Nuciferine prevents obesity by activating brown adipose tissue.

Increasing evidence suggests that brown adipose tissue (BAT) plays an important role in obesity and related diseases. Increasing the amount or activity of BAT could prevent obesity. Therefore, a safe and effective method of activating BAT is urgently required. Here, we evaluated the potential effects of lotus leaf extract (LLE) on BAT function. We found that LLE substantially increased UCP1 mRNA and protein levels as well as thermogenic protein expression in primary brown adipocytes. Additionally, LLE treatment reduced diet-induced obesity and improved glucose homeostasis owing to BAT activation and increased energy expenditure. We found that nuciferine, an active ingredient of LLE, could dose-dependently activate BAT in vitro and in vivo, alleviate diet-induced obesity, and improve glucose homeostasis by increasing energy expenditure. Mechanistically, we found that nuciferine induced PPARG coactivator 1 alpha (PGC1-α) expression, which is a key gene involved in mitochondrial biogenesis promoter activity, by directly binding to RXRA. Furthermore, RXRA knockdown abolished expression of the nuciferine-induced mitochondrial and thermogenesis-related gene in primary brown adipocytes. In summary, we found that LLE and nuciferine have a notable effect on BAT activation and highlight the potential applications of the main component of LLE in preventing obesity and treating metabolic disorders.

mTOR hypoactivity leads to trophectoderm cell failure by enhancing lysosomal activation and disrupting the cytoskeleton in preimplantation embryo.

BACKGROUND: Metabolic homeostasis is closely related to early impairment of cell fate determination and embryo development. The protein kinase mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism in the body. Inhibition of mTOR signaling in early embryo causes postimplantation development failure, yet the mechanisms are still poorly understood. METHODS: Pregnancy mice and preimplantation mouse embryo were treated with mTOR inhibitor in vivo and in vitro respectively, and subsequently examined the blastocyst formation, implantation, and post-implantation development. We used immunofluorescence staining, RNA-Seq smart2, and genome-wide bisulfite sequencing technologies to investigate the impact of mTOR inhibitors on the quality, cell fate determination, and molecular alterations in developing embryos. RESULTS: We showed mTOR suppression during preimplantation decreases the rate of blastocyst formation and the competency of implantation, impairs the post implantation embryonic development. We discovered that blocking mTOR signaling negatively affected the transformation of 8-cell embryos into blastocysts and caused various deficiencies in blastocyst quality. These included problems with compromised trophectoderm cell differentiation, as well as disruptions in cell fate specification. mTOR suppression significantly affected the transcription and DNA methylation of embryos. Treatment with mTOR inhibitors increase lysosomal activation and disrupts the organization and dynamics of the actin cytoskeleton in blastocysts. CONCLUSIONS: These results demonstrate that mTOR plays a crucial role in 8-cell to blastocyst transition and safeguards embryo quality during early embryo development.

Astragaloside-IV induces the differentiation of bone marrow mesenchymal stem cells into osteoblasts through NMUR2-mediated Wnt/ß-catenin pathway.

Aim: Given that astragaloside-IV (As-IV) has the ability to promote osteogenic differentiation, its mechanism is worthy of exploration. Methods: The effect of As-IV on rat tibial defects was examined by histopathological staining and MiR-CT scan. The alkaline phosphatase (ALP) content, osteogenic differentiation-related gene expressions, and mineralized nodule formation in bone marrow mesenchymal stem cells (BMSCs) were detected. Results: As-IV repaired tibial defects of rats. As-IV or neuromedin receptor 2 (NMUR2) overexpression elevated ALP content, mineralized nodules, osteogenic differentiation-related genes, ß-catenin and NMUR2 levels, the effects of which were reversed by NMUR2 silencing or Wnt/ß-catenin pathway inhibitors. Conclusion: As-IV regulates the Wnt/ß-catenin pathway through NMUR2 to promote the repair of tibial defects in rats and the differentiation of BMSCs into osteoblasts.

Temperature-dependent photoluminescence properties of single defects in AlGaN micropillars.

Single-photon emitters (SPEs) are attractive as integrated platforms for quantum applications in technologically mature wide-bandgap semiconductors since their stable operation at room temperature or even at high temperatures. In this study, we systematically studied the temperature dependence of the SPE in AlGaN micropillar by experiment. The photoluminescence (PL) spectrum, PL intensity, radiative lifetime and second-order autocorrelation function measurements are investigated over the temperature range from 303 to 373 K. The point defects of AlGaN show strong zero phonon line in the wavelength range of 800-900 nm and highly antibunched photon emission even up to 373 K. Our study reveals a possible mechanism for linewidth broadening in AlGaN SPE at high temperatures. This indicates a possible key for on-chip integration applications based on this material operating at high temperatures.

High-Efficiency Differentiation of Human Pluripotent Stem Cells to Hematopoietic Stem/Progenitor Cells in Random Positioning Machine Bioreactors.

Human pluripotent stem cells (PSCs) are known to differentiate into almost all the blood lineage cells in vitro and hold a great promise for studying human early hematopoietic development and have a huge potential in the treatment of hematological disorders. Although several methods of hematopoietic stem/progenitor cell (HSPC) differentiation have been developed, the HSPC yields achieved using these strategies are not yet available for clinical application. Recently, bioreactor-based devices and biochemical factors synergistically have been used to induce hematopoietic differentiation and showed a potential role in hematopoiesis. This chapter describes a protocol for using a random positioning machine bioreactor to culture human PSCs and the large-scale production of HPCs. Techniques for characterizing the differentiated cells and assessing the efficiency of hematopoietic differentiation in the bioreactor with immunostaining and flow cytometry are also presented.

Research Status, Synthesis and Clinical Application of Recently Marketed and Clinical BCR-ABL Inhibitors.

Tyrosine kinases expressed by BCR-ABL fusion genes can cause changes in cell proliferation, adhesion, and survival properties, which are the main causes of chronic myelogenous leukemia (CML). Inhibiting the activity of BCR-ABL tyrosine kinase has become one of the effective methods for the treatment of chronic myelogenous leukemia. Initially, imatinib was the first small molecule of BCR-ABL tyrosine kinases inhibitors (TKIs) for the effective treatment of chronic myelogenous leukemia. Later, due to the emergence of various BCR-ABL mutations, especially T315I mutation, imatinib developed strong resistance. The second-generation kinase inhibitors dasatinib and nilotinib were able to overcome most of the mutation resistance but not T315I mutations. Therefore, in order to further overcome the problem of drug resistance, new types of KTIs such as flumatinib and radotinib have been developed, providing more options for clinical treatment. Some new drugs have entered clinical trials. In this review, two new BCRABL inhibitors (flumatinib and radotinib) and five new BCR-ABL inhibitors have been introduced into the clinical market in recent years. We reviewed their research status, synthesis methods, and clinical applications.

Identification of Hub mRNAs and lncRNAs in Atrial Fibrillation Using Weighted Co-expression Network Analysis With RNA-Seq Data.

Atrial fibrillation (AF)/paroxysmal AF (PAF) is the main cause of cardiogenic embolism. In recent years, the progression from paroxysmal AF to persistent AF has attracted more and more attention. However, the molecular mechanism of the progression of AF is unclear. In this study, we performed RNA sequencing for normal samples, paroxysmal AF and persistent AF samples to identify differentially expressed gene (DEG) and explore the roles of these DEGs in AF. Totally, 272 differently expressed mRNAs (DEmRNAs) and 286 differentially expressed lncRNAs (DElncRNAs) were identified in paroxysmal AF compared to normal samples; 324 DEmRNAs and 258 DElncRNAs were found in persistent atrial fibrillation compared with normal samples; and 520 DEmRNAs and 414 DElncRNAs were identified in persistent AF compared to paroxysmal AF samples. Interestingly, among the DEGs, approximately 50% were coding genes and around 50% were non-coding RNAs, suggesting that lncRNAs may also have a crucial role in the progression of AF. Bioinformatics analysis demonstrated that these DEGs were significantly related to regulating multiple AF associated pathways, such as the regulation of vascular endothelial growth factor production and binding to the CXCR chemokine receptor. Furthermore, weighted gene co-expression network analysis (WGCNA) was conducted to identify key modules and hub RNAs and lncRNAs to determine their potential associations with AF. Five hub modules were identified in the progression of AF, including blue, brown, gray, turquoise and yellow modules. Interestingly, blue module and turquoise module were significantly negatively and positively correlated to the progression of AF respectively, indicating that they may have a more important role in the AF. Moreover, the hub protein-protein interaction (PPI) networks and lncRNA-mRNA regulatory network were constructed. Bioinformatics analysis on the hub PPI network in turquoise was involved in regulating immune response related signaling, such as leukocyte chemotaxis, macrophage activation, and positive regulation of α-ß T cell activation. Our findings could clarify the underlying molecular changes associated fibrillation, and provide a useful resource for identifying AF marker.

Astragaloside IV Improves Tibial Defect in Rats and Promotes Proliferation and Osteogenic Differentiation of hBMSCs through MiR-124-3p.1/STAT3 Axis.

Astragaloside IV (AST-IV) facilitates the proliferation and migration of osteoblast-like cells. We sought to explore the effect and potential mechanism of AST-IV on regeneration of tibial defects. To reveal the effect of AST-IV on regeneration of tibial defects in rat, HE staining and microcomputed tomography (µCT) were performed on tibial bone. The binding relationship between miR-124-3p.1 and STAT3 was analyzed by TargetScan V7.2 and a dual-luciferase reporter assay. Human bone marrow mesenchymal stromal/stem cells (hBMSCs) were identified by morphological observation and flow-cytometric analysis. To reveal the effect and mechanism of AST-IV on phenotypes of hBMSCs, hBMSCs were treated with AST-IV, miR-124-3p.1 mimic, and pcDNA-STAT3, and cell viability, cell cycle, ALP activity, and calcium deposition of hBMSCs in vitro were determined by MTT, flow-cytometric analysis, ELISA, and Alizarin red staining, respectively. The expressions of osteoblast marker molecules (RUNX2, OCN, Smad4), miR-124-3p.1, and STAT3 were indicated by RT-qPCR and Western blot. AST-IV decreased miR-124-3p.1 expression, increased STAT3 expression in tibial bone defects, and promoted regeneration of tibial bone defects in a concentration-dependent manner. The hBMSCs appeared spindle-shaped and were positive for CD105, but negative for CD34. MiR-124-3p.1 negatively regulated STAT3 expression in hBMSCs under osteogenic conditions. AST-IV promoted viability, cell cycle, ALP activity, and osteogenic differentiation of hBMSCs along with increased expressions of osteoblast marker molecules, which was partially reversed by miR-124-3p.1 overexpression. However, the effect of miR-124-3p.1 overexpression on hBMSCs was also partially reversed by STAT3 overexpression. AST-IV improves tibial defects in rats and promotes proliferation and osteogenic differentiation of hBMSCs through the miR-124-3p.1/STAT3 axis.

Simulated Microgravity Potentiates Hematopoietic Differentiation of Human Pluripotent Stem Cells and Supports Formation of 3D Hematopoietic Cluster.

Microgravity has been shown to induces many changes in proliferation, differentiation and growth behavior of stem cells. Little is known about the effect of microgravity on hematopoietic differentiation of pluripotent stem cells (PSCs). In this study, we used the random position machine (RPM) to investigate whether simulated microgravity (SMG) allows the induction of hematopoietic stem/progenitor cell (HSPC) derived from human embryonic stem cells (hESCs) in vitro. The results showed that SMG facilitates hESCs differentiate to HSPC with more efficient induction of CD34+CD31+ hemogenic endothelium progenitors (HEPs) on day 4 and CD34+CD43+ HSPC on day 7, and these cells shows an increased generation of functional hematopoietic cells in colony-forming unit assay when compared with normal gravity (NG) conditions. Additionally, we found that SMG significantly increased the total number of cells on day 4 and day 7 which formed more 3D cell clusters. Transcriptome analysis of cells identified thousands of differentially expressed genes (DEGs) between NG and SMG. DEGs down-regulated were enriched in the axonogenesis, positive regulation of cell adhesion, cell adhesion molecule and axon guidance, while SMG resulted in the up-regulation of genes were functionally associated with DNA replication, cell cycle, PI3K-Akt signaling pathway and tumorigenesis. Interestingly, some key gene terms were enriched in SMG, like hypoxia and ECM receptor interaction. Moreover, HSPC obtained from SMG culture conditions had a robust ability of proliferation in vitro. The proliferated cells also had the ability to form erythroid, granulocyte and monocyte/macrophage colonies, and can be induced to generate macrophages and megakaryocytes. In summary, our data has shown a potent impact of microgravity on hematopoietic differentiation of hPSCs for the first time and reveals an underlying mechanism for the effect of SMG on hematopoiesis development.

Development of mouse preimplantation embryos in space.

The development of life beyond planet Earth is a long-standing quest of the human race, but whether normal mammalian embryonic development can occur in space is still unclear. Here, we show unequivocally that preimplantation mouse embryos can develop in space, but the rate of blastocyst formation and blastocyst quality are compromised. Additionally, the cells in the embryo contain severe DNA damage, while the genome of the blastocysts developed in space is globally hypomethylated with a unique set of differentially methylated regions. The developmental defects, DNA damage and epigenetic abnormalities can be largely mimicked by the treatment with ground-based low-dose radiation. However, the exposure to simulated microgravity alone does not cause major disruptions of embryonic development, indicating that radiation is the main cause for the developmental defects. This work advances the understanding of embryonic development in space and reveals long-term extreme low-dose radiation as a hazardous factor for mammalian reproduction.

MicroRNA-7-5p Promotes Cisplatin Resistance of Cervical Cancer Cells and Modulation of Cellular Energy Homeostasis by Regulating the Expression of the PARP-1 and BCL2 Genes.

BACKGROUND Resistance to cisplatin results in recurrence or relapse of cervical cancer in women. An understanding of the mechanisms of cisplatin resistance will be important to improve the efficacy of cisplatin treatment. The aim of this study was to investigate the role of microRNA-7-5p (mir-7-5p) in cisplatin-resistant cervical cancer cells in vitro. MATERIAL AND METHODS The expression levels of miR-7-5p were detected in cisplatin-resistant cervical cancer cells, HeLa, and SiHa cells (HPV16-positive), and in clinical tissue samples, using miR-7-5p inhibition and a luciferase reporter assay. Fifteen paired cervical cancer tissue samples and adjacent normal cervical tissues were obtained from 15 patients who underwent surgery for cervical cancer. Western blot and flow cytometry were used to investigate cell apoptosis. The expression of mir-7-5p was detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS The level of miR-7-5p was increased in cisplatin-resistant HeLa and SiHa cervical cancer cells. Increased expression of miR-7-5p inhibited DNA repair by modulating the expression of poly (ADP-ribose) polymerase 1 (PARP-1), reducing energy consumption, and promoting autophagy via suppression of the expression of Bcl-2. These findings supported that increasing energy generation and reducing energy consumption, resulted in miR-7-5p maintaining energy homeostasis during cisplatin treatment. CONCLUSIONS The findings of this study showed that there was a protective role of miR-7-5p in cervical cancer cells treated with cisplatin and that miR-7-5p expression maintained energy homeostasis in cisplatin-resistant cervical cancer cells. However, miR-7-5p reduced energy consumption via inhibiting PARP-1 expression, and miR-7-5p increased energy generation by suppressing the expression of Bcl-2.

Effect of microgravity on proliferation and differentiation of embryonic stem cells in an automated culturing system during the TZ-1 space mission.

OBJECTIVE: Despite a great number of studies analysing the effects of microgravity on stem cell proliferation and differentiation, few of them have focused on real-time imaging estimates in space. Herein, we utilized the TZ-1 cargo spacecraft, automatic cell culture equipment and live cell imaging techniques to examine the effects of real microgravity on the proliferation and differentiation of mouse embryonic stem cells (mESCs). MATERIALS AND METHODS: Oct4-GFP, Brachyury-GFP mESC and Oct4-GFP mESC-derived EBs were used as experimental samples in the TZ-1 spaceflight mission. These samples were seeded into chambers, cultured in an automatic cell culture device and were transported into space during the TZ-1 mission. Over 15 days of spaceflight, bright field and fluorescent images of cell growth were taken in micrography, and the medium was changed every day. Real-time image data were transferred to the ground for analysis. RESULTS: Space microgravity maintains stemness and long-term survival of mESCs, promising 3D aggregate formation. Although microgravity did not significantly prevent the migration of EBs on the ECM substrate, it did prevent terminal differentiation of cells. CONCLUSIONS: This study demonstrates that space microgravity might play a potential role in supporting 3D cell growth and maintenance of stemness in embryonic stem cells, while it may negatively affect terminal differentiation.

Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs.

The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA 1 . Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress2,3 and metabolic disorders4-6. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m5C, m2G) in sperm 30-40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA 'coding signature' that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m5C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.

Expansion of Hair Follicle Stem Cells Sticking to Isolated Sebaceous Glands to Generate in Vivo Epidermal Structures.

Hair follicle stem cells (HFSCs) are considered one of the useful donor cell types for skin regenerative medicine owing to their robust proliferative capacity and multipotency. However, methods for easily and effectively obtaining HFSCs from a limited skin biopsy are still lacking. Here we report a novel approach for obtaining a subpopulation of HFSCs from a small skin sample from the rat tail, which uses the sebaceous glands (SGs) to capture the adjacent HFSCs. By means of organ culture, keratinocytes were expanded from the detached SGs, which also included adherent HFSCs from the hair follicle that could be passaged at the single-cell level. These SG-captured keratinocytes strongly expressed the basal layer markers K14, integrin α6, and p63; the bulge stem cell marker K15; and the upper isthmus stem cell marker Plet1. Furthermore, we reconstituted new epidermis, hair follicles, and SGs from the SG-captured keratinocytes using an easily operated, modified skin reconstitution assay based on silicone gel sheeting. This study suggests that the SGs could be an accessible capturer to harvest the adjacent HFSC subpopulation, particularly when the donor tissue is limited.

Exogenous R-Spondin1 Induces Precocious Telogen-to-Anagen Transition in Mouse Hair Follicles.

R-spondin proteins are novel Wnt/ß-catenin agonists, which signal through their receptors leucine-rich repeat-containing G-protein coupled receptor (LGR) 4/5/6 and substantially enhance Wnt/ß-catenin activity. R-spondins are reported to function in embryonic development. They also play important roles in stem cell functions in adult tissues, such as the intestine and mammary glands, which largely rely on Wnt/ß-catenin signaling. However, in the skin epithelium and hair follicles, the information about R-spondins is deficient, although the expressions and functions of their receptors, LGR4/5/6, have already been studied in detail. In the present study, highly-enriched expression of the R-spondin family genes (Rspo1/2/3/4) in the hair follicle dermal papilla is revealed. Expression of Rspo1 in the dermal papilla is specifically and prominently upregulated before anagen entry, and exogenous recombinant R-spondin1 protein injection in mid-telogen leads to precocious anagen entry. Moreover, R-spondin1 activates Wnt/ß-catenin signaling in cultured bulge stem cells in vitro, changing their fate determination without altering the cell proliferation. Our pioneering study uncovers a role of R-spondin1 in the activation of cultured hair follicle stem cells and the regulation of hair cycle progression, shedding new light on the governance of Wnt/ß-catenin signaling in skin biology and providing helpful clues for future treatment of hair follicle disorders.

[Mechanism of serotonin-promoted synthesis of osteoblast type I collagen].

OBJECTIVE: To explore the mechanism of serotonin-promoted osteoblast differentiation. METHODS: Expression levels of collagen I and lysyl oxidase (LOX) in osteoblast were measured by RT-PCR after treated by (50, 100, 200 and 400 ng/L) serotonin. LOX siRNA effect was measured by Western blot, and protein levels of collagen I were determined by ELISA after treated by serotonin. Expression levels of Smad2 and Smad3 in osteoblasts were also measured by RT-PCR after treated by serotonin.Moreover, expression levels of LOX were measured by RT-PCR after Smad3 was knockout. RESULTS: Serotonin promoted collagen I and LOX expression. The expression level of collagen I was significantly decreased by LOX siRNA. Furthermore, serotonin up-regulated the expression of Smad2 and Smad3 in osteoblasts, and the expression level of LOX was inhibited by Smad3 siRNA. CONCLUSION: Serotonin promoted collagen I expression by activating Smads signaling pathway and up-regulating the LOX expression.

Spatiotemporal Expression of p63 in Mouse Epidermal Commitment.

The embryonic surface ectoderm is a simple flat epithelium consisting of cells that express the cytokeratins K8/K18. Before stratification, K5/K14 expression substitutes K8/K18 expression, marking the event called epidermal commitment. Previous studies show that the transcription factor p63 plays an essential role in epidermal commitment. However, detailed expression information of p63 during early epidermal development in mice is still unclear. We systematically studied the expression pattern of p63 in mouse epidermal commitment, together with K8 and K5. We show that p63 expression could be detected as early as E8.5 in mouse embryos preceding epidermal commitment. p63 expression first appears near the newly formed somites and the posterior part of the embryo, further expanding to the whole embryonic surface with particular enrichment in the first branchial arches and the limb buds. ΔNp63 is the major class of isoforms expressed in this period. Relative expression intensity of p63 depends on the embryonic position. In summary, there is a sequential and regular expression pattern of K8, p63 and K5 in mouse epidermal commitment. Our study not only contributes to understanding the early events during epidermal development but also provides a basal tool to study the function of p63 in mammals.

[EFFECT OF WNT6 IN PROLIFERATION, DIFFERENTIATION, AND MIGRATION OF BONE MARROW MESENCHYMAL STEM CELLS].

OBJECTIVE: To explore the potential role of WNT6 in the proliferation, differentiation, and migration of bone marrow mesenchymal stem cells (BMSCs). METHODS: Mouse BMSCs were cultured to the cell fusion of 30%-50%, and divided into different groups. WNT6 knockdown included 3 experiment groups: cells transfected with WNT6 specific short hairpin RNA (shRNA) (group A1), cells transfected with control shRNA group (group B1), and non- transfected cells (group C1). WNT6 over-expression included 3 groups: cells transfected with WNT6 recombinant plasmid (group A2), cells transfected with blank vector (group B2 ), and non-transfected cells (group C2). After transfection, the stably transfected cells were cultured for 48 hours. Cell morphology was observed under inverted microscope; real-time fluorescent quantitative PCR was used to analyze WNT6 mRNA levels; Western blot was used to detect WNT6 and Ki67 protein expressions; cell proliferation was assayed by MTT method, and cell migration was detected by Transwell assay. After cells were cultured in osteogenic differentiation medium for 12 days, the alkaline phosphatase (ALP) activity and calcium deposits were detected by biochemical determination. RESULTS: The inverted microscope observation showed that the cell morphology were similar among groups A1, B1, C1, and A2, B2, C2. The WNT6 mRNA and protein levels, Ki67 protein level, cell proliferation, cell migration, ALP activity, and calcium deposition in group Al were all significantly lower than those in groups B1 and C1 (P < 0.05), but there was no significant difference between groups B1 and C1 (P > 0.05). On the contrary, the above indexes in group A2 were all significantly higher than those in groups B2 and C2 (P < 0.05), but no significant difference was shown between groups B2 and C2 (P > 0.05). CONCLUSION: WNT6 can promote the proliferation and migration, as well as can enhance osteogenic differentiation ability in mouse BMSCs.