Strengthening and Toughening CNTs/Mg Composites by OpTimizing the Grinding Time of Magnesium Powder.

In this paper, CNT/Mg composites with high compressive properties were prepared by using Ni-plated CNT and pure magnesium powder as raw materials through the grinding of magnesium powder, ball-milling mixing and hot-pressing sintering. The effect of grinding time for finer magnesium powder on the microstructure and properties of the final composites was studied mainly by SEM, XRD, HRTEM and compression tests. The results show that with the prolongation of milling time, the magnesium particle size decreases gradually and the CNT dispersion becomes more uniform. Moreover, the nickel layer on the surface of CNT reacts with highly active broken magnesium powder in the sintering process to generate MgNi2 intermediate alloy, which significantly improves interface bonding. The strength and fracture strain of composites are significantly increased by the combined action of the uniform distribution of CNTs and strong interface bonding from the MgNi2 phase. The compressive strength, yield strength and fracture strain of the composites, prepared with a 60 h grinding of magnesium powder, reached 268%, 272% and 279% of those in composites without the grinding of magnesium powder.

Strengthening Ni-Coated CNT/Mg Composites by Optimizing the CNT Content.

The dispersion of carbon nanotubes (CNTs) is the bottleneck in CNT-reinforced metal matrix composites. In this work, CNT/Mg composites were prepared by grinding Mg powder and then dispersing CNTs via ball milling and hot pressing. The uniform distribution of Ni-coated CNTs in the matrix was achieved by optimizing the content of CNTs. Scanning electron microscope, high-resolution transmission electron microscopy and X-ray diffraction, optical microscopy, and compression tests were employed. With the CNT content being less than 1%, the CNTs can be evenly distributed in CNT/Mg composites, resulting in a sharp increase in strength. However, with the higher CNT content, the CNTs gradually cluster, leading decreased fracture strain and strength. Furthermore, the coated Ni in the CNTs reacts with the magnesium matrix and completely transforms into Mg2Ni, significantly enhancing the interface bonding. This strong interface bonding and the diffusely distributed Mg2Ni in the matrix significantly strengthen the CNT/Mg composite.

Influence of Soft Phase and Carbon Nanotube Content on the Properties of Hierarchical AZ61 Matrix Composite with Isolated Soft Phase.

Carbon nanotube-reinforced magnesium matrix (CNTs/Mg) composite has great application potential in the transportation industry, but the trade-off between strength and ductility inhibits its widespread application. In order to balance the strength and plasticity of the composite, in this work, on the basis of the AZ61 matrix composite homogeneously reinforced by Ni-coated CNTs (hard phase), 30 vol.% large-size AZ61 particles are introduced as an isolated soft phase to fabricate hierarchical CNTs/AZ61 composites. The compression tests show the fracture strain and compressive strength of this composite increases by 54% and 8%, respectively, compared with homogeneous CNTs/AZ61 composite. During deformation, the hard phase is mainly responsible for bearing the load and bringing high strength, due to the precipitation of the Mg17Al12 phase, uniformly dispersed CNT and strong interfacial bonding of the CNTs/Mg interface through nickel plating and interfacial chemical reaction. Furthermore, the toughening of the soft phase results in high ductility. With the increase in CNT content, the compressive strength of composites is nearly unchanged but the fracture strain gradually decreases due to the stress concentration of CNT and its agglomeration.

Effect of CNT Content on Microstructure and Properties of CNTs/Refined-AZ61 Magnesium Matrix Composites.

Carbon nanotubes (CNTs) reinforced magnesium matrix composites have great application potential in the transportation industry, but the low absolute strength is the main obstacle to its application. In this paper, copper-coated CNTs and AZ61 powder were used as raw materials to prepare CNTs/refined-AZ61 composites with good interfacial bonding, uniformly dispersed CNTs and fine grains by the process of ball milling refinement of AZ61 powder, ball milling dispersion and hot-pressing sintering. When the volume fraction of CNTs is less than or equal to 1 vol.%, CNTs can be uniformly dispersed and the yield strength and compressive strength of composites increase with higher CNT content. When the volume fraction of CNTs is 1 vol.%, the compressive strength and yield strength of composites reach 439 MPa and 361 MPa, respectively, which are 14% and 9% higher than those of matrix composites with nearly the same value of fracture strain. When the volume fraction of CNTs is greater than 1 vol.%, with the increase in CNT content, CNT clustering becomes more and more serious, resulting in a decrease in the strength and fracture strain of composites.

Correlation of plasma TSG-6 with cardiac function, myocardial fibrosis, and prognosis in dilated cardiomyopathy patients with heart failure. / 血浆TSG-6æ°´å¹³ä¸Žæ‰©å¼ åž‹å¿ƒè‚Œç— å¿ƒåŠ›è¡°ç«­æ‚£è€ å¿ƒåŠŸèƒ½ã€å¿ƒè‚Œçº¤ç»´åŒ–åŠé¢„åŽçš„ç›¸å ³æ€§.

OBJECTIVES: Tumor necrosis factor α stimulated gene 6 (TSG-6) protein is an inflammation-inducing protein. In recent years, TSG-6 protein has been found to play an anti-inflammatory and anti-fibrosis role in a variety of disease models. The level of TSG-6 protein in circulating blood is considered to be a biological indicator for the evaluation of acute coronary syndrome, severe infection, and other diseases, and it is closely related to the prognosis. The clinical correlation between TSG-6 protein and dilated cardiomyopathy (DCM) patients with heart failure has not been reported. This study aims to investigate the changes of plasma TSG-6 protein levels in cardiomyopathy patients with heart failure and its correlation with cardiac function, myocardial fibrosis, and prognosis. METHODS: Based on the prospective studies, a number of 90 DCM patients with heart failure were selected as a DCM heart failure group from Dec.1, 2019 to Sept.1, 2020. Thirty-nine healthy people were served as a control group. Plasma TSG-6, Collagen Ⅰ, Collagen III, and α-smooth muscle actin (α-SMA) were measured with ELISA test. Echocardiography was used to evaluate the structure and function of the heart. DCM patients with heart failure were followed up for 3 months. The patients were assigned into 2 groups according to whether they had major adverse cardiovascular events (MACE). The general clinical data, plasma TSG-6, Collagen Ⅰ, Collagen III, and α-SMA protein levels were compared between the control group and the DCM heart failure group. At the same time, the correlation between plasma TSG-6 protein level and cardiac function grade, myocardial fibrosis or prognosis of patients in the DCM heart failure group was analyzed. RESULTS: Compared with the control group, the heart rate, TSG-6, Collagen Ⅰ, Collage III, α-SMA, hemoglobin, atrial natriuretic peptide (NT-proBNP), hypersensitive C-reactive protein, aspartate aminotransferase, serum creatinine, lactate dehydrogenase, and left ventricular end diastolic diameter (LVEDD) increased significantly (all P<0.001). High-density lipoprotein, left ventricular short axis shortening rate (LVFS), and left ventricular ejection fraction (LVEF) decreased significantly in the DCM heart failure group (all P<0.001). Plasma levels of TSG-6 were positively correlated with NT-proBNP, Collagen Ⅰ, Collagen III, α-SMA, and LVEDD (all P<0.001), while they were negatively correlated with LVFS and LVEF (all P<0.001). With the increase of NYHA heart function classification, plasma levels of TSG-6, Collagen Ⅰ, Collagen III, and α-SMA increased significantly (all P<0.001). The increases in plasma levels of NT-proBNP and TSG-6 was associated with poor prognosis in DCM patients with heart failure (all P<0.05). The sensitivity and specificity of plasma NT-proBNP for evaluating the prognosis of DCM heart failure were 76.2% and 68.1%, respectively. The sensitivity and specificity of plasma TSG-6 for evaluating the prognosis of DCM heart failure were 95.2% and 66.7%, respectively. The sensitivity and specificity of plasma TSG-6 combined with NT-proBNP for prognostic evaluation of DCM heart failure were 85.7% and 81.2%, respectively. The specificity of plasma TSG-6 combined with NT-proBNP for the prognosis of heart failure was better than that of NT-proBNP or TSG-6 alone (P<0.001). CONCLUSIONS: The plasma levels TSG-6 in DCM patients with heart failure increase significantly, and the plasma levels TSG-6 could be used as a new predictor for cardiac function, myocardial fibrosis, and prognosis.

Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish.

Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F1 offspring. Notably, most of these maternal mutants displayed either sgRNA site-spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.

A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish.

Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(zpc:zcas9) with oocyte-specific cas9 expression, we have successfully obtained maternal or maternal-zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target dvl2 and dvl3a simultaneously and introduced this construct to the genome of Tg(zpc:zcas9) by meganuclease I-Sce I. As expected, we confirmed the existence of Mdvl2;Mdvl3a embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZdvl2;MZdvl3a embryos were also obtained by crossing the mutant carrying mosaic F0 female with dvl2+/-;dvl3a-/- male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs.

Rbm24 controls poly(A) tail length and translation efficiency of crystallin mRNAs in the lens via cytoplasmic polyadenylation.

Lens transparency is established by abundant accumulation of crystallin proteins and loss of organelles in the fiber cells. It requires an efficient translation of lens messenger RNAs (mRNAs) to overcome the progressively reduced transcriptional activity that results from denucleation. Inappropriate regulation of this process impairs lens differentiation and causes cataract formation. However, the regulatory mechanism promoting protein synthesis from lens-expressed mRNAs remains unclear. Here we show that in zebrafish, the RNA-binding protein Rbm24 is critically required for the accumulation of crystallin proteins and terminal differentiation of lens fiber cells. In the developing lens, Rbm24 binds to a wide spectrum of lens-specific mRNAs through the RNA recognition motif and interacts with cytoplasmic polyadenylation element-binding protein (Cpeb1b) and cytoplasmic poly(A)-binding protein (Pabpc1l) through the C-terminal region. Loss of Rbm24 reduces the stability of a subset of lens mRNAs encoding heat shock proteins and shortens the poly(A) tail length of crystallin mRNAs encoding lens structural components, thereby preventing their translation into functional proteins. This severely impairs lens transparency and results in blindness. Consistent with its highly conserved expression in differentiating lens fiber cells, the findings suggest that vertebrate Rbm24 represents a key regulator of cytoplasmic polyadenylation and plays an essential role in the posttranscriptional control of lens development.

Tumacrophage: macrophages transformed into tumor stem-like cells by virulent genetic material from tumor cells.

Tumor-associated macrophages are regarded as tumor-enhancers as they have key roles in the subversion of adaptive immunity and in inflammatory circuits that promote tumor progression. Here, we show that cancer cells can subvert macrophages yielding cells that have gained pro-tumor functions. When macrophages isolated from mice or humans are co-cultured with dead cancer cell line cells, induced to undergo apoptosis to mimic chemotherapy, up-regulation of pro-tumor gene expression was identified. Phagocytosis of apoptotic cancer cells by macrophages resulted in their transformation into tumor stem (initiating)-like cells, as indicated by the expression of epithelial markers (e.g., cytokeratin) and stem cell markers (e.g., Oct4) and their capability to differentiate in vitro and self-renew in serum-free media. Moreover, we identified a subset of monocytes/macrophages cells in the blood of cancer (breast, ovarian and colorectal) patients undergoing chemotherapy that harbor tumor transcripts. Our findings uncover a new role for macrophages in tumor development, where they can be transformed into tumor-like cells, potentially by horizontal gene transfer of tumor-derived genes, thus, by taking advantage of chemotherapy, these transformed macrophages promote tumor metastasis by escaping immune surveillance.

Staurosporine Induced Apoptosis May Activate Cancer Stem-Like Cells (CD44(+)/CD24(-)) in MCF-7 by Upregulating Mucin1 and EpCAM.

Malignant tumors recur after chemotherapy. A small population of cancer stem-like cells within tumors is now generally considered the prime source of the recurrence. To better understand how cancer stem-like cells induce relapse after fractionated chemotherapy, we examined changes in the CD44(+)/CD24(-) cancer stem-like cells population and behavior using the breast cancer cell line MCF-7. Our results show that apart from an increase in the CD44(+)/CD24(-) population, proliferation and clone formation, but not migration, were enhanced after recovery from apoptosis induced by two pulses of staurosporine (STS). The distribution of cells in the cell cycle differed between acutely induced apoptosis and fractionated chemotherapy. Sorted CD44(+)/CD24(-) stem-like cells from MCF-7 cells recovered from STS treatment possessed greater proliferation abilities. We also observed that mucin1 (MUC1) and Epithelial cell adhesion molecule (EpCAM) were up-regulated in abundance coincidently with proliferation and clone formation enhancement. Our findings suggest that fractionated chemotherapy induced apoptosis could stimulate cancer stem-like cell to behave with a stronger malignant property than cancer cells themselves and MUC1 and EpCAM are important factors involving in this process. By demonstrating changes in cancer stem cell during chemotherapy and identifying the crucial factors, we potentially can target them, to eradicate tumors and overcome cancer relapse.

Exposure of tumor-associated macrophages to apoptotic MCF-7 cells promotes breast cancer growth and metastasis.

Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer. To clarify the mechanisms underlying the crosstalk between TAMs and cancer stem cells (CSCs) in breast cancer recurrence and metastasis, we used a co-culture model of macrophages and apoptotic human breast cancer cell line MCF-7 cells to investigate the effects of TAMs on MCF-7 in vitro and in vivo. Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay. The macrophages exposed to apoptotic cells also induce an increase in the proportion of CD44+/CD24- cancer stem-like cells, as well as their proliferative ability accompanied with an increase in mucin1 (MUC1) expression. During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-ß1 and HIF-1α. Our results indicate that when cancer cells endure chemotherapy induced apoptosis, macrophages in their microenvironment can then activate cancer stem cells to promote cancer growth and metastasis by secreting IL-6, which activates STAT3 phosphorylation to regulate the transcription of its downstream target genes.

Role of glucokinase in the subcellular localization of glucokinase regulatory protein.

Glucokinase (GCK) is the rate-limiting enzyme of liver glucose metabolism. Through protein-protein interactions, glucokinase regulatory protein (GCKR) post-transcriptionally regulates GCK function in the liver, and causes its nuclear localization. However the role of GCK in regulating GCKR localization is unknown. In the present study, using in vitro and in vivo models, we examined the levels of GCK and GCKR, and their subcellular localization. We found that total cellular levels of GCKR did not vary in the in vivo models, but its subcellular localization did. In animals with normal levels of GCK, GCKR is mainly localized to the nuclei of hepatocytes. In seven-day old rats and liver-specific Gck gene knockout mice (animals that lack or have reduced levels of GCK protein), GCKR was found primarily in the cytoplasm. The interaction of GCK with GCKR was further examined using in vitro models where we varied the levels of GCK and GCKR. Varying the level of GCK protein had no effect on total cellular GCKR protein levels. Taken together, our results indicate that GCK is important for the localization of GCKR to the nucleus and raises the possibility that GCKR may have functions in addition to those regulating GCK activity in the cytoplasm.

An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology.

Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid ß (Aß) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aß abundance. What is more, the activity of the three key secretases in Aß formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.

Resistin disrupts glycogen synthesis under high insulin and high glucose levels by down-regulating the hepatic levels of GSK3ß.

The effect of mouse resistin on hepatic insulin resistance in vivo and in vitro, and its possible molecular mechanism were examined. Focusing on liver glycogen metabolism and gluconeogenesis, which are important parts of glucose metabolism, in primary cultures of rat hepatocytes we found that glycogen content was significantly lower (P<0.05) after treatment with recombinant murine resistin only in the presence of insulin plus glucose stimulation. Protein levels of factors in the insulin signaling pathway involved in glycogen synthesis were examined by Western blot analysis, with the only significant change observed being the level of phosphorylated (at Ser 9) glycogen synthase kinase-3ß (GSK-3ß) (P<0.001). No differences in the protein levels for the insulin receptor ß (IRß), insulin receptor substrates (IRS1 and IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) or their phosphorylated forms were observed between control and resistin treated primary rat hepatocytes. In a mouse model with high liver-specific expression of resistin, fasting blood glucose levels and liver glycogen content changed. Fasting blood glucose levels were significantly higher (P<0.001) in the model mice, compared to the control mice, while the glycogen content of the liver tissue was about 60% of that of the control mice (P<0.05). The gluconeogenic response was not altered between the experimental and control mice. The level of phosphorylated GSK-3ß in the liver tissue was also decreased (P<0.05) in the model mice, consistent with the results from the primary rat hepatocytes. Our results suggest that resistin reduces the levels of GSK-3ß phosphorylated at Ser 9 leading to impaired hepatic insulin action in primary rat hepatocytes and in a mouse model with high liver-specific expression of resistin.

Differential expression of genes associated with the progression of renal disease in the kidneys of liver-specific glucokinase gene knockout mice.

Liver glucokinase (GCK) deficient mice possess mild renal complications associated with diabetes. To investigate the progression of kidney disease and identify candidate genes involved in the pathogenesis of renal damage, we examined changes in tissue structure and gene expression in the kidneys of liver-specific GCK knockout (gckw/-) mice and age-matched normal wild-type control (gckw/w) mice as they aged. Suppression subtractive hybridization (SSH) was used to identify candidate genes that showed a pattern of differential expression between kidneys of gckw/- and gckw/w mice at 60 weeks of age. Differential expression of the candidate genes was examined by real-time qPCR in liver-specific gckw/- and gckw/w mice at 16, 26, 40, 60, and 85 weeks of age. Among the candidate genes, only glutathione peroxidase-3 (GPX3) was confirmed to show differential expression by qPCR in the 60-week old mice, however two others genes, MALAT1 and KEG, showed significant changes at other ages. This study shows that liver-specific glucokinase deficient mice display changes in kidney morphology by 40 weeks of age, and that renal complication may be correlated with a reduction in GPX3 levels. Since decreased GPX3 mRNA expression was observed at 26 weeks, which is younger than the age when pathological changes can be seen in kidney biopsies, GPX3 may serve as an early marker for kidney damage.