine residues to the N-terminus of ApoEp (6KApoEp) directly inhibited apoE binding to N-terminal APP and markedly limited apoE- and ApoEp-mediated Aß generation, presumably through decreasing APP cellular membrane trafficking and p44/42 mitogen-activated protein kinase phosphorylation. Moreover, while promoting apoE interaction with APP by ApoEp exacerbated Aß and tau brain pathologies in 3XTg-AD mice, disrupting this interaction by 6KApoEp ameliorated cerebral Aß and tau pathologies, neuronal apoptosis, synaptic loss, and hippocampal-dependent learning and memory impairment in 5XFAD mice without altering cholesterol, low-density lipoprotein receptor, and apoE expression levels. CONCLUSIONS: These data suggest that disrupting apoE interaction with N-terminal APP may be a novel disease-modifying therapeutic strategy for AD.

[Application of gold nanoparticles-based lateral flow strip for dengue virus detection based on strand-displacement amplification].

OBJECTIVE: To investigate the rapid and accurate method for the detection of dengue virus (DENV) by using nicking enzyme assisted strand-displacement amplification (SDA) combined with gold nanoparticles-based lateral flow strip. METHODS: Total RNA of the virus was extracted by using magnetic beads method and transcribed to cDNA for SDA detection system. Nicking enzyme-assisted method was used for detecting DENV, and agarose gel electrophoresis was used for analyzing the sensitivity of SDA amplification products. A gold nanoparticles-based lateral flow strip was developed based on the principle of nucleic acid base complementary pairing to design the test line and control line. The gold particles were prepared by using sodium citrate reduction method for gold nanoparticles-based lateral flow strip construction. RESULTS: The sensitivity of the SDA method was 10 fmol/L, and the sensitivity of gold nanoparticles-based lateral flow strip based on SDA method was also 10 fmol/L. In a linear range from 10 fmol/L to 1012 fmol/L, the corresponding linear correlation coefficient (R2) of DENV was 0.98. The specificity of nanoparticles-based lateral flow strip based on SDA for DENV detection was high, which was no crossing with other control groups. CONCLUSIONS: A gold nanoparticles-based lateral flow strip based on SDA method for DENV detection has been established, which is convenient, fast, and the result is visible to naked eyes.

Mesoporous Graphitic Carbon-Encapsulated Fe2 O3 Nanocomposite as High-Rate Anode Material for Sodium-Ion Batteries.

A mesoporous graphitic carbon-encapsulated Fe2 O3 nanocomposite is synthesized as a superior anode material for sodium-ion batteries. A threefold strategy is adopted to achieve a high rate performance. First, the mesoporous structure with high specific surface area and large pore volume facilitates the transfer of electrolyte and accommodates the large volume change. Secondly, graphitic carbon encapsulation further improves the electronic conductivity of the nanocomposite. Finally, ultrafine Fe2 O3 nanocrystals effectively shorten the Na+ diffusion length. Consequently, this nanocomposite exhibits stable and fast Na+ storage, thus leading to excellent rate capability and cyclability. Pseudocapacitive behavior is found to dominate in the redox reactions, accounting for the outstanding rate and cycling performance. In addition, full cells, assembled with O3-Na0.9 [Cu0.22 Fe0.30 Mn0.48 ]O2 as cathodes, present good electrochemical performance.

DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells.

Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of Rex1, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFß pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies.

Effects of WT1 down-regulation on oocyte maturation and preimplantation embryo development in pigs.

The Wilms' tumour 1 (WT1) gene originally identified as a tumour suppressor associated with WTs encodes a zinc finger-containing transcription factor that is expressed in multiple tissues and is an important regulator of cellular and organ growth, proliferation, development, migration and survival. However, there is a deficiency of data regarding the expression and function of WT1 during oocyte maturation and preimplantation embryonic development. Herein, we sought to define the expression characteristics and functions of WT1 during oocyte maturation and preimplantation embryonic development in pigs. We show that WT1 is expressed in porcine oocytes and at all preimplantation stages in embryos generated by ICSI. We then evaluated the effects of down-regulating WT1 expression at germinal vesicle and early ICSI stages using a recombinant plasmid (pGLV3-WT1-shRNA). Down-regulation of WT1 did not affect oocyte maturation but significantly decreased preimplantation embryonic development and increased apoptosis in blastocysts. These results indicate that WT1 plays important roles in the development of porcine preimplantation embryos.

Effects of WT1 gene downregulation on apoptosis in porcine fetal fibroblasts.

Wilms' tumor gene 1 (WT1) is located on chromosome 11p13. Besides a role in the development of Wilms' tumor, specific mutations in the Zn finger region are found in Denys-Drash syndrome and Frasier syndrome, both characterized by urogenital abnormalities, sometimes in combination with Wilms' tumor. Our past study shows that WT1 is expressed in porcine kidney fibroblasts (PKFs) and swine testis cells (ST cells) and is essential for the maintenance of the development and survival of PKFs and ST cells. But we do not know whether WT1 gene was expressed in porcine fetal fibroblasts or not. To further explore whether WT1 was expressed in porcine fetal fibroblasts (PFFs) and its contribution to cell apoptosis, RT-PCR, immunocytochemical staining, and Western blot were used to detect the expression of WT1, the recombinant plasmids of pLV3-WT1 short hairpin ribonucleic acid (shRNA) were used to downregulate the WT1 gene in porcine fetal fibroblasts, and the role of WT1 in cell proliferation was examined by apoptosis analysis also. Our results indicated that WT1 was expressed in PFFs, the pLV3-WT1 shRNA dramatically reduced the expression of WT1, and downregulation of WT1 directly led to early cell apoptosis by downregulating the expression of antiapoptotic gene Bcl-2 and upregulating the expression of proapoptotic gene Bax in PFFs. Our results demonstrate that WT1 is also essential for the maintenance of the survival of PFFs.

miRNAs promote generation of porcine-induced pluripotent stem cells.

The pigs have similarities of organ size, immunology and physiology with humans. Porcine-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. Here, we established piPSCs induced from porcine fetal fibroblasts by the retroviral overexpression of Oct4, Sox2, Klf4, and c-Myc. The piPSCs not only express pluripotent markers but also have the capacity for differentiation in vivo and in vitro, including EB and teratoma formation. We supplemented microRNAs during the induction process because miR-302a, miR-302b, and miR-200c have been reported to be highly expressed in human and mouse embryonic stem cells and in iPSCs. In this study, we found that the overexpression of miR-302a, miR-302b, and miR-200c effectively improved the reprogramming efficiency and reduced the induction time for piPSCs in the OSKM and OSK induction systems. Due to the similar induction efficiency of 4F-induced piPSCs or of three factors combined with miR-302a, miR-302b, and miR-200c (3F-miRNA-induced piPSCs), we recommend the addition of miRNAs instead of c-Myc to reduce the tumorigenicity of piPSCs.

Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells.

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1 (OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, MYC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and MYC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.

The effects of the WT1 gene on apoptosis and development-related gene expression in porcine kidney fibroblasts and swine testis cells.

The Wilm's tumor 1 gene (WT1) encodes zinc finger proteins that function as tumor suppressors, and play important roles in the development of the genito-urinary system and other organs. Its precise function in the development of porcine tissues and organs, which is an attractive transplantation resource for certain human diseases, is still unclear. Here, we sought to define the role of WT1 in porcine kidney and testis tissues using porcine kidney fibroblasts (PKFs) and swine testis (ST) cells as in vitro models, both of which express WT1. The recombinant plasmids pLV3-WT1 shRNA and pIRES2 -WT1-EGFP were constructed to respectively down- and up-regulate the WT1 gene in porcine cells. The role of WT1 in cell proliferation was investigated by RT-PCR, immunocytochemical staining, apoptosis analysis, and Western blot. The pLV3-WT1 shRNA dramatically reduced WT1 expression at both the transcription and protein levels. The down-regulation of WT1 directly led to early cell apoptosis, and changes in Sf1, Sox9, and Gdnf gene expression in PKFs and ST cells. In contrast, up-regulation of WT1 gave no obvious phenotype in ST cells. Our results demonstrate that WT1 is essential for the survival of PKFs and ST cells because it regulates apoptosis- and development-related genes in the cells; however, no obvious effect was observed when WT1 was over-expressed.

Proliferative capacity and pluripotent characteristics of porcine adult stem cells derived from adipose tissue and bone marrow.

Direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) provides an invaluable resource for regenerative medicine. Because of some ethical and logistical barriers, human iPSCs cannot be used to generate a chimera, which is one of markers representing pluripotency. As the most attractive model for preclinical studies, pigs offer another path to improve clinical medicine. In this study, porcine adult stem cells (pASCs), including adipose mesenchymal stem cells (AMSCs) and bone marrow mesenchymal stem cells (BMSCs), were collected and cultured under the same conditions in vitro. Real-time PCR, immunocytochemical staining, apoptosis analysis, and induced differentiation and reprogramming techniques were used to investigate the proliferative capacity and pluripotent characteristics of pASCs. Our results showed that both AMSCs and BMSCs displayed a similar immunophenotype, and their proliferative capacity appeared as a downward trend as the cell passage number increased. The cell proliferative capacity of AMSCs was significantly lower than that of BMSCs (p<0.05). Moreover, each type of pASCs went through 20 passages without undergoing alterations in the expression of reprogramming transcriptional factors (Oct4, Sox2, c-Myc, and Nanog). All pASCs had adipogenic and osteogenic differentiation potential. In addition, they also could be reprogrammed to pig induced pluripotent stem cells (piPSCs) with similar time and efficiency. In conclusion, porcine BMSCs had a higher proliferative capacity than AMSCs, and the pluripotency of pASCs was stable in long-term culture.