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fluorescence inverted microscope, inverted microscope, upright microscope, microscope, epifluorescent microscope, microscope, microscope, microscope, microscope, fluorescent microscope, widefield microscope, inverted microscope, microscope, microscope, microscope, microscope, inverted microscope, microscope, microscope, inverted microscope, inverted microscope, fluorescence microscope, microscope, fluorescence microscope, microscope, inverted microscope, microscope, microscope, microscope, inverted microscope, inverted microscope, microscope, microscope, laser, microscope magnification, microscope, microscope, inverted microscope, microscope, microscope, microscope, epifluorescence microscope, inverted phase/epifluorescent microscope
This model was found at
6323 locations
The model is used in
64 countries
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About the Nikon Eclipse E200

The model Nikon Eclipse E200 was found in 6323 unique locations in 64 countries where it was mentioned from 2003 until recentlyIt is used by scientists in various research fields such as Molecular Biology, Cell Biology, General Medicine, Biochemistry, and General Biochemistry, Genetics and Molecular Biology. The model is also used in Genetics, Cancer Research, Molecular Medicine, Biomedical Engineering, Oncology, Immunology, General Chemistry, Bioengineering, Biomaterials, Cellular and Molecular Neuroscience, Biophysics, Organic Chemistry, Pharmacology, Physiology, Biotechnology, General Neuroscience, Physical and Theoretical Chemistry, General Physics and Astronomy, General Materials Science, Pharmaceutical Science, Spectroscopy, Microbiology, Developmental Biology, Immunology and Allergy, and Catalysis.
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Research that uses the Nikon Eclipse E200

Helena M. Viola, Ashay A. Shah, Victoria P. A. Johnstone, Henrietta Cserne Szappanos, Mark P. Hodson, Livia C. Hool, Proceedings of the National Academy of Sciences, 117, 23113-23124 (37), 2020
Significance Hypertrophic cardiomyopathy affects 1:500 of the general population. Current drug therapy is used to manage symptoms in patients. There is an unmet need for treatments that can prevent the cardiomyopathy. Here we identify biomarkers of hypertrophic cardiomyopathy resulting from causing cardiac troponin I mutation Gly203Ser, and present a safe, nontoxic, preventative approach for the treatment of associated cardiomyopathy.
Liangkun Xie, Zheng Guan, Mingzhu Zhang, Sha Lyu, Nattawut Thuaksuban, Suttatip Kamolmattayakul, Thongchai Nuntanaranont, Takashi Yazawa, BioMed Research International, 2020, 1-13, 2020

Human dental pulp stem cells (DPSCs) hold great promise in bone regeneration. However, the exact mechanism of osteogenic differentiation of DPSCs remains unknown, especially the role of exosomes played in. The DPSCs were cultured and received osteogenic induction; then, exosomes from osteogenic-induced DPSCs (OI-DPSC-Ex) at different time intervals were isolated and sequenced for circular RNA (circRNA) expression profiles. Gradually, increased circular lysophosphatidic acid receptor 1 (circLPAR1) expression was found in the OI-DPSC-Ex coincidentally with the degree of osteogenic differentiation. Meanwhile, results from osteogenic differentiation examinations showed that the OI-DPSC-Ex had osteogenic effect on the recipient homotypic DPSCs. To investigate the mechanism of exosomal circLPAR1 on osteogenic differentiation, we verified that circLPAR1 could competently bind to hsa-miR-31, by eliminating the inhibitory effect of hsa-miR-31 on osteogenesis, therefore promoting osteogenic differentiation of the recipient homotypic DPSCs. Our study showed that exosomal circRNA played an important role in osteogenic differentiation of DPSCs and provided a novel way of utilization of exosomes for the treatment of bone deficiencies.

Jureepon Roboon, Tsuyoshi Hattori, Hiroshi Ishii, Mika Takarada-Iemata, Dinh Thi Nguyen, Collin D. Heer, Denis O'Mealley, Charles Brenner, Yasuhiko Yamamoto, Hiroshi Okamoto, Haruhiro Higashida, Osamu Hori, 2020
Abstract Background Neuroinflammation is initiated by the activation of the brain’s innate immune system in response to an inflammatory challenge. Insufficient control of neuroinflammation leads to enhanced or prolonged pathology in neurological conditions, including multiple sclerosis, traumatic brain injury, and Alzheimer’s disease. Nicotinamide adenine dinucleotide (NAD+) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that the deletion of CD38, an enzyme that converts NAD+ to calcium-mobilizing second messengers, increased NAD+ levels in the brain and suppressed neuroinflammation, glial activation, and demyelination in a cuprizone-induced demyelination model mouse. However, the direct effects of CD38 and NAD+ on neuroinflammation have not been clarified. Here, we investigated the effect of CD38 inhibition and NAD+ replacement in lipopolysaccharide (LPS)-induced neuroinflammation in mice. Methods To induce neuroinflammation, LPS (10 µg) was injected into the lateral cerebral ventricle of wild-type (WT) and CD38 knockout (KO) male ICR mice. Apigenin, a flavonoid with CD38 inhibitory activity, (40 mg/kg) or nicotinamide riboside (NR), an NAD+ precursor, (400 mg/kg) was administered intraperitoneally, once per day for 7 consecutive days, followed by LPS injection 6 h after the final administration of apigenin or NR. NAD+ levels in the hippocampus were measured, and neuroinflammation and neuronal damage in the hippocampus were assessed by qPCR, western blotting, and immunohistochemical analysis. In cell culture, mouse primary astrocytes and microglia were treated with apigenin (50 µM), NAD+ (200 µM), NR (200 µM), or 78c (0.5 µM; a specific CD38 inhibitor), 4 h before LPS (100 ng/mL) stimulation. Proinflammatory cytokine expression and NF-kB nuclear translocation were assessed by qPCR and immunocytochemical analysis, respectively. Results CD38 expression in the cortex and hippocampus increased after LPS administration. Inflammatory responses and glial activation after LPS injection were significantly lower in CD38 KO mice than in WT mice. Pre-administration of apigenin or NR for 7 d increased NAD+ levels in the brain and significantly suppressed the induction of cytokines and chemokines after LPS administration in mice. Moreover, LPS-induced glial activation and neurodegeneration were significantly suppressed under the same conditions. In cell culture, LPS-induced inflammatory responses were suppressed by treatment of primary astrocytes or microglia with apigenin, NAD+, NR, or 78c. Finally, all these compounds suppressed the translocation of p65 to the nucleus by LPS in cultured microglia. Conclusions CD38-mediated neuroinflammation is linked to NAD+ consumption, and CD38 inhibition and NR supplementation may be beneficial for preventing neuroinflammation in pathological conditions.
Zhao-huan Zhang, Xiu-ling Liu, Yun-yi Zhu, Hai Huang, Xiao-hui Xu, BioMed Research International, 2020, 1-10, 2020
Cell division-related proteins are essential for the normal development and differentiation of cells and may be related to the occurrence of cancer and the drug resistance mechanism of cancer cells. The mitotic kinesin-like protein 1 (MKLP1) is a kinesin protein that has been involved in the assembly of the midzone/midbody during mitosis and cytokinesis. In this study, we found that the tail domain of MKLP1 exhibited an autoinhibitory effect on its motor activity. Overexpression of the tail domain in HEK293 cells blocked cytokinesis and caused bi-/multinucleation. It is possible that protein binding to the MKLP1 tail relieves this autoinhibition and induces the motility of MKLP1. We used the GST pull-down assay followed by the LC-MS/MS analysis and identified 54 MKLP1 tail domain-specific binding proteins. Further, we confirmed the MS result by coimmunoprecipitation and FRET that a serine/threonine kinase, p21-activated kinase 2 (PAK2), binding to MKLP1. Endogenous PAK2 expression was found to be identical to that of MKLP1 in HEK293 cells during cytokinesis. Finally, functional studies indicated that when PAK2 expression was downregulated by siRNA, MKLP1 underwent a change in its localization away from the midbody, and cell cytokinesis was subsequently impeded. This study presents a novel regulatory mechanism that PAK2 promotes the activation of MKLP1 and contributes to complete cell cytokinesis.
Shengjie Shi, Xiaoge Zhou, Jingjing Li, Lutong Zhang, Yamei Hu, Yankun Li, Gongshe Yang, Guiyan Chu, 2020
Abstract Background: Granulosa cells proliferation and estradiol synthesis significantly affect follicular development. The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows, indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on granulosa cells are unclear. In this study, miR-214-3p was transfected into porcine ovarian granulosa cells to investigate its functions in terms of proliferation and estradiol synthesis via flow cytometry, CCK-8 assay, EdU staining, ElisA, Real-Time PCR, and Western blot analyses. We also identified the targets of miR-214-3p via Luciferase Reporter Assay. Results: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells. We also found that miR-214-3p up-regulates the expression of cell cycle genes including Cell cycle protein B (Cyclin B), Cell cycle protein D (Cyclin D), Cell cycle protein E (Cyclin E), and Cyclin-dependent kinase 4 (CDK4) at the transcription and translation levels, while down-regulating the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), and steroidogenic acute regulatory protein (StAR) (i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively. Conclusions: Our findings suggest that miR-214-3p plays an important role in the functional regulation of porcine granulosa cells and therefore may be a target gene for regulating follicular development.
Daibang Nie, Jianying Zhang, Yiqin Zhou, Jiuyi Sun, Wang Wang, James H.-C. Wang, Stem Cells International, 2021, 1-10, 2021
The elderly population is prone to tendinopathy due to aging-related tendon changes such as cellular senescence and a decreased ability to modulate inflammation. Aging can render tendon stem/progenitor cells (TSCs) into premature senescence. We investigated the effects of rapamycin, a specific mTOR inhibitor, on the senescence of TSCs. We first showed that after treatment with bleomycin in vitro, rat patellar TSCs (PTSCs) underwent senescence, characterized by morphological alterations, induction of senescence-associated β-galactosidase (SA-β-gal) activity, and an increase in p53, p21, and p62 protein expression. Senescence of PTSCs was also characterized by the elevated expression of MMP-13 and TNF-α genes, both of which are molecular hallmarks of chronic tendinopathy. We then showed that rapamycin treatment was able to reverse the above senescent phenotypes and increase autophagy in the senescent PTSCs. The activation of autophagy and senescence rescue was, at least partly, due to the translocation of HMGB1 from the nucleus to the cytosol that functions as an autophagy promoter. By reducing TSC senescence, rapamycin may be used as a therapeutic to inhibit tendinopathy development in the aging population by promoting autophagy.
Heui Min Lim, Jongsung Lee, Myeong Jin Nam, See-Hyoung Park, Oxidative Medicine and Cellular Longevity, 2021, 1-19, 2021
Acetylshikonin, a naphthoquinone, is a pigment compound derived from Arnebia sp., which is known for its anti-inflammatory potential. However, its anticarcinogenic effect has not been well investigated. Thus, in this study, we focused on investigating its apoptotic effects against HCT-15 and LoVo cells, which are human colorectal cancer cells. MTT assay, cell counting assay, and colony formation assay have shown acetylshikonin treatment induced cytotoxic and antiproliferative effects against colorectal cancer cells in a dose- and time-dependent manner. DNA fragmentation was observed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Also, the increase of subG1 phase in cell cycle arrest assay and early/late apoptotic rates in annexin V/propidium iodide (PI) double staining assay was observed, which indicates an apoptotic potential of acetylshikonin against colorectal cancer cells. 2 ,7 -Dichlorofluorescin diacetate (DCF-DA) staining was used to evaluate reactive oxygen species (ROS) generation in acetylshikonin-treated colorectal cancer cells. Fluorescence-activated cell sorting (FACS) analysis showed that acetylshikonin induced an increase in reactive oxygen species (ROS) levels and apoptotic rate in a dose- and time-dependent manner in HCT-15 and LoVo cells. In contrast, cotreatment with N-acetyl cysteine (NAC) has reduced ROS generation and antiproliferative effects in colorectal cancer cells. Western blotting analysis showed that acetylshikonin treatment induced increase of cleaved PARP, γH2AX, FOXO3, Bax, Bim, Bad, p21, p27, and active forms of caspase-3, caspase-7, caspase-9, caspase-6, and caspase-8 protein levels, while those of inactive forms were decreased. Also, the expressions of pAkt, Bcl-2, Bcl-xL, peroxiredoxin, and thioredoxin 1 were decreased. Furthermore, western blotting analysis of cytoplasmic and nuclear fractionated proteins showed that acetylshikonin treatment induced the nuclear translocation of FOXO3, which might result from DNA damage by the increased intracellular ROS level. This study represents apoptotic potential of acetylshikonin against colorectal cancer cells via translocation of FOXO3 to the nucleus and upregulation of ROS generation.
Sanjay Kumar Tiwari, Suping Wang, Yannan Huang, Xuedong Zhou, Hockin H. K. Xu, Biao Ren, Xian Peng, Yan Xiao, Mingyun Li, Lei Cheng, BioMed Research International, 2021, 1-10, 2021
Quaternary ammonium methacrylates (QAMs) are useful antimicrobial compounds against oral bacteria. Here, we investigated the effects of two QAMs, dimethylaminododecyl methacrylate (DMADDM) and dimethylaminohexadecyl methacrylate (DMAHDM), on biofilm formation, survival and development of tolerance by biofilm, and survival and development of tolerance against QAMs after prolonged starvation. Enterococcus faecalis (E. faecalis), Streptococcus gordonii (S. gordonii), Lactobacillus acidophilus (L. acidophilus), and Actinomyces naeslundii (A. naeslundii) were used. Minimum inhibitory concentration (MIC) of QAMs against multispecies biofilm was determined. Biofilm formed under sub-MIC was observed by crystal violet staining and confocal laser scanning microscopy (CLSM). Metabolic activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactic acid production measurement. Development of tolerance was determined by MIC values before and after exposure to QAMs or after prolonged starvation. It was found that E. faecalis and S. gordonii could survive and form biofilm under sub-MIC of QAMs. Lactic acid production from biofilms formed under sub-MIC was significantly higher than control specimens ( p < 0.05 ). The exposure to sub-MIC of QAMs promoted biofilm formation, and prolonged starvation or prolonged contact with sub-MIC helped bacteria develop tolerance against killing by QAMs.
HyangI Lim, Do Kyung Kim, Tae-Hyeon Kim, Kyeong-Rok Kang, Jeong-Yeon Seo, Seung Sik Cho, Younghee Yun, Ye-yong Choi, Jungtae Leem, Hyoun-Woo Kim, Geon-Ung Jo, Chan-Jin Oh, Deuk-Sil Oh, Hong-Sung Chun, Jae-Sung Kim, Oxidative Medicine and Cellular Longevity, 2021, 1-16, 2021
Osteoarthritis (OA) is the most common degenerative joint disease with chronic joint pain caused by progressive degeneration of articular cartilage at synovial joints. Acteoside, a caffeoylphenylethanoid glycoside, has various biological activities such as antimicrobial, anti-inflammatory, anticancer, antioxidative, cytoprotective, and neuroprotective effect. Further, oral administration of acteoside at high dosage does not cause genotoxicity. Therefore, the aim of present study is to verify the anticatabolic effects of acteoside against osteoarthritis and its anticatabolic signaling pathway. Acteoside did not decrease the viabilities of mouse fibroblast L929 cells used as normal cells and primary rat chondrocytes. Acteoside counteracted the IL-1β-induced proteoglycan loss in the chondrocytes and articular cartilage through suppressing the expression and activation of cartilage-degrading enzyme such as matrix metalloproteinase- (MMP-) 13, MMP-1, and MMP-3. Furthermore, acteoside suppressed the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in the primary rat chondrocytes treated with IL-1β. Subsequently, the expression of proinflammatory cytokines was decreased by acteoside in the primary rat chondrocytes treated with IL-1β. Moreover, acteoside suppressed not only the phosphorylation of mitogen-activated protein kinases in primary rat chondrocytes treated with IL-1β but also the translocation of NFκB from the cytosol to the nucleus through suppression of its phosphorylation. Oral administration of 5 and 10 mg/kg acteoside attenuated the progressive degeneration of articular cartilage in the osteoarthritic mouse model generated by destabilization of the medial meniscus. Our findings indicate that acteoside is a promising potential anticatabolic agent or supplement to attenuate or prevent progressive degeneration of articular cartilage.
Jing Gao, Lingjuan Shi, Jiayi Chen, Weizhi Lu, Jingtai Cai, Wei Zhao, Bao Zhang, 2020
Abstract Background: Zika virus is among the most widely transmitted arboviruses in the world and closely associated with diseases, such as encephalitis, fetal microcephaly, and Guillain–Barré syndrome. The pathogenic mechanism of the virus has not been fully elucidated, and there are no vaccines or specific drugs targeting the virus. To address these issues, the application of reverse genetics is needed for viral reconstruction and reproduction.Methods: Polymerase chain reaction (PCR) was used to merge the full-length Zika virus genome, CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into a pBAC11 vector through recombination to produce recombinant pBAC-ZIKA-EGFP. The ZIKA–EGFP was rescued by transfection of 293T cells with pBAC-ZIKA-EGFP, and at 7-days post-transfection, the supernatant (P0 generation) was passed through a 0.45-μm membrane and used to infect Vero cells (to produce the P1 generation). Fluorescence-based quantitative PCR, 50% tissue culture infectious dose, and plaque assays were used to measure differences in replication ability and pathogenicity relative to the rescue virus (ZIKA–WT), the sequence of which is consistent with that of the wild-type Zika virus. Additionally, caffeic acid phenethyl ester (CAPE), a nuclear factor kappaB (NF-kB) inhibitor, was used to examine its effect on viral replication.Results: The results showed that ZIKA–EGFP could effectively infect Vero cells, SH-SY5Y cells and C6/36 cells, and cause cytopathic effects on them. ZIKA–EGFP exhibited stable replication and EGFP expression during cell passage for at least six generations, with no significant difference in replication ability relative to the ZIKA–WT. Fluorescent cell foci were observed in the plaque assay while the ZIKA–EGFP was in the absence of phage plaque formation. The inhibition of NF-kB inhibitor on ZIKA-EGFP was observed by fluorescence microscopy, which was consistent with the results of fluorescence quantitative PCR.Conclusions: We constructed an infectious clone of the full-length genome of Zika virus which could replicate with stable EGFP expression in eukaryotic cells during passage. The infectious clone, remaining main characteristics of wild type ZIKA virus could be appied on the studies of reverse genetics, drug screening and gene function of ZIKA virus.
HyangI Lim, Do Kyung Kim, Tae-Hyeon Kim, Kyeong-Rok Kang, Jeong-Yeon Seo, Sung Sik Cho, Younghee Yun, Ye-yong Choi, Jungtae Leem, Hyoun-Woo Kim, Geon-Ung Cho, Chan-Jin Oh, Deuk-Sil Oh, Hong-Sung Chun, jae-sung kim, 2020
Abstract Objectives To verify the anti-catabolic effects of acteoside [α-L-rhamnosyl-(1->3)-β-D-glucoside] against osteoarthritis and its anti-catabolic signaling pathway. Methods Primary rat chondrocytes were isolated enzymatically from the articular cartilage of rat knee joint. Cytotoxicity of acteoside was assessed by MTT and Cell Live/Dead assay. Proteoglycan content was measured by dimethylmethylene blue assay. The proteoglycan loss was assessed by histological analysis using safranin-O & fast green staining after ex vivo organ culture of articular cartilage. The alteration of catabolic factors such as cartilage degrading enzymes, pro-inflammation cytokines, and inflammatory mediators were assessed by qPCR, qRT-PCR, gelatin zymography, western blot, and cytokine array. Cellular signaling pathways were investigated by western blot and nucleus translocation. Acteoside was orally administrated to osteoarthritic animals generated by the destabilization of medial meniscus at the knee joint of mice for 8 weeks. Thereafter, proteoglycan loss was assessed by safranin-O & fast green staining. Results Acteoside did not decrease the viabilities of mouse fibroblast L929 cells used as a normal cells and primary rat chondrocytes. Acteoside counteracted the IL-1β-induced proteoglycan loss in the chondrocytes and articular cartilage through suppressing the expression and activation of cartilage-degrading enzyme such as matrix metalloproteinase (MMP)-13, MMP-1, and MMP-3. Furthermore, acteoside suppressed the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in the primary rat chondrocytes treated with IL-1β. Subsequently, the expression of pro-inflammatory cytokines was decreased by acteoside in the primary rat chondrocytes treated with IL-1β. Moreover, acteoside suppressed not only the phosphorylation of mitogen-activated protein kinases in primary rat chondrocytes treated with IL-1β but also the translocation of NFκB from the cytosol to the nucleus through suppression of its phosphorylation. Oral administration of 5 and 10 mg/kg acteoside attenuated the progressive degeneration of articular cartilage in the osteoarthritic mouse model generated by destabilization of the medial meniscus. Conclusion Our findings indicate that acteoside is a promising potential anti-catabolic agent or supplement to attenuate or prevent progressive degeneration of articular cartilage.
Mei Shi, Li-Na Zhao, Rui Ma, Hua Yang, Chang-Hao Liu, Jing Hu, 2020
Abstract Background Distant metastasis remained the major cause of mortality in nasopharyngeal carcinoma (NPC) receiving radiotherapy despite favorable locoregional control in the past decades. Tumor microenvironment plays a critical role in tumor progression by secreting factors that regulate cancer cell metastasis. We explored the role of secreted protein Slit2 in NPC metastasis and the underlying mechanisms. Methods Colony formation assay were determined the opitimal dose. Using real-time PCR, western blotting, and ELISA, we detected the expression of Slit2 in NPC cells and in supernatant. Cell migration assay and cell invasion assay were examined the role of Slit2 in promoting metastasis of NPC cell lines. Western blotting, immunofluorescence staining, transwell and Co-IP assay et al. were performed to explore the detailed molecular mechanism of Slit2 in NPC. Finally, we estimated the effects on Slit2-shRNA NPC cells in vivo tumor metastasis experiment Results We found that irradiated dying NPC cells could secrete Slit2 to enhance the metastatic ability of surviving NPC cells. Mechanistic study showed that Slit2 interacted with its cognate Robo1 receptor, which activated PI3K/Akt pathway and enhanced the nuclear translocation of β-catenin to promote NPC metastasis. Finally, we demonstranted that knockdown of Slit2 in NPC cells significantly suppressed lung and liver metastasis. Conclusion Slit2, a secreted factor released from irradiated dying NPC cells could promote metastasis of living/resistent NPC cells through PI3K/AKT/β-catenin pathway. Tumor microenvironment altered by irradiated dying cancer cells may be promising target to treat cancer metastasis.
XiaoMei Huang, ZeXun Mo, YuJun Li, Hua He, KangWei Wang, NingNing Xu, WeiHong Guo, WanNa Tang, Shuquan Wei, 2020
Abstract Background Nuclear factor kappa-B (NF-κB) activation increased the expression of cytokines and further lead to lung injury was considered the main mechanism of acute lung injury (ALI). Here, we focus on exploring the potential regulatory mechanism between long noncoding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) and NF-κB on LPS-induced ALI. Methods A549 cells were then divided into 4 groups: HOTAIR group, NC group, si-HOTAIR group and si-NC group. These 4 groups were then treated with 1μg/mL lipopolysaccharides (LPS) or without LPS at 37°C for 24 h. The expression level of cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6) and LncRNA HOTAIR were evaluated by quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and Enzyme-linked immunosorbent assay (ELISA). Western Blot analysis was adopted for evaluating the level of p-IκBα/IκBα and p-p65/p65. Nuclear translocation of p65 was observed by immunofluorescence staining. Results qRT-PCR and ELISA assay showed that the expression of cytokines (IL-1β, IL-6 and TNF-α) and inflammatory gene HOTAIR was remarkably increased with LPS treatment (p < 0.01). Over-expression of HOTAIR significantly increased the expression of cytokines (including IL-1β, IL-6 and TNF-α) and NF-κB pathway associated proteins (including p-IκBα/IκBα and p-p65/p65), while knockdown of HOTAIR had the opposite effect (p < 0.01). The immunofluorescence assay showed that the level of p65 in the nucleus was significantly higher in the HOTAIR group and significantly lowers in the si-HOTAIR group (p < 0.01). Conclusion HOTAIR may play a pro-inflammatory response through NF-κB pathway in LPS-induced ALI, which may provide a perspective for further understanding the pathogenic mechanism of ALI.
Anastasia Kariagina, Olena Morozova, Reyhane Hoshyar, Mark D. Aupperlee, Mitchell A. Borin, Sandra Z. Haslam, Richard C. Schwartz, 2020
Abstract Background Benzophenone-3 is a putative endocrine disrupting chemical and common active ingredient in sunscreens and personal care products. The potential of endocrine disrupting chemicals to act as agonists or antagonists in critical hormonally regulated processes, such as mammary gland development and mammary tumorigenesis, demands evaluation of their potential in promoting breast cancer. We previously demonstrated promotion of mammary tumorigenesis by a diet high in saturated animal fat. This study examines the activity of benzophenone-3 in a dietary context to provide insight into its potential role in promoting breast cancer, and how diet might influence this. Methods Mammary tumorigenesis was studied in a BALB/c mouse Trp53-null transplantation model. Three-week-old mice were fed low-fat or high-fat diets, and at ten weeks of age were switched to high-fat or low-fat diets, respectively, while other mice were maintained continuously on low-fat diet. Mice additionally were treated continuously with or without benzophenone-3. The level of benzophenone-3 exposure yielded levels in urine similar to that observed in humans subjected to heavy topical exposure of benzophenone-3-containing commercial sunscreen. Results Benzophenone-3 had complex effects that were dependent upon diet and tumor histopathology. Benzophenone-3 was protective in regard to epithelial tumorigenesis in mice fed low-fat diet and was promotional for epithelial tumorigenesis in mice fed high-fat diet restricted to adulthood. It increased tumor cell proliferation, decreased tumor cell apoptosis, and increased tumor vascularity in a manner dependent on specific dietary regimen and tumor histopathology. Protective effects were not always concordant with a decrease in properties associated with tumor progression. Notably, although benzophenone-3 seemed protective for tumorigenesis in mice fed low-fat diet, spindle cell tumors that arose in these mice showed increased proliferation and decreased apoptosis. Conclusions Benzophenone-3 elicits promotional and protective effects on mammary tumorigenesis dependent upon diet and tumor histopathology. However, even in instances of an ostensibly protective effect, other parameters suggest potential for greater risk. This points to a need for further studies of benzophenone-3 in both animal models and humans as a potential breast cancer risk factor, as well as a more general need to evaluate endocrine disrupting chemicals in the context of varying diets.
Zimo Zhao, Ling Yang, Dan Zhang, Zi Zheng, Ning Li, Qianjun Li, Maosheng Cui, PeerJ, 8, e9913, 2020
The MPF and MAPK genes play crucial roles during oocyte maturation processes. However, the pattern of MPF and MAPK gene expression induced by melatonin (MT) and its correlation to oocyte maturation quality during the process of porcine oocyte maturation in vitro remains unexplored. To unravel it, in this study, we cultured the porcine oocytes in maturation medium supplemented with 0, 10−6, 10−9, and 10−12 mol/L melatonin. Later, we analyzed the MPF and MAPK gene expression levels by RT-PCR and determined the maturation index (survival and maturation rate of oocytes). The GSH content in the single oocyte, and cytoplasmic mitochondrial maturation distribution after porcine oocyte maturation in vitro was also evaluated. We also assessed the effects of these changes on parthenogenetic embryonic developmental potential. The oocytes cultured with 10−9mol/L melatonin concentration showed higher oocyte maturation rate, and MPF and MAPK genes expression levels along with better mitochondrial distribution than the 0, 10−6, and 10−12 mol/L melatonin concentrations (p < 0.05). No significant difference was observed in the survival rates when the oocytes were cultured with different melatonin concentrations. The expression of the MPF gene in the oocytes cultured with 10−6 mol/L melatonin was higher than with 10−12 and 0 mol/L melatonin, and the expression of the MAPK gene in 10−6 and 10−12 group was higher than the control (p < 0.05). As far as the embryonic developmental potential is concerned, the cleavage and blastocyst rate of oocytes cultured with 10−6 and 10−9 mol/L melatonin was significantly higher than the 10−12 mol/L melatonin and control. In conclusion, 10−9–10−6 mol/L melatonin significantly induced the MPF and MAPK gene expression; besides, it could also be correlated with GSH content of single oocyte, mitochondrial maturation distribution, and the first polar body expulsion. These changes were also found to be associated with parthenogenetic embryo developmental potential in vitro.
Margarida Rodrigues, Payel Bhattacharjee, Ann Brinkmalm, Dung Do, Colin Pearson, Suman De, Aleks Ponjavic, Juan Varela, Francesco Ruggeri, Isabelle Baudrexel, Ji Lee, Alexander Carr, Klara Kulenkampff, Tuomas Knowles, Henrik Zetterberg, Thomas Snaddon, Sonia Gandhi, Steven Lee, David Klenerman, 2020
Abstract The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. We introduce the structure-specific chemical antibody; a Y shaped, bioinspired small molecule with a dimeric region to mimic avidity, and an attachment region to mimic the Fc region. Our probe, capture molecule for amyloid precipitation (CAP-1), consists of a derivative of Pittsburgh compound B (dimer) to target the cross β-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we targeted the amyloid structure of protein aggregates in human cerebrospinal fluid, isolated them for analysis and then characterised them using single-molecule fluorescence imaging and mass spectrometry. AP allows unbiased determination of the molecular composition and structural features of the in vivo aggregates, formed in neurodegenerative diseases, that are present in biofluids.
Shushan Li, Sabine Stöckl, Christoph Lukas, Julia Götz, Marietta Herrmann, Marianne Federlin, Susanne Grässel, Frontiers in Bioengineering and Biotechnology, 8, 2020
Background: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) provide a promising therapeutic approach in the cell-based therapy of osteoarthritis (OA). However, several disadvantages evolved recently, including immune responses of the host and regulatory hurdles, making it necessary to search for alternative treatment options. Extracellular vesicles (EVs) are released by multiple cell types and tissues into the extracellular microenvironment, acting as message carriers during intercellular communication. Here, we investigate putative protective effects of hBMSC-derived EVs as a cell-free approach, on IL-1β-stimulated chondrocytes obtained from OA-patients.Methods: EVs were harvested from the cell culture supernatant of hBMSCs by a sequential ultracentrifugation process. Western blot, scanning electron microscopy (SEM), and nanoparticle tracking analysis (NTA) were performed to characterize the purified particles as EVs. Intracellular incorporation of EVs, derived from PHK26-labeled hBMSCs, was tested by adding the labeled EVs to human OA chondrocytes (OA-CH), followed by fluorescence microscopy. Chondrocytes were pre-stimulated with IL-1β for 24 h, followed by EVs treatment for 24 h. Subsequently, proliferation, apoptosis, and migration (wound healing) were analyzed via BrdU assay, caspase 3/7 assay, and scratch assay, respectively. With qRT-PCR, the relative expression level of anabolic and catabolic genes was determined. Furthermore, immunofluorescence microscopy and western blot were performed to evaluate the protein expression and phosphorylation levels of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB as components of pro-inflammatory signaling pathways in OA-CH.Results: EVs from hBMSCs (hBMSC-EVs) promote proliferation and reduce apoptosis of OA-CH and IL-1β-stimulated OA-CH. Moreover, hBMSC-EVs attenuate IL-1β-induced reduction of chondrocyte migration. Furthermore, hBMSC-EVs increase gene expression of PRG4, BCL2, and ACAN (aggrecan) and decrease gene expression of MMP13, ALPL, and IL1ß in OA-CH. Notably, COL2A1, SOX9, BCL2, ACAN, and COMP gene expression levels were significantly increased in IL-1β+ EV groups compared with those IL-1β groups without EVs, whereas the gene expression levels of COLX, IL1B, MMP13, and ALPL were significantly decreased in IL-1β+ EV groups compared to IL-1β groups without EVs. In addition, the phosphorylation status of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling molecules, induced by IL-1β, is prevented by hBMSC- EVs.Conclusion: EVs derived from hBMSCs alleviated IL-1β-induced catabolic effects on OA-CH via promoting proliferation and migration and reducing apoptosis, probably via downregulation of IL-1ß-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways. EVs released from BMSCs may be considered as promising cell-free intervention strategy in cartilage regenerative medicine, avoiding several adverse effects of cell-based regenerative approaches.
Peng Zhang, Kang Yang, Ziyu Zhou, Xingrong Zhu, Wenchao Li, Chuanliang Cao, Kui Zhou, Lan Liao, Fanrong Ai, Frontiers in Bioengineering and Biotechnology, 8, 2020
Graft reconstruction of the mandible is an important approach that aims at improving the appearance and functionality of defected mandibles. The traditional implant materials are generally bioinert, non-degradable, and that they lack favorable pore structures for cell proliferation, which limit their clinical application. In this study, we used boron-containing bioactive glass which was combined with a three-dimensional (3D) printing technology to construct an osteoinductive implant scaffold, according to the imaging instructions of CT scan on bone defects. Here, the boron-containing bioglass scaffold (B-BGs) was prepared through sol-gel processing and a 3D print technique. Different boron content of borosilicate bioglass was prepared by incorporating B2O3 (molar: 19.4 and 38.8%) into 58S bioglass to replace parts of SiO2. For fabricated mandible implants through three-dimensional 3D printing of B-BGs (size: 8 × 2 mm; pore size: 250 μm) modified with borosilicate bioglass powder and sodium alginate. Notably, the compressive strength of the B-BGs was about 3.8 Mpa, which supported mandibular activity. Subsequently, the excellent biocompatibility of B-BGs was confirmed using cytotoxicity in vitro studies. Finally, data from in vivo experiments demonstrated that the B-BGs could promote bone regeneration and they could almost get completely degraded within 4 weeks. Our results showed that the boron-containing bioglass could repair mandibular defects.
Tanja Niedermair, Christoph Lukas, Shushan Li, Sabine Stöckl, Benjamin Craiovan, Christoph Brochhausen, Marianne Federlin, Marietta Herrmann, Susanne Grässel, Frontiers in Bioengineering and Biotechnology, 8, 2020
Background: Studies with extracellular vesicles (EVs), including exosomes, isolated from mesenchymal stem cells (MSC) indicate benefits for the treatment of musculoskeletal pathologies as osteoarthritis (OA) and osteoporosis (OP). However, little is known about intercellular effects of EVs derived from pathologically altered cells that might influence the outcome by counteracting effects from “healthy” MSC derived EVs. We hypothesize, that EVs isolated from osteoblasts of patients with hip OA (coxarthrosis/CA), osteoporosis (OP), or a combination of both (CA/OP) might negatively affect metabolism and osteogenic differentiation of bone-marrow derived (B)MSCs.Methods: Osteoblasts, isolated from bone explants of CA, OP, and CA/OP patients, were compared regarding growth, viability, and osteogenic differentiation capacity. Structural features of bone explants were analyzed via μCT. EVs were isolated from supernatant of naïve BMSCs and CA, OP, and CA/OP osteoblasts (osteogenic culture for 35 days). BMSC cultures were stimulated with EVs and subsequently, cell metabolism, osteogenic marker gene expression, and osteogenic differentiation were analyzed.Results: Trabecular bone structure was different between the three groups with lowest number and highest separation in the CA/OP group. Viability and Alizarin red staining increased over culture time in CA/OP osteoblasts whereas growth of osteoblasts was comparable. Alizarin red staining was by trend higher in CA compared to OP osteoblasts after 35 days and ALP activity was higher after 28 and 35 days. Stimulation of BMSC cultures with CA, OP, and CA/OP EVs did not affect proliferation but increased caspase 3/7-activity compared to unstimulated BMSCs. BMSC viability was reduced after stimulation with CA and CA/OP EVs compared to unstimulated BMSCs or stimulation with OP EVs. ALP gene expression and activity were reduced in BMSCs after stimulation with CA, OP, and CA/OP EVs. Stimulation of BMSCs with CA EVs reduced Alizarin Red staining by trend.Conclusion: Stimulation of BMSCs with EVs isolated from CA, OP, and CA/OP osteoblasts had mostly catabolic effects on cell metabolism and osteogenic differentiation irrespective of donor pathology and reflect the impact of tissue microenvironment on cell metabolism. These catabolic effects are important for understanding differences in effects of EVs on target tissues/cells when harnessing them as therapeutic drugs.
Elizabeth M. Boazak, Rebecca King, Jiaxing Wang, Cassandra M. Chu, Aaron M. Toporek, Joseph M. Sherwood, Darryl R. Overby, Eldon E. Geisert, C. Ross Ethier, Frontiers in Bioengineering and Biotechnology, 9, 2021
The biomechanical properties of the cornea and sclera are important in the onset and progression of multiple ocular pathologies and vary substantially between individuals, yet the source of this variation remains unknown. Here we identify genes putatively regulating corneoscleral biomechanical tissue properties by conducting high-fidelity ocular compliance measurements across the BXD recombinant inbred mouse set and performing quantitative trait analysis. We find seven cis-eQTLs and non-synonymous SNPs associating with ocular compliance, and show by RT-qPCR and immunolabeling that only two of the candidate genes, Smarce1 and Tns4, showed significant expression in corneal and scleral tissues. Both have mechanistic potential to influence the development and/or regulation of tissue material properties. This work motivates further study of Smarce1 and Tns4 for their role(s) in ocular pathology involving the corneoscleral envelope as well as the development of novel mouse models of ocular pathophysiology, such as myopia and glaucoma.
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