Quality assessment of variable collagen tissues of sea cucumber (Stichopus japonicus) body wall under different heat treatment durations by label-Free proteomics analysis.

The microstructure of the body wall, body wall composition, and collagen fibers of sea cucumber (Stichopus japonicus) under different heating times (1 h, 4 h, 12 h, and 24 h) was investigated based on heat treatment at 80 °C. A Label-Free proteomics technique was applied to study the proteomic changes in the body wall of sea cucumbers under 4 and 12 h of heat treatment. Compared with the fresh group, 981 proteins were found to be differentially expressed proteins (DEPs) after heat treatment at 80 °C (4 h), and 1110 DEPs were observed after heat treatment at the same temperature for 12 h. There were 69 DEPs associated with mutable collagenous tissues (MCTs) structures. The results of correlation analysis showed that 55 DEPs were correlated with sensory properties, among which A0A2G8KRV2 was significantly correlated with hardness and SEM image texture features (SEM_Energy, SEM_Correlation, SEM_Homogeneity, and SEM_Contrast). These findings could be conducive to further comprehension of the structural changes and mechanisms of quality loss in the body wall of sea cucumbers at different heat treatment times.

Effects of TYROBP Deficiency on Neuroinflammation of a Alzheimer's Disease Mouse Model Carrying a PSEN1 p.G378E Mutation.

Objective To study the effects of TYRO protein kinase-binding protein (TYROBP) deficiency on learning behavior, glia activation and pro-inflammatory cycokines, and Tau phosphorylation of a new Alzheimer's disease (AD) mouse model carrying a PSEN1 p.G378E mutation.Methods A new AD mouse model carrying PSEN1 p.G378E mutation was built based on our previously found AD family which might be ascribed to the PSEN1 mutation, and then crossed with TYROBP deficient mice to produce the heterozygous hybrid mice (PSEN1G378E/WT; Tyrobp+/-) and the homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/-). Water maze test was used to detect spatial learning and memory ability of mice. After the mice were sacrificed, the hippocampus was excised for further analysis. Immunofluorescence was used to identify the cell that expresses TYROBP and the number of microglia and astrocyte. Western blot was used to detect the expression levels of Tau and phosphorylated Tau (p-Tau), and ELISA to measure the levels of pro-inflammatory cytokines. Results Our results showed that TYROBP specifically expressed in the microglia of mouse hippocampus. Absence of TYROBP in PSEN1G378E mutation mouse model prevented the deterioration of learning behavior, decreased the numbers of microglia and astrocytes, and the levels of interleukin-6, interleukin-1ß and tumor necrosis factor-α in the hippocampus (all P < 0.05). The ratios of AT8/Tau5, PHF1/Tau5, pT181/Tau5, pT231/Tau5 and p-ERK/ERK were all higher in homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/- mice) compared with PSEN1G378E/G378E mice (all P < 0.05). Conclusions TYROBP deficiency might play a protective role in the modulation of neuroinflammation of AD. However, the relationship between neuroinflammation processes involving microglia and astrocyte activation, and release of pro-inflammatory cytokines, and p-Tau pathology needs further study.

Correlation between metastasis-associated gene 1 expression and tumor-associated macrophages in non-small cell lung cancer.

The role of metastasis-associated gene 1 (MTA1) in the metastasis of non-small cell lung cancer (NSCLC) has been proved, but its role in the tumor microenvironment is still insufficient. The study was performed to explore the correlation between MTA1 and tumor-associated macrophages (TAMs) in NSCLC. The expression profile data of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were downloaded from TCGA database. The tumor-infiltrating immune cells in each LUAD and LUSC patient were estimated using the CIBERSORT method. Then, the online TIMER database containing multiple algorithms was used to analyze the relationship between MTA1 and TAMs. Besides, correlations between MTA1 and TAMs markers were also explored. Additionally, the immunohistochemistry staining of MTA1 protein and CD206 was performed in 75 NSCLC tissue specimens. Associations of MTA1 and CD206 with the clinicopathological characteristics were analyzed, as well as the correlation between MTA1 and CD206. Based on different algorithms, MTA1 expression was correlated with the distribution of infiltrating immune cells in the tumor microenvironment and negatively correlated with tumor immune-stromal score. MTA1 was associated with TAMs markers according to TCGA database. In 75 NSCLC tissue specimens, the positive rate of MTA1 was 60.00% (45/75), which of CD206 was 42.67% (32/75). The MTA1 expression was significantly correlated with T stage, lymph node metastasis, and TNM stage. The CD206 expression was significantly correlated with T stage, lymph node metastasis, TNM stage, and tumor type. Additionally, we found that MTA1 was positively correlated with CD206 in NSCLC and LUSC. In NSCLC, MTA1 expression was correlated with the infiltrations of different types of macrophages and the expression of TAMs markers, as well as the M2-TAMs marker CD206, suggesting that MTA1-promoting tumor metastasis may mediate the infiltration of different types of macrophages in the tumor microenvironment.

Effects of TYROBP Deficiency on Neuroinflammation of a Alzheimer's Disease Mouse Model Carrying a PSEN1 p.G378E Mutation / 中国医学科学杂志(英文版)

Objective To study the effects of TYRO protein kinase-binding protein (TYROBP) deficiency on learning behavior, glia activation and pro-inflammatory cycokines, and Tau phosphorylation of a new Alzheimer's disease (AD) mouse model carrying a PSEN1 p.G378E mutation.Methods A new AD mouse model carrying PSEN1 p.G378E mutation was built based on our previously found AD family which might be ascribed to the PSEN1 mutation, and then crossed with TYROBP deficient mice to produce the heterozygous hybrid mice (PSEN1G378E/WT; Tyrobp+/-) and the homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/-). Water maze test was used to detect spatial learning and memory ability of mice. After the mice were sacrificed, the hippocampus was excised for further analysis. Immunofluorescence was used to identify the cell that expresses TYROBP and the number of microglia and astrocyte. Western blot was used to detect the expression levels of Tau and phosphorylated Tau (p-Tau), and ELISA to measure the levels of pro-inflammatory cytokines. Results Our results showed that TYROBP specifically expressed in the microglia of mouse hippocampus. Absence of TYROBP in PSEN1G378E mutation mouse model prevented the deterioration of learning behavior, decreased the numbers of microglia and astrocytes, and the levels of interleukin-6, interleukin-1β and tumor necrosis factor-α in the hippocampus (all P < 0.05). The ratios of AT8/Tau5, PHF1/Tau5, pT181/Tau5, pT231/Tau5 and p-ERK/ERK were all higher in homozygous hybrid mice (PSEN1G378E/G378E; Tyrobp-/- mice) compared with PSEN1G378E/G378E mice (all P < 0.05). Conclusions TYROBP deficiency might play a protective role in the modulation of neuroinflammation of AD. However, the relationship between neuroinflammation processes involving microglia and astrocyte activation, and release of pro-inflammatory cytokines, and p-Tau pathology needs further study.

Effect of Heat Input on Porosity Defects in a Fiber Laser Welded Socket-Joint Made of Powder Metallurgy Molybdenum Alloy.

Porosity defects are still a challenging issue in the fusion welding of molybdenum and its alloys due to the pre-existing interior defects associated with the powder metallurgy process. Fiber laser welding of end plug and cladding tube made of nanostructured high-strength molybdenum (NS-Mo) alloy was performed in this work with an emphasis on the role of welding heat input. The distribution and morphology of porosity defects in the welded joints were examined by computed tomography (CT) and scanning electron microscopy (SEM). Preliminary results showed that laser welding of NS-Mo under low heat input significantly reduced the porosity defects in the fusion zone. The results of computed tomography (CT) showed that when the welding heat input decreased from 3600 J/cm (i.e., 1200 W, 0.2 m/min) to 250 J/cm (i.e., 2500 W, 6 m/min), the porosity ratio of the NS-Mo joints declined from 10.7% to 2.1%. Notable porosity defects under high heat input were related to the instability of the keyhole, expansion and the merging of bubbles in the molten pool, among which the instability of the keyhole played the dominant role. The porous defects at low heat input were generated as bubbles released from the powder metallurgy base metal (BM) did not have enough time to overflow and escape.

Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stromal Cells May Attenuate Sarcopenia in Aged Mice Induced by Hindlimb Suspension.

BACKGROUND Since the use of human umbilical cord Wharton's Jelly derived mesenchymal stromal cells (hWJ-MSCs) to treat sarcopenia has not been explored, we studied the effects of hWJ-MSCs in aged male C57BL/6J mice with sarcopenia induced by hindlimb suspension, and explored the potential mechanism. MATERIAL AND METHODS Hindlimb suspension was used to induce sarcopenia in 24-month-old C57BL/6J mice and green fluorescent protein-tagged hWJ-MSCs and controls were transplanted into mice via tail vein or local intramuscular injection. After hWJ-MSC transplantation, changes in whole body muscle strength and endurance, gastrocnemius muscle weight and myofiber cross-sectional area (CSA) were studied. Proliferation of skeletal muscle stem cell, apoptosis, and chronic inflammation were also investigated. RESULTS We demonstrated that whole body muscle strength and endurance, gastrocnemius muscle mass, and CSA were significantly increased in hWJ-MSC-transplanted mice than in controls (P<0.05). In hWJ-MSC-transplanted mice, apoptotic myonuclei was reduced, and BrdU and Pax-7 expression indices of gastrocnemius muscles were increased (P<0.05). Tumor necrosis factor (TNF)-α and interleukin (IL)-6 were downregulated, and IL-4 and IL-10 were upregulated (P<0.05). CONCLUSIONS hWJ-MSCs may ameliorate sarcopenia in aged male C57BL/6J mice induced by hindlimb suspension, and this may be via activation of resident skeletal muscle satellite cells, reduction of apoptosis, and less chronic inflammation.

[Effect of TEB-415, a Derivative of Imatinib, on Multiple Myeloma].

OBJECTIVE: To investigate the growth inhibitory effect of Imatinib derivative TEB-415 on various multiple myeloma (MM) cell lines, such as U226, H929, RPMI8226, MM1R and MM1S. METHODS: TEB-415, a derivative of Imatinib was synthesized by modifying the chemical structure of Imatinib. MM cell lines (U226, H929, RPMI8226, MM1R and MM1S) were treated with TEB-415, Imatini and Bortezomib of various concentrations. Cells were grown for 72 hours and the growth rate was measured by CCK-8 method, cell morphology was observed and the IC50 was calculated. RESULTS: TEB-415 could inhibit H929 and RPMI8226 growth significantly. When the concentration of TEB-415 was <0.1 nmol/L, >50% H929 cells died. The IC50 of Imatinib was 0.123 mol/L while the IC50 of Bortezomib was 0.03 nmol/L. In RPMI8226 cell line, when the concentration of TEB-415 was 11.9 mol/L, more than 50% of cells died. In contrast, when RPMI8266 were treated with Imatinib of the concentration of 12.8 mol/L, cells grew normally. CONCLUSION: In comparison to Imatinib, TEB-415, a derivative of Imatinib, can kill H929 MM cells much effectively, its effecacy is only inferior to Bortezomib. RPMI8226, an MM cell line is insensitive to Imatinib, but still sensitive to TEB-415 and its growth can be inhibited by TEB-415.

SIRT6 protects cardiomyocytes against ischemia/reperfusion injury by augmenting FoxO3α-dependent antioxidant defense mechanisms.

SIRT6, a member of the NAD(+)-dependent class III deacetylase sirtuin family, has been revealed to play important roles in promoting cellular resistance against oxidative stress. The formation of reactive oxygen species (ROS) and oxidative stress are the crucial mechanisms underlying cellular damage and dysfunction in cardiac ischemia/reperfusion (I/R) injury, but the role of SIRT6 in I/R-induced ROS and oxidative stress is poorly understood. In this study, by using heterozygous SIRT6 knockout (SIRT6(+/-)) mice and cultured neonatal cardiomyocyte models, we investigated how SIRT6 mediates oxidative stress and myocardial injury during I/R. Partial knockout (KO) of SIRT6 aggravated myocardial damage, ventricular remodeling, and oxidative stress in mice subjected to myocardial I/R, whereas restoration of SIRT6 expression by direct cardiac injection of adenoviral constructs encoding SIRT6 reversed these deleterious effects of SIRT6 KO in the ischemic heart. In addition, partial deletion of the SIRT6 gene decreased myocardial functional recovery following I/R in a Langendorff perfusion model. Similarly, the protective effects of SIRT6 were also observed in cultured cardiomyocytes following hypoxia/reoxygenation. Intriguingly, SIRT6 was noticed to up-regulate AMP/ATP and then activate the adenosine 5'-monophosphate-activated protein kinase (AMPK)-forkhead box O3α (FoxO3α) axis and further initiated the downstream antioxidant-encoding gene expression (manganese superoxide dismutase and catalase), thereby decreasing cellular levels of oxidative stress and mediating cardioprotection in the ischemic heart. These results suggest that SIRT6 protects the heart from I/R injury through FoxO3α activation in the ischemic heart in an AMP/ATP-induced AMPK-dependent way, thus upregulating antioxidants and suppressing oxidative stress.

Synergistic effects of co-administration of suicide gene expressing mesenchymal stem cells and prodrug-encapsulated liposome on aggressive lung melanoma metastases in mice.

The success of conventional suicide gene therapy for cancer treatment is still limited because of lack of efficient delivery methods, as well as poor penetration into tumor tissues. Mesenchymal stem cells (MSCs) have recently emerged as potential vehicles in improving delivery issues. However, these stem cells are usually genetically modified using viral gene vectors for suicide gene overexpression to induce sufficient therapeutic efficacy. This approach may result in safety risks for clinical translation. Therefore, we designed a novel strategy that uses non-viral gene vector in modifying MSCs with suicide genes to reduce risks. In addition, these cells were co-administrated with prodrug-encapsulated liposomes for synergistic anti-tumor effects. Results demonstrate that this strategy is effective for gene and prodrug delivery, which co-target tumor tissues, to achieve a significant decrease in tumor colonization and a subsequent increase in survival in a murine melanoma lung metastasis model. Moreover, for the first time, we demonstrated the permeability of MSCs within tumor nests by using an in vitro 3D tumor spheroid model. Thus, the present study provides a new strategy to improve the delivery problem in conventional suicide gene therapy and enhance the therapeutic efficacy. Furthermore, this study also presents new findings to improve our understanding of MSCs in tumor-targeted gene delivery.

[An improved method for generating integration-free human induced pluripotent stem cells].

The genome instability and tumorigenicity of induced pluripotent stem cells (iPSC) hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells (CB MNC), to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre-stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV (spleen focus forming virus) promoter under the hypoxic condition (3%), CB MNC could be mostly efficiently reprogrammed with the efficiency 0.12%. Furthermore, the results showed that erythroblasts (CD36(+)CD71(+)CD235a(low)) were the cells that are reprogrammed with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.

[Efficient reprogramming of human cord blood CD34(+) cells for formation of induced pluripotent stem cells with non-integrating plasmid system].

This study was to establish the episomal vector reprogramming method to reprogram iPSC from human cord blood (CB) CD34(+) cells. The non-integrating plasmids of pEB-C5 and pEB-Tg were transfected into short-term cultured CB CD34(+) cells by using the nucleofector, so as to demonstrate efficient reprogramming of CB CD34(+) cells. Within 14 days of one-time transfection by two plasmids together, up to 200 iPSC-like colonies per 2 million transfected CB CD34(+) cells were generated. The results showed that the pluripotency of iPSC-derived CB CD34(+) cells was similar to that of hESC and the karyotypes of iPSC were normal. In addition, no vector integration was found in iPSC of 9th and 10th passages. Furthermore, hiPSC formed teratoma with three embryonic germ layers. It is concluded that the integration-free method to generate human iPSC from CB CD34(+) cells is reliable and can provide new ways for both research and future clinical applications.

Detection of bovine central nervous system tissue as bovine spongiform encephalopathy risk material by real-time reverse transcriptase-PCR in raw and cooked beef products.

There is increasing evidence of the association of the new variant of Creutzfeldt-Jacob disease (nvCJD) in humans with bovine spongiform encephalopathy (BSE) in cattle. Many countries established legislation of banning central nervous system (CNS) tissues, which are regarded as BSE-specified risk materials (SRM), in human food supply because of the potential transmission of BSE to humans. A real-time reverse transcriptase-PCR assay using the bovine glial fibrillary acidic protein (GFAP) mRNA template for the detection of CNS tissues in raw and cooked beef products was developed in this study. The results showed that (1) this method can detect CNS tissues from bovine and ovine origins, but not from porcine and avian ones; (2) GFAP mRNA can only be detected from brain and spinal cords rather than other tissues; (3) the GFAP mRNA was detectable in CNS tissues even after dilution to 0.001%; and (4) the assay was unaffected by heat treatment at 100 degrees C for 30 min or storage at room temperature for 4 days, and at 4 degrees C for at least 15 days.

[Detection of beef contaminated by bovine central nervous system tissue by enzyme immunoassay].

OBJECTIVE: To study influencing factors of detection of bovine central nervous system (CNS) tissue contaminated beef by enzyme immunoassay (EIA), and the method was applied to the detection of imported beef and domestic beef of China. METHODS: Raw beef homogenates containing different concentrations of raw CNS tissue and the same samples which were heated were detected after different time by RIDASCREEN(r) Risk Material 10/5 and RIDASCREEN(r) Probennahme- zubehor Sampling tools kits. PBS suspension and sample dilution buffer (SDB) suspension of bovine brain tissue with the same concentration of the standard were detected. Beef from USA and domestic market of China were then detected by the kits. RESULTS: The kits could detect both raw and heated CNS tissue in the products with high sensitivity. The absorbance values (AV) increased with the concentrations of CNS in samples. Heating and increasing of time could decrease the absorbance values of the samples which contain CNS tissue. The AV of the PBS suspension of bovine brain tissue was higher than the SDB suspension and the AV of both were higher than the AV of standard of the same concentration. No CNS tissue was detected from all imported beef. No CNS tissue was detected in all samples from domestic market of China except for foxtail. CONCLUSION: The EIA method has high sensitivity for detection of bovine CNS tissue contaminated beef with the glial fibrillary acidic protein (GFAP) as accurate target substance. Heating and increasing of time can lead to decreasing of the AV of samples. Improper slaughter process can lead to contamination of bovine products by bovine CNS tissue.